METHODS AND COMPOSITIONS INVOLVING PLANTARICIN EF (PLNEF)

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 62/664,803, filed April 30, 2018, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

BACKGROUND

[0002] Diets high in fat and sugars can result in obesity and a condition known as“leaky” gut. Translocation of microbial products from the intestine to extra-intestinal organs drives metabolic dysfunction. Probiotic Lactobacillus are associated with improved metabolic health and intestinal barrier function, however the bacterial effectors resulting in these outcomes are unknown.

SUMMARY

[0003] In one aspect, the disclosure features a method of reducing gut inflammation in a subject in need thereof, comprising administering directly or indirectly to the subject a plantaricin E peptide (PlnE peptide) comprising a sequence that is substantially identical to a sequence of FNRGGYNF GKS VRHVVD AIGS V AGIRGILKSIR (SEQ ID NO: l) and a plantaricin F peptide (PlnF peptide) comprising a sequence that is substantially identical to a sequence of VFHAYS ARGVRNNYKS AV GP ADWVIS AVRGFIHG (SEQ ID NO:2), wherein the gut inflammation in the subject is reduced.

[0004] In some embodiments of this aspect, the method improves gut barrier function in the subject.

[0005] In another aspect, the disclosure features a method of improving gut barrier function in a subject in need thereof, comprising administering directly or indirectly to the subject a plantaricin E peptide (PlnE peptide) comprising a sequence that is substantially identical to a sequence of FNRGGYNF GKS VRHVVD AIGS V AGIRGILKSIR (SEQ ID NO: l) and a plantaricin F peptide (PlnF peptide) comprising a sequence that is substantially identical to a sequence of VFHAYS ARGVRNNYKS AVGP AD WVISAVRGFIHG (SEQ ID NO:2), wherein the gut barrier function in the subject is improved.

[0006] In some embodiments of this aspect, the method reduces gut inflammation in the subject. [0007] In some embodiments of the methods described herein, the subject is diagnosed with a disease associated with gut inflammation and/or compromised gut barrier function.

[0008] In another aspect, the disclosure features a method of treating or preventing a disease associated with gut inflammation and/or compromised gut barrier function in a subject in need thereof, comprising administering directly or indirectly to the subject a plantaricin E peptide (PlnE peptide) comprising a sequence that is substantially identical to a sequence of FNRGGYNF GKS VRHVVD AIGS V AGIRGILKSIR (SEQ ID NO: l) and a plantaricin F peptide (PlnF peptide) comprising a sequence that is substantially identical to a sequence of VFHAYSARGVRNNYKSAVGPADWVISAVRGFIHG (SEQ ID NO:2).

[0009] In some embodiments of the methods described herein, the disease associated with gut inflammation and/or compromised gut barrier function is selected from the group consisting of inflammatory bowel disease ( e.g ., Crohn's disease and ulcerative colitis), gastrointestinal infectious viral and bacterial diseases, celiac disease, microscopic colitis, diarrheal disease, bile acid malabsorption, irritable bowel disease, and colon cancer. In some embodiments, the diseases associated with gut inflammation and/or compromised gut barrier function further include, e.g., graft-versus-host disease, atherosclerosis, cardiovascular disease, irritable Bowel Syndrome (IBS), and necrotizing enterocolitis.

[0010] In some embodiments, the methods described herein improve epithelial barrier function in the gut of the subject. In some embodiment, the methods increase the expression level of one or more ileal tight junction proteins (e.g., the ileal tight junction protein ZO-l). In some embodiments, the expression level of the ileal tight junction protein is increased at least 1% (e.g, at least 2%, 4%, 6%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%) relative to the expression level of the ileal tight junction protein of a control subject that is not administered directly or indirectly the PlnE peptide and the PlnF peptide. In some embodiments, the methods increase the expression level of interleukin 23 (IL-23) in the subject. In some embodiments, the the expression level of IL-23 is increased at least 1% (e.g., at least 2%, 4%, 6%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%) relative to the expression level of the IL-23 of a control subject that is not administered directly or indirectly the PlnE peptide and the PlnF peptide. In some embodiments, the methods treat or prevent an autoimmune disorder in the subject. In particular embodiments, the autoimmune disorder may be associated with and/or caused by gut inflammation and/or compromised gut barrier function in the subject. [0011] In another aspect, the disclosure features a method of treating or preventing an autoimmune disease in a subject in need thereof, comprising administering directly or indirectly to the subject a plantaricin E peptide (PlnE peptide) comprising a sequence that is substantially identical to a sequence of FNRGGYNF GKS VRHVVD AIGS V AGIRGILKSIR (SEQ ID NO: l) and a plantaricin F peptide (PlnF peptide) comprising a sequence that is substantially identical to a sequence of

VFH AY S ARGVRNNYKS AV GP AD WVI S AVRGFIHG (SEQ ID NO:2). In some embodiments of this aspect, the method reduces gut inflammation and/or improves gut barrier function of the subject.

[0012] In another aspect, the disclosure features a method of reducing body weight of a subject in need thereof, comprising administering directly or indirectly to the subject a plantaricin E peptide (PlnE peptide) comprising a sequence that is substantially identical to a sequence of FNRGGYNF GKS VRHVVD AIGS V AGIRGILKSIR (SEQ ID NO: l) and a plantaricin F peptide (PlnF peptide) comprising a sequence that is substantially identical to a sequence of VFH AY S ARGVRNNYKS A V GP AD WVI S AVRGFIHG (SEQ ID NO:2).

[0013] In some embodiments, the subject is on a high-fat diet (HFD). Further, the subject may be overweight. The subject may have a body mass index (BMI) of 25 or greater (e.g., a BMI of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35).

[0014] In some embodiments, the weight of the subject is reduced at least 2% (e.g., at least 4%, 6%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%) relative to the weight of the subject prior to the subject is administered directly or indirectly the PlnE peptide and the PlnF peptide.

[0015] In some embodiments, the method of this aspect reduces the body weight gain of the subject by at least 2% (e.g., at least 4%, 6%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%) relative to the body weight gain of the subject prior to the subject is administered directly or indirectly the PlnE peptide and the PlnF peptide.

[0016] In some embodiments of the methods described herein, the PlnE peptide comprises a sequence having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99% sequence identity) to the sequence of SEQ ID NO: l. In some embodiments, the PlnF peptide comprises a sequence having at least 75% sequence identity (e.g, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99% sequence identity) to the sequence of SEQ ID NO:2.

[0017] In some embodiments, the PlnE peptide and the PlnF peptide to be administered to the subject in any one of the methods described herein are in a pharmaceutical composition, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers or excipients.

[0018] In some embodiments of the methods described herein, the subject is administered a population of probiotic bacteria (e.g., a population of Lactobacillus plantarum probiotic bacteria) expressing the PlnE peptide and the PlnF peptide. In some embodiments, the population of probiotic bacteria (e.g., the population of Lactobacillus plantarum probiotic bacteria) may be modified as a population of recombinant probiotic bacteria expressing the PlnE peptide and the PlnF peptide.

[0019] In some embodiments of the methods described herein, the PlnE peptide and PlnF peptide are conjugated to each other either directly or by way of a linker. For example, the C- terminus of the PlnE peptide is conjugated to the N-terminus of the PlnF peptide either directly or by way of the linker. In another example, the C-terminus of the PlnF peptide is conjugated to the N-terminus of the PlnE peptide either directly or by way of the linker. In further embodiments, the PlnE peptide and PlnF peptide administered directly or indirectly to the subject in any one of the methods described herein are protease resistant.

[0020] In some embodiments of the methods described herein, the methods reduce food intake and/or increases satiety of the subject.

[0021] In another aspect, the disclosure features a pharmaceutical composition comprising a PlnE peptide comprising a sequence that is substantially identical to a sequence of FNRGGYNF GKS VRHVVD AIGS V AGIRGILKSIR (SEQ ID NO: l) and a PlnF peptide comprising a sequence that is substantially identical to a sequence of VFHAYSARGVRNNYKSAVGPADWVISAVRGFIHG (SEQ ID NO:2), and one or more pharmaceutically acceptable carriers or excipients.

[0022] In another aspect, the disclosure features a pharmaceutical composition comprising a population of probiotic bacteria (e.g., a population of Lactobacillus plantarum probiotic bacteria) expressing a PlnE peptide comprising a sequence having at least 75% sequence identity (e.g, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99% sequence identity) to the sequence of

FNRGGYNF GKS VRHVVD AIGS V AGIRGILKSIR (SEQ ID NO: l) and a PlnF peptide comprising a sequence having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99% sequence identity) to the sequence of VFHAYS ARGVRNNYKS AV GPADWVIS AVRGFIHG (SEQ ID NO:2), and one or more pharmaceutically acceptable carriers or excipients. In some embodiments of this aspect, the PlnE peptide and PlnF peptide are conjugated to each other by way of a linker. For example, the C-terminus of the PlnE peptide is conjugated to the N-terminus of the PlnF peptide by way of the linker. In another example, the C-terminus of the PlnF peptide is conjugated to the N- terminus of the PlnE peptide by way of the linker. Further, the PlnE peptide and PlnF peptide may be protease resistant.

[0023] In another aspect, the disclosure features a pharmaceutical composition comprising a population of probiotic bacteria (e.g., a population of Lactobacillus plantarum probiotic bacteria) expressing a PlnE peptide comprising a sequence that is substantially identical to a sequence of FNRGGYNFGKSVRHVVDAIGSV AGIRGILKSIR (SEQ ID NO: l) and a PlnF peptide comprising a sequence that is substantially identical to a sequence of VFHAYS ARGVRNNYKSAVGP AD WVIS AVRGFIHG (SEQ ID NO:2), and one or more pharmaceutically acceptable carriers or excipients.

[0024] In some embodiments of the pharmaceutical compositions described herein, the PlnE peptide comprises a sequence having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99% sequence identity) to the sequence of SEQ ID NO: l. In some embodiments, the PlnF peptide comprises a sequence having at least 75% sequence identity (e.g, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99% sequence identity) to the sequence of SEQ ID NO:2. In some embodiments, the PlnE peptide and PlnF peptide are conjugated to each other by way of a linker. For example, the C-terminus of the PlnE peptide is conjugated to the N-terminus of the PlnF peptide by way of the linker. In another example, the C-terminus of the PlnF peptide is conjugated to the N-terminus of the PlnE peptide by way of the linker. Further, the PlnE peptide and PlnF peptide may be protease resistant.

[0025] In some embodiments of the pharmaceutical compositions described herein, the pharmaceutical composition further comprises one or more protease inhibitors. [0026] In some embodiments of the pharmaceutical compositions described herein, the pharmaceutical composition is formulated as an encapsulated tablet. In some embodiments, the pharmaceutical composition is formulated for oral administration.

Definitions

[0027] As used herein, the terms“plantaricin E peptide” or“PlnE peptide” refers to one of two peptides in plantaricin, which is a type of a bacteriocin. Plantaricin includes two peptides: PlnE peptide and PlnF peptide. A PlnE peptide may be a wild-type PlnE peptide which is produced by bacteria Lactobacillus plantarum ( L . plantarum) and has the sequence of FNRGGYNF GKS VRHVVD AIGS V AGIRGILKSIR (SEQ ID NO: l). A PlnE peptide may be a variant of the wild-type PlnE peptide and have a sequence that is substantially identical to the sequence of SEQ ID NO: 1.

[0028] As used herein, the terms“plantaricin F peptide” or“PlnF peptide” refers to one of two peptides in plantaricin, which is a type of a bacteriocin. Plantaricin includes two peptides: PlnE peptide and PlnF peptide. A PlnF peptide may be a wild-type PlnF peptide which is produced by bacteria Lactobacillus plantarum {L. plantarum) and has the sequence of VFHAYS ARGVRNNYKS AV GP ADWVIS AVRGFIHG (SEQ ID NO:2). A PlnF peptide may be a variant of the wild-type PlnF peptide and have a sequence that is substantially identical to the sequence of SEQ ID NO:2.

[0029] As used herein, the terms“plantaricin EF” or“PlnEF” refers to both PlnE peptide and PlnF peptide. One or both peptides may be the wild-type peptide or a variant thereof that has a sequence substantially identical to the sequence of the wild-type peptide.

[0030] As used herein, the terms“gut” refers to the organs, glands, tracts, and anatomical systems that are responsible for the transfer and digestion of food, absorption of nutrients, and excretion of waste. In humans, the gut comprises the gastrointestinal (GI) tract, which starts at the mouth and ends at the anus, and additionally comprises the esophagus, stomach, small intestine, and large intestine. The upper gastrointestinal tract comprises the esophagus, stomach, and duodenum of the small intestine. The lower gastrointestinal tract comprises the remainder of the small intestine, e.g., the jejunum and ileum, and all of the large intestine, e.g., the cecum, colon, rectum, and anal canal.

[0031] As used herein, the term“reduces gut inflammation” refers to reducing, ameliorating, and/or preventing inflammation of the gut. Symptoms associated with gut inflammation include, but are not limited to, abdominal pain, vomiting, diarrhea, constipation, bloating, abdominal distension, rectal bleeding, and internal cramps. Methods described herein may reduce one or more symptoms of gut inflammation.

[0032] As used herein, the term“improves gut barrier function” refers the strengthening, enhancement, and/or protection of the gut barrier to ensure that it functions properly to contain undesirable contents within the lumen of the gut while also to allow a certain level of permeability such that nutrients can passed and be absorbed by the body. For example, the intestinal mucosa is a type of gut barrier that protects the mucosal tissues and circulatory system from exposure to pro-inflammatory molecules, such as microorganisms, toxins, and antigens. The central component of the gut barrier is the epithelial layer, which provides physical separation between the lumen and the body. When the gut barrier function or the epithelial barrier function is compromised, there may be increased permeability in the gut ( e.g ., intestines) such that harmful contents may leak to the rest of the body.

[0033] As used herein, the term“high-fat diet” or“HFD” refers to a diet that is high in fats and simple sugars. Such a diet may lead to excessive weight gain, be a risk factor for type 2 diabetes, cardiovascular diseases, and other disorders. In some embodiments, a HFD may include at least 40% fat, e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% fat.

[0034] As used herein, the term“overweight” refers to a condition of having more body fat than is optimally healthy. The degree to which a person is overweight is generally described by the body mass index (BMI). Overweight is defined as a BMI of 25 or more.

[0035] As used herein, the term“body mass index” refers to a measure of a person's weight taking into account their height. It is given by the formula: BMI = (a person's weight (mass) in kilograms) / [(the person's height in metres) ² ]. The units therefore are kg/m ² , but BMI measures are typically used and written without units. BMI provides a significantly more accurate representation of body fat content than simply measuring a person's weight.

[0036] As used herein, the term“probiotic bacteria” or“probiotic” refers to live, non- pathogenic bacteria, which can confer health benefits to a host organism (e.g., a human), /. e.. by expressing one or more proteins or small molecules that are beneficial to the host organism. [0037] As used herein, the term“administering directly” refers to administering to a subject in the methods described herein a PlnE peptide comprising a sequence that is substantially identical to a sequence of SEQ ID NO: l and a PlnF peptide comprising a sequence that is substantially identical to a sequence of SEQ ID NO:2, in which the peptides are already expressed as amino acid chains. In some embodiments, the PlnE peptide and the PlnF peptide, both already expressed as amino acid chains, may be directly administered to the subject in a pharmaceutical composition.

[0038] As used herein, the term“administering indirectly” refers to administering to a subject in the methods described herein a population of probiotic bacteria that, once inside the gut of the subject, expresses a PlnE peptide comprising a sequence that is substantially identical to a sequence of SEQ ID NO: l and a PlnF peptide comprising a sequence that is substantially identical to a sequence of SEQ ID NO:2.

[0039] In the methods described herein, the PlnE peptide and the PlnF peptide may be administered directly or indirectly to the subject. In either route, the peptides are delivered to the gut ( e.g intestines) of the subject. In some embodiments, the peptides may be formulated and delivered to the gut of the subject as a foodstuff or a suppository. In other embodiments, the peptides may be formulated and delivered to the gut of the subject as a fecal transplant.

[0040] As used herein, the term “protease-resistant” refers to a peptide comprising a modification that renders the peptide less susceptible to cleavage by a protease than a corresponding non-modified wild-type peptide. In specific embodiments, a protease-resistant peptide may be a PlnE peptide or a PlnF peptide that has an amino acid substitution, relative to a wild-type peptide sequence, at an R or a K residue.

[0041] As used herein, the term "substantial identity" or "substantially identical," used in the context of nucleic acids or polypeptides, refers to a sequence that has at least 50% sequence identity with a reference sequence. Alternatively, percent identity can be any integer from 50% to 100%. In some embodiments, a sequence is substantially identical to a reference sequence if the sequence has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the reference sequence as determined using the methods described herein; preferably BLAST using standard parameters, as described below. [0042] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0043] A comparison window includes reference to a segment of any one of the number of contiguous positions, e.g., a segment of at least 10 residues. In some embodiments, the comparison window has from 10 to 600 residues, e.g., about 10 to about 30 residues, about 10 to about 20 residues, about 50 to about 200 residues, or about 100 to about 150 residues, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

[0044] Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=l, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).

[0045] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, an amino acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test amino acid sequence to the reference amino acid sequence is less than about 0.01, more preferably less than about 10 ⁵ , and most preferably less than about 10 ²⁰ .

[0046] As used herein, the terms“peptide” and“polypeptide” are used interchangeably and describe a single polymer in which the monomers are amino acid residues which are joined together through amide bonds. A peptide is intended to encompass any amino acid sequence, either naturally occurring, recombinant, or synthetically produced.

[0047] As used herein, the term“polynucleotide” refers to an oligonucleotide, or nucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single- or double-stranded, and represent the sense or anti-sense strand. A single polynucleotide is translated into a single polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

[0048] FIGS. 1A and 1B show that PlnEF alleviates the effects of pro-inflammatory cytokines on epithelial barrier integrity and IL-8 production in Caco-2 cells. Caco-2 monolayers were either untreated (black bars/symbols) or basally stimulated with IFN-g (100 ng/mL) for 24 hours before stimulation with TNF-a (10 ng/mL) (white bars/symbols). Plantaricin was added or omitted before cytokine exposure. FIG. 1A: FITC-dextran 4kDa (2 mg/mL) was added into the apical side of Caco-2 monolayer, and basolateral supernatants were measured for fluorescence at indicated time points. FIG. 1B: Basolateral concentrations of IL-8 were measured 2 hr after cytokine induction. In FIG. 1A, differences between groups were in some cases more significant (P =¾ 0.001), but only the highest P value ( U 0.01) is shown for clarity. Note: In FIG. 1B, although not shown, all treatments result in statistically significant increases in IL-8 production when compared to non-treated cells (P s _¾ 0.01). [0049] FIGS. 2 A and 2B show that PlnEF prevents cytokine-induced decreases in transepithelial resistance. Caco-2 monolayers were either untreated (black bars) or basally stimulated with IFN-g (100 ng/mL) for 24 h and then with TNF-a (10 ng/mL) (white bars). Plantaricin EF (PlnEF) was added or omitted before cytokine exposure. FIG. 2A: Comparison of PlnEF to individual PlnE and PlnF peptides. FIG. 2B: Comparison of PlnEF to the PlnA peptide. TER was significantly lower for all Caco-2 cells exposed to IFN-g and TNF-a compared to the non-treated controls (P < 0.05). Data represent at least four independent experiments. Number of transwells per treatment: No treatment n = 23, IFN n = 25, PlnEF n = 24, PlnA n = 9, PlnE n = 9, PlnF n = 9. Statistics were calculated with Kruskal-Wallis test and Dunn’s multiple comparisons test (*, P<0.05; ***, P0.001; ****, P0.0001) showing differences as compared to IFN-g and TNF-a treated wells.

[0050] FIGS. 3A-3C show that LP-feeding during HF diet significantly improves production of ZO-l in ileum tissues. FIG. 3 A: ZO-l was quantified in 3D space using Imaris software and data are presented relative to nuclei volume. FIG. 3B: ZO-l transcripts were quantified relative to HF control mice. FIG. 3C: Representative images of ileal tissue ZO-l and DAPI- stained nuclei.

[0051] FIGS. 4A-4D show that supplementation with LP but not the MU strain reduces weight gain in DIO mice. FIG. 4A: Oral glucose tolerance was conducted in week 9. Data are presented as area under curve from plots of glucose concentrations in blood taken over 2 hr after oral challenge. FIG. 4B: Difference in individual mouse weights at the beginning and end of the 9 week study. FIG. 4C: Average body weight of feeding groups + sem over time. Asterisks denote significant differences in body weight between HF and HF-LP fed mice. (*, P =¾ 0.5). FIG. 4D: Cumulative food intake over nine weeks. For A, B, and D one-way ANOVA with Tukey’s multiple comparisons test was used, for C a repeated ANOVA was used. (*, PO.05; ***, P0.001).

[0052] FIGS. 5A and 5B show that LP supplementation during HF diet decreases adipose associated inflammation. FIG. 5A: PA-l and FIG. 5B: leptin were measured with multiplex ELISA and data are presented relative to total protein concentrations.

[0053] FIGS. 6A-6D show that IL-23-axis is subtly elevated in intestinal compartment in response to LP but not MU supplementation. IL-23 and IL-22 were measured with multiplex ELISA and data are presented as relative to total protein concentration. [0054] FIGS. 7A-7E show that consumption of a HFD alters the intestinal microbiota regardless of L. plantarum- feeding as shown by 16S rRNA marker sequencing of the intestinal microbiota. FIG. 7A: Fecal samples were collected from all mice before study began (chow) and HF controls at 6 and 9 weeks. Samples were also taken from FIG. 7B: ileum, FIG. 7C: cecum, and FIG. 7D: feces at week 9. FIG. 7E: Percentage of total reads assigned to L. plantarum at each intestinal site. The pie charts contain the average percentage of reads assigned at order level.

[0055] FIGS. 8A-8D show that HFD significantly alters bacterial composition of the fecal microbiota as shown by 16S rRNA marker gene sequencing of the fecal microbiota. FIG 8A: PD whole tree a-diversity analysis at sequencing depths of 5,000 and 10,000 (asymptote of the rarefaction curve). Chow indicates all mice before beginning of the study, and HF-groups indicates all mice irrespective of bacteria feeding. FIG. 8B: PCoA of fecal microbial community. Feeding group differences confirmed by conducting ANOSIM (R = 0.615, P =¾ 0.001). FIG. 8C: Percentage of total reads assigned at order level. Chow indicates all mice before beginning the study, and HFD indicates only HF-control mice at 6 and 9 weeks of HF- feeding. FIG. 8D: Percentage of reads assigned at genus level. Labeled as in FIG. 8C.

[0056] FIGS. 9A-9E show that L. plantarum supplementation does not alter the HFD- associated microbiota of the ileum as shown by 16S rRNA marker gene sequencing of ileal contents. FIG. 9A: PD whole tree a-diversity analysis. Monte-Carlo simulation with 999 permutations indicated the asymptotes of the curves are not-significantly different P = 0.82. FIG. 9B: PCoA of ileal microbial community. No clustering effects from feeding group could be assigned, ANOSIM R = 0.014, P = 0.289. FIG. 9C: Percentage of reads assigned at order level. FIG. 9D: Percentage of reads assigned at family level. FIG. 9E: Percentage of reads assigned at genus level. Figure legend is shared between FIGS. 9A and 9B.

[0057] FIGS. 10A-10E show that L. plantarum supplementation does not alter the HFD- associated microbiota of the cecum as shown by 16S rRNA marker gener sequencing of cecal contents. FIG. 10A: PD whole tree a-diversity analysis. Monte-Carlo simulation with 999 permutations indicated the asymptotes of the curves are not-significantly different P = 0.96. FIG. 10B: PCoA of cecal microbial community. No clustering effects from feeding group could be assigned, ANOSIM R = -0.00935, P = 0.538. FIG. 10C: Percentage of reads assigned at order level. FIG. 10D: Percentage of reads assigned at family level. FIG. 10E: Percentage of reads assigned at genus level. Figure legend is shared between FIGS. 10A and 10B. [0058] FIGS. 11A-11E show that L. plantarum supplementation does not alter the HFD- associated microbiota of feces as shown by 16S rRNA marker gene sequencing of fecal contents. FIG. 11 A: PD whole tree a-diversity analysis. Monte-Carlo simulation with 999 permutations indicated the asymptotes of the curves are not-significantly different P = 0.93. FIG. 11B: PCoA of fecal microbiota. No clustering effects from feeding group could be assigned, ANOSIM R = 0.014, P = 0.28. FIG. 11C: Percentage of reads assigned at order level. FIG. 11D: Percentage of reads assigned at family level. FIG. 11E: Percentage of reads assigned at genus level. Figure legend is shared between FIGS. 11 A and 11B.

[0059] FIGS. 12A-12C show that PlnEF -biosynthesis does not alter the relative abundance of indigenous Lactobacillus in vivo in the ileum (FIG. 12 A), cecum (FIG. 12B), and feces (FIG. 12C). Operational taxonomical unit sequences recovered from 16S rRNA analysis were used as an input sequence and aligned against the National Center for Biotechnology Information database using BLAST to determine most related species.

[0060] FIGS. 13A-13C show that LP and MU strains were recovered at similar levels throughout the intestinal tract at the ileum (FIG. 13 A), cecum (FIG. 13B), and feces (FIG. 13C). qPCR of intestinal content DNA extracts with genus and species-specific primers.

[0061] FIGS. 14A-14D show that Lactobacillus- feeding does not alter composition of cecal or colon metabolites. FIG. 14A: PCoA of Bray-Curtis distances between cecal metabolomes. FIG. 14B: PCoA of Bray-Curtis distances between colon metabolomes. Concentrations of FIG. 14C: cholate and FIG. 14D: threonine in the ceca and colons, respectively.

[0062] FIG. 15 shows the phylogenetic tree of corC nucleotide sequences from L. pentosus strains. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model with distances shown under branches. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the nodes.

[0063] FIG. 16 shows the multiple-sequence alignment of CorC amino acid sequences. Amino acid sequences LP965 (Y216-G351) and Oenococcus oeni CorC 30CO (A211-G346) were aligned using MUSCLE (93).

[0064] FIG. 17 shows the growth of spontaneous plantaricin EF resistant mutants of LP965. L. plantarum NCIMB 700965 (WT) and PlnEF -resistant isolates were grown in MRS in the presence of 25 nM PlnEF or 1000 nM PlnA. The avg ± stdev of n = 3 replicates per strain is shown.

[0065] FIG. 18 shows the heterologous expression of LP956 and EF.A corC in L. casei BL23. Cells were grown in MRS with 50 nM of PlnEF. pJIMDHl contains corC from LP965 and pJIMDH2 contains corC from strain EF.A. The avg ± stdev of n = 3 replicates per strain is shown.

[0066] FIG. 19 shows the consensus sequence of conserved residues in the proximal CBS domain of the LP965 CorC protein. Consensus sequences were identified using the LP965 CorC protein as a template aligned against the CBS domain of closely related CorC proteins. The alignment revealed a highly conserved region where the substitution G334V mutation was found (indicated as a black star).

[0067] FIGS. 20A-20C show a structural model of the LP965 CorC protein. FIG. 20A: Top- down view of proposed homodimer formed by tandem CBS domains. FIGS. 20B: Top-down and FIG. 20C: side-views of (monomer) LP965 CorC protein region from Y216 to G351 shown in ribbon representation and colored by rainbow scheme from N-terminus region to C- terminus region. All sidechains are shown in stick representation, except for G334, which is shown in space-filling representation.

[0068] FIG. 21 shows the conservation of CorC in L. pentosus. Letter height indicates conserved CorC amino acid sequences detected in nine L. pentosus strains. Black stars are located over amino acid residues associated with increased PlnEF-resistance in L. pentosus EL8 and EL24. Double stars indicate variable amino acid residues that were not associated with PlnEF-resistance. The arrow shows the location of the G334V mutation in L. plantarum EF.A. Black underlines show the four predicted transmembrane domains (amino acids 6 to 30, 65 to 83, 103 to 123, and 143 to 165). Grey underlines show the two CBS domains (amino acids 229 to 289 and 294 to 351). The red underline shows the transport-associated domain (amino acids 362 to 441).

[0069] FIG. 22 shows that plantaricin EF decreases L. plantarum tolerance to magnesium stress. LP8826 (black hashes) and the LP8826 AplnEF mutant strain LM0420 (open circles) were grown in MRS supplemented with 500 mM MgSCri. The avg ± stdev of n = 3 replicates per strain is shown. [0070] FIG. 23 shows the effects of PlnEF on cellular concentrations of ATP. Cells of LP965 and the PlnEF -resistant isolate EF.A were initially energized with 10 mM glucose (arrow at 1 min). At 10 min (second arrow), 25 nM of PlnEF, 25 mM nisin, or water (NT) was added to each culture. The avg ± SD of n = 3 replicates is shown.

[0071] FIG. 24 shows the effects of PlnEF on intracellular concentrations of ATP. Cells of WT and PlnEF-resistant isolates were initially energized with 10 mM glucose (arrow at 1 min). At 10 min (second arrow), 25 nM of PlnEF, 25 mM nisin, or water (NT) was added to each culture. The avg ± SD of n = 3 replicates is shown.

[0072] FIG. 25 shows the effects of PlnEF on extracellular concentrations of ATP. WT and PlnEF-resistant isolates of LP965 were initially energized with 10 mM glucose (arrow at 1 min). At 10 min (second arrow), 25 nM of PlnEF, 25 mM nisin, or water (NT) was added to each culture. The avg ± SD of n = 3 replicates is shown.

[0073] FIGS. 26A and 26B show that supplementation with L. plantarum is associated with improved fasting glucose responses. FIG. 26A: Oral glucose tolerance was conducted in week nine. FIG 26B: Insulin concentrations in sera at time of necropsy. Statistics were calculated with one-way ANOVA and Tukey’s multiple comparisons test. (*, PO.05; **, PO.01 HF vs. HF-LP; $, PO.05; $$, PO.01 HF vs. HF-MU).

[0074] FIGS. 27A-27C show that LP does not alter feed efficiency or visceral fat accumulation. FIG. 27A: Feed efficiency was calculated as body weight gained over nine weeks divided by kilocalories of food ingested. FIG. 27B: Mesenteric and FIG. 27C: epididymal fat pads were removed and weighed at the time of necropsy.

[0075] FIG. 28 shows that LP supplementation does not alter adipose markers of inflammation. Adipose proteins were measured by ELISA and are presented relative to total protein concentrations.

DETAILED DESCRIPTION OF THE EMBODIMENTS

I. Introduction

[0076] Diets high in fats and simple sugars (HFD) can result in excessive weight gain, a risk factor for type 2 diabetes, cardiovascular diseases, and other disorders. Symptoms of impaired physiologic function resulting from HFD are due in part to decreased gut barrier integrity observed in diet-induced obese (DIO) mice [1] and humans [2] Translocation of bacterial lipopolysaccharide (LPS) and other microbial products to sites distal from the gut (e.g., adipose tissues) results in low-grade, systemic inflammation and endotoxemia [1,3,4] Therefore, strategies that target intestinal barrier integrity have great potential to improve pathologies associated with HFD, even without significant dietary restrictions.

[0077] The ingestion of living bacteria, such as strains of Lactobacillus and Bifidobacterium as probiotics is an emerging approach to prevent or attenuate the effects of obesity [5,6] Strains of Lactobacillus plantarum in particular were found to reduce weight gain and liver and adipose tissue inflammation in DIO mice [7,8] and obese humans [9,10] Unlike some other Lactobacillus species, L. plantarum is also associated with leanness [11]. As recently shown for the commensal species Akkermansia muciniphila [12], an important route through which intestinal bacteria can benefit human health is by inducing improvements to intestinal epithelial barrier integrity. This possibility has also been demonstrated in numerous studies on L. plantarum (e.g., strain NCIMB8826). Ingestion of L. plantarum (e.g., strain NCIMB8826) resulted in significant increases in the production of tight junction proteins in the duodenum of healthy adults [13,14] The capacity of this strain to alter the intestinal epithelium was confirmed in healthy and Simian Immunodeficiency Virus (SlV)-infected rhesus macaques [15] Introduction of L. plantarum into the rhesus macaque ileum rapidly improved epithelial tight junctions and resulted in the reversal of the IL-l induced gut epithelial damage caused by SIV. These findings indicate that probiotic L. plantarum might also prevent weight-gain and related impairments by improving gut barrier integrity.

[0078] In silico genotype-phenotype matching previously indicated that a bacteriocin locus encoding plantaricin EF (PlnEF), the cognate immunity protein (Plnl), and the extracellular transporter (PlnG) gave L. plantarum the capacity to alter cytokine synthesis by human peripheral blood mononuclear cells [16] and dendritic cells (DCs) [17] Compared to wild- type L. plantarum (e.g., strain NCIMB8826), plantaricin-deficient mutants induced the production of TNF-a and IL-10 as well as higher IL10 / IL12 ratios [16,17], thereby indicating that PlnEF might directly interact with the mucosal immune system. Moreover, research suggests that PlnEF may bind to magnesium channels on a cell’s surface to cause membrane leakage and cell death. The bacterial gene corC, encoding a putative magnesium efflux pump, may serve as the molecular receptor for PlnEF. By combining data from sequencing and the sensitivity assays, it was found that sequence divergence of the corC gene was associated with altered sensitivities to PlnEF. More recently, it was observed that a plnEFI- deficient mutant of L. plantarum NCIMB8826 was less effective at reducing markers of colonic inflammation associated with chemically-induced colitis in mice. Because plnEFI operon expression was significantly increased in the gastrointestinal (GI) tracts in human subjects and mice as opposed to laboratory culture media [19,20], this L. plantarum bacteriocin might have a more direct effect on the mammalian intestinal ecosystem.

[0079] PlnEF is a well-characterized bacteriocin that induces cell membrane disruption in closely-related bacterial cells [21] Like other bacteriocins, PlnEF is ribosomally synthesized and potent in the picomolar to nanomolar range [22] Bacteriocins are distinguished from antibiotics by their small size (<30 kDa) and narrow inhibitory spectra, typically against relatives from the same or highly-related species [23] The human gut microbiota has the genetic potential to produce thousands of bacteriocins [24,25], although strains of Lactobacillus have been of particular interest for their capacity to modify the gut microbiota by bacteriocin biosynthesis [26,27] Lactobacillus salivarius bacteriocin biosynthetic capacity was shown to be required to prevent Listeria monocytogenes infection in mice [28] and to module the gut microbiota in pigs [29] and healthy [29] and DIO mice [26]

[0080] Because of the known associations of /.. plantarum NCIMB8826 plnEFI with anti inflammatory responses in vitro and in vivo, Lactobacillus bacteriocins could directly support epithelial barrier function and thereby counter the effects of HFD either by directly interacting with the intestinal epithelium and/or altering the gut microbiota. Therefore, PlnEF peptides were evaluated for the capacity to increase epithelial barrier integrity after direct application in cell culture and in DIO mice upon feeding L. plantarum wild-type and NplnEFI strains. Mouse weight gain and food intake were measured for nine weeks after which host intestinal and systemic responses as well as intestinal microbiota and metabolites were assessed.

[0081] The disclosure provides that a secreted bacteriocin, plantaricin EF (PlnEF), produced exclusively by strains of Lactobacillus plantarum {L. plantarum), may attenuate the response of human colonic epithelial cells to inflammatory cytokine challenge. The examples demonstrate that weight gain was reduced and quantities of the major tight junction protein Zonula Occludins-1 in ileal tissue were increased in diet-induced obese (DIO) mice fed L. plantarum (PlnEF+) but not those fed a PlnEF-mutant strain. Further, L. plantarum (PlnEF+ and PlnEF-) did not alter the intestinal microbiota and metabolomes, indicating that the probiotic acted directly on the host.

[0082] PlnEF was investigated for maintaining epithelial barrier integrity in vitro and in a mouse model of diet-induced obesity. Design PlnEF peptides were applied to Caco-2 cell monolayers challenged with TNF-a and IFN-g. Transepithelial resistance, macromolecular permeability, and basolateral production of IL-8 were measured. Next, C57BL/6J mice on a high-fat diet (HFD) were administered wild-type L. plantarum NCMIB8826 or an isogenic AplnEFI mutant every 48 h for 9 weeks. Quantities of ileal tight junction protein zonula ocludins-l (ZO-l), anthropometry, oral glucose tolerance (OGT), adipose and intestinal tissue pro-inflammatory cytokines and proteins, and the intestinal microbiota and metabolome were investigated. The results demonstrated that PlnEF prevented cytokine-induced losses to Caco- 2 cell para- and transcellular permeability and reduced IL-8 levels. Only mice fed wild-type L. plantarum increased production of ileal ZO-l. Those mice also gained less weight and had a lower food intake. The responses were independent of OGT, as both NCMIB8826 and the AplnEFI mutant fed mice had improved OGT compared to sham controls. Although bacteriocins have antibacterial properties, the ileal, cecal, and fecal microbiota and cecocolic metabolomes were unchanged between mice fed either wild-type L. plantarum or the AplnEFI mutant. Probiotic L. plantarum may have the potential to ameliorate the effects of obesogenic diets via a mechanism that involves PlnEF induced maintenance of the intestinal epithelium.

[0083] PlnEF peptides may have the therapeutic potential to promote intestinal barrier integrity and their biosynthetic capacity may be used for improved selection of probiotic strains intended for body weight management and/or for reducing gut inflammation.

II. Plantaricin EF

[0084] Bacteriocins are peptides or proteins with antimicrobial activity directed against related species. Plantaricin is the name given to the group of bacteriocins produced by Lactobacillus plantarum {L. plantarum). Plantaricin EF includes two peptides, the 33 residue plantaricin E (PlnE) peptide and the 34 residue plantaricin F (PlnF) peptide. PlnE has a pair of a-helices in both the N- and C-terminal ends, while PlnF has a single long central a-helix (71). Both helices of PlnE are amphiphilic, while the PlnF helix is polar at the N-terminal end and amphiphilic at the C-terminus. Both peptides contain a GXXXG motif characteristic of class lib bacteriocins, and nuclear magnetic resonance analysis of this motif was shown to provide an interaction point between the peptides (72).

[0085] As described herein, a PlnE peptide may be a wild-type PlnE peptide which is produced by L. plantarum and has the sequence of FNRGGYNFGKSVRHVVDAIGSVAGIRGILKSIR (SEQ ID NO: l). In other embodiments, a PlnE peptide may be a variant of the wild-type PlnE peptide and have a sequence that is substantially identical to the sequence of SEQ ID NO: l. For example, a PlnE peptide may comprise a sequence having at least 75% sequence identity ( e.g ., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99% sequence identity) to the sequence of SEQ ID NO: l. A wild-type PlnE peptide having the sequence of SEQ ID NO: l may be modified to include one or more amino acid substitutions, deletions, and/or insertions, while maintaining substantially similar functions and properties as those of the wild-type PlnE peptide.

[0086] A PlnF peptide may be a wild-type PlnF peptide which is produced by L. plantarum and has the sequence of VFHAYS ARGVRNNYKS AV GPADWVIS AVRGFIHG (SEQ ID NO:2). In other embodiments, a PlnF peptide may be a variant of the wild-type PlnF peptide and have a sequence that is substantially identical to the sequence of SEQ ID NO:2. For example, a PlnF peptide may comprise a sequence having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99% sequence identity) to the sequence of SEQ ID NO:2. A wild-type PlnF peptide having the sequence of SEQ ID NO:2 may be modified to include one or more amino acid substitutions, deletions, and/or insertions, while maintaining substantially similar functions and properties as those of the wild-type PlnF peptide.

[0087] In methods of reducing gut inflammation, improving gut barrier function, treating diseases associated with gut inflammation and/or compromised gut barrier function, and/or reducing body weight as described herein, a subject may be administered directly or indirectly a PlnE peptide and a PlnF peptide. In some embodiments, the PlnE peptide and the PlnF peptide may be directly administered in the same pharmaceutical composition. In some embodiments, the PlnE peptide and the PlnF peptide may be directly administered substantially simultaneously in two separate pharmaceutical compositions with one pharmaceutical composition comprising the PlnE peptide and the other pharmaceutical composition comprising the PlnF peptide.

[0088] In other embodiments of the methods described herein, a subject may be administered indirectly a PlnE peptide and a PlnF peptide. For example, the subject may be administered a population of probiotic bacteria (e.g., a population of L. plantarum) that express the PlnE peptide and the PlnF peptide.

[0089] In some embodiments of the methods described herein, a subject may be administered directly or indirectly a PlnE peptide and a PlnF peptide that are conjugated to each other either directly or by way of a linker. For example, the C-terminus of the PlnE peptide may be conjugated to the N-terminus of the PlnF peptide either directly or by way of a linker. In another example, the C-terminus of the PlnF peptide may be conjugated to the N- terminus of the PlnE peptide either directly or by way of a linker.

Linker

[0090] A linker between a PlnE peptide and a PlnF peptide may be a peptide including 2-200 amino acids. Suitable linkers are known in the art, and include, for example, linkers containing flexible amino acid residues such as glycine and serine. In certain embodiments, a linker may contain motifs, e.g., multiple or repeating motifs, of GS, GGS, GGG, GGGGS (SEQ ID NO:4), GGSG (SEQ ID NO:5), or SGGG (SEQ ID NO:6). In certain embodiments, a linker may contain 2 to 12 amino acids including motifs of GS, e.g., GS, GSGS (SEQ ID NO:7), GSGSGS (SEQ ID NO: 8), GSGSGSGS (SEQ ID NO:9), GSGSGSGSGS (SEQ ID NO: lO), or GSGSGSGSGSGS (SEQ ID NO: 11). In certain other embodiments, a linker may contain 3 to 12 amino acids including motifs of GGS, e.g., GGS, GGSGGS (SEQ ID NO: 12), GGS GGS GGS (SEQ ID NO: 13), and GGS GGS GGS GGS (SEQ ID NO: 14). In yet other embodiments, a linker can contain 4 to 12 amino acids including motifs of GGSG (SEQ ID NO:5), e.g., GGSG (SEQ ID NO:5), GGSGGGSG (SEQ ID NO: 15), or GGSGGGSGGGSG (SEQ ID NO: 16). In other embodiments, a linker may contain motifs of GGGGS (SEQ ID NO:4), e.g., GGGGS GGGGS GGGGS (SEQ ID NO: 17). In other embodiments, a linker may also contain amino acids other than glycine and serine, e.g., GGGGAGGGG (SEQ ID NO: 18). The length of the linker and the amino acids used can be adjusted depending on the two peptide components involved and the degree of flexibility desired in the final conjugated polypeptide. The length of the linker can be adjusted to ensure proper protein folding and avoid aggregate formation.

Pharmacokinetics and Protease-Resistance

[0091] In some embodiments, the PlnE peptide and PlnF peptide may be protease resistant. The PlnE peptide and/or the PlnF peptide may be modified to improve their pharmacokinetic properties and resist protease degradation. In some embodiments, the PlnE peptide and/or the PlnF peptide may be fused with a serum protein-binding peptide, e.g., an albumin-binding peptide, to improve the pharmacokinetics, e.g., half-life, of the peptides. As one example, albumin-binding peptides that can be used to improve the half-life of the PlnE peptide and/or the PlnF peptide are generally known in the art. In one embodiment, the albumin-binding peptide includes the sequence DICLPRWGCLW (SEQ ID NO: 19). Without being bound to a theory, it is expected that inclusion of an albumin-binding peptide may lead to prolonged retention of the PlnE peptide and the PlnF peptide through their binding to serum albumin. In other embodiments, modifications of the PlnE peptide and/or the PlnF peptide involving PEGylation and glycosylation may also improve the half-life of the peptides. For example, polyethylene glycol (PEG) moieties of various sizes are available in the art and may be attached to a terminal or an internal amino acid of the PlnE peptide or the PlnF peptide. A PEG moiety may react with the amino groups on the side-chain of a lysine and the N-terminus.

[0092] Introduction of PEG or long chain polymers of PEG increases the effective molecular weight of the PlnE peptide or the PlnF peptide, for example, to prevent rapid filtration of the peptide into the urine. In some embodiments, a lysine residue in the peptide sequence is conjugated to PEG directly or through a linker. Such linker can be, for example, a Glu residue or an acyl residue containing a thiol functional group for linkage to the appropriately modified PEG chain. An alternative method for introducing a PEG chain is to first introduce a cysteine residue at the C-terminus of the peptide or at solvent exposed residues such as replacements for Arg or Lys residues. This Cys residue may then be site-specifically attached to a PEG chain containing, for example, a maleimide function. Methods for incorporating PEG or long chain polymers of PEG are well-known in the art (described, for example, in Veronese, F. M., et al, Drug Disc. Today 10: 1451-8 (2005); Greenwald, R. B., et al.. Adv. Drug Deliv. Rev. 55: 217- 50 (2003); Roberts, M. J., et al, Adv. Drug Deliv. Rev., 54: 459-76 (2002)), the contents of which is incorporated herein by reference.

[0093] In some embodiments, the PlnE peptide or PlnF peptide may be made protease- resistant by substituting at least one amino acid in the C-terminal portion of the peptide. The substitution may be at an R or K residue so that the peptide has increased resistance, e.g., to trypsin-like proteases. Any amino acid may be substituted for an R or K in a protease-resistant PlnE peptide or PlnF peptide. In some embodiments, a substitution may be a polar amino acid, e.g., H, N, Q, S, T, A, or Y. In some embodiments, a substitution may be H, N, Q, S, T, or Y. In some embodiments, a substitution may be S or Q.

III. Target of Plantaricin EF

[0094] Also described herein is the identification of the bacterial receptor CorC, a putative magnesium/cobalt efflux membrane protein, for plantaricin EF (PlnEF) on a PlnEF-sensitive strain of L. plantarum. The importance of the extracellular portion of CorC for PlnEF sensitivity was confirmed by genetic and phenotypic comparisons. This knowledge will result in an improved understanding of the ecological significance of plantaricin EF and use of bacteriocins to improve human health.

[0095] Lactic acid bacteria (LAB) bacteriocins kill closely related bacteria by mechanisms that result in the permeabilization of the cell membrane (61). Some broad-spectrum bacteriocins, such as nisin, are indiscriminate in cell-killing and bind to extracellular components shared across taxa. For example, nisin binds to lipid II, the final precursor in peptidoglycan synthesis (64). Bacteriocins with a narrow-inhibitory spectrum are known to target species- or genus-specific extracellular proteins (65). Initial success at identification of such receptors employed comparative genomics of spontaneous bacteriocin-resistant LAB species (65). In this way, the receptor for the pediocin-like bacteriocin leucocin A (targeting a mannose-specific phosphotransferase), the leaderless bacteriocin enterocin Kl (targeting a stress response membrane-bound Zn-dependent protease) (66) and the two-peptide bacteriocins lactococcin G (targeting undecaprenyl pyrophosphate phosphatase) (65), and plantaricin JK (targeting an uncharacterized protein in the amino acid-polyamine-organocation (APC) transporter protein family) were identified (67, 68).

[0096] Characterization of the molecular orientation that the PlnE peptide and the PlnF peptide adopt in artificial, cellular membrane-mimicking micelles revealed the most likely conformation is an anti-parallel coupling with the C-terminus of PlnE pointed into the cellular membrane and the N-terminus of PlnF closer to the extracellular environment (72). In summary, in a forward genetics approach, spontaneous PlnEF-resistant mutants of the PlnEF indicator strain L. plantarum NCIMB 700965 (LP965) were isolated. Genome comparisons resulted in the identification of a single gene annotated as a magnesium/cobalt efflux membrane protein CorC. Most isolates contained a single point mutation (G334V) in an extracellular cysteine-beta-synthase (CBS) domain in the C-terminal region of CorC. Heterologous expression of LP965 corC in the PlnEF-resistant strain Lactobacillus casei BL23 resulted in increased PlnEF sensitivity. This was not found for strain BL23 containing the G334V CorC mutant protein. In silico template-based modeling of the LP965 CorC CBS domains indicated that the G334V mutation resides in a coil located between two beta-sheets. Further support for the role of CorC was found upon comparing PlnEF sensitive and resistant strains of Lactobacillus pentosus and localization of amino acid variations among resistant strains to the extracellular, C-terminus of the CorC protein. Although the growth of LP965 corC mutants was not affected by high Mg ²⁺ or Co ²⁺ concentrations, endogenous production of PlnEF by L. plantarum was associated with decreased tolerance to exogenous MgSCri. These results strongly indicate that PlnEF induces cell killing in L. plantarum and closely related Lactobacillus species by targeting Mg ²⁺ homeostasis regulated by CorC.

[0097] As described in further detail herein (see Example 2), a forward genetics approach using genome comparisons was adopted to identify the PlnEF receptor in the sensitive strain Lactobacillus plantarum NCIMB 700965 (LP965). This strain has been used as an indicator for plantaricin biosynthesis (73, 74) and lacks a functional immunity protein Plnl (due to IS30 transposon, 75). Isolation of PlnEF -resistant mutants of strain LP965 led to the identification of corC, a magnesium and cobalt efflux protein, as a target of PlnEF. This finding was confirmed by heterologous expression of the wild-type and mutant LP965 copies of corC, localization of the variant amino acids in CorC by in silico template-based modeling, comparisons of PlnEF resistance and CorC amino acid variation in Lactobacillus pentosus strains, and measurements of growth in response to altered magnesium and cobalt concentrations.

[0098] Antimicrobial activities of bacteriocins result from interactions of bacteriocin peptides with receptors located on the surface of sensitive cells. Although a variety of LAB bacteriocins have been characterized, only a limited number of receptors for these peptides are known. The finding that the receptor for the L. plantarum bacteriocin PlnEF is CorC, a putative membrane-bound, magnesium efflux protein is in agreement with previous studies on PlnEF showing that this bacteriocin causes cation efflux (80). The activity of PlnEF is distinct from Plantaricin JK which induces anion efflux and for which the cell surface receptor was identified as a protein in the amino acid-polyamine-organocation (APC) transporter family (68). The combination of the two plantaricins is known to result in potent bactericidal effects (80).

[0099] Maintaining physiological magnesium homeostasis is essential for stabilizing cellular membranes, ribosomes, and enzymatic reactions. Magnesium transport systems in bacteria are typically composed of a CorA protein, understood to be an influx pore, and an accompanying efflux protein (81). Magnesium efflux proteins have been tentatively described in Salmonella as CorB, CorC, and CorD (81), although the ion specificity for Cor-like proteins is questionable in other taxa such as Pseudomonas which may also transport Zn ²⁺ (82). The genomes of both LP965 and the well-characterized reference strain L. plantarum WCFS1 (a single colony isolate of L. plantarum NCIMB 8826 for which the genome sequence is known (83)) contain a single copy of corC, and two copies of corA. Although both LP965 and the PlnEF-resistant LP965 mutants grew equally well in the presence of high MgSOr concentrations, those conditions resulted in impaired growth of the PlnEF-producing strain, LP8826, compared to the isogenic, PlnEF-deficient LM0420 mutant. These findings suggest that the endogenous production of PlnEF can magnify the effects of magnesium stress, likely by inhibiting the function of CorC. Because LP8826 also produces the Plnl immunity protein, the reduced growth of that strain in the presence of high magnesium concentrations shows that the protection provided by Plnl against the bacteriocin is not complete or is compromised with added Mg ²⁺ stress.

[0100] Levels of resistance to PlnEF observed for the LP965 mutants were less than found for other bacteriocin systems. For example, the MIC50 values of L. lactis mutants resistant to the bacteriocin lactococcin G were at least 1,000 to 10,000 times greater than the wild-type strain MIC50 value (65). This discrepancy could be due to mutations resulting in truncations of the Upp receptor protein among lactococcin G - resistant mutants (65), whereas only a single amino acid substitution was found in the LP965 CorC mutants. Because no other CorC mutants were found, and the inability to delete corC in the genetically-amenable strain LP8826, it is possible that CorC is an essential protein required for L. plantarum growth. To this regard, a Staphylococcus aureus CorC-like protein (SA00657) was recently characterized and found to be essential for growth in the presence of elevated magnesium concentrations (84).

[0101] To circumvent the use of potentially lethal CorC mutants in L. plantarum, L. casei was used for heterologous expression (Example 2). L. casei BL23 is naturally, more resistant to PlnEF than LP965. Although the L. casei BL23 genome contains a corC homolog, the protein it encodes shares only 61% amino acid identity to CorC in LP965, and this strain also lacks other plantaricin associated genes. Exposure of L. casei transformants expressing CorC from either LP965 or the EF.A mutant to PlnEF confirmed that sensitivity to the bacteriocin is at least partially dependent on the presence of a CorC protein similar to the one contained in LP965 and that the G334V mutation facilitates increased PlnEF resistance.

[0102] The CorC G334V mutation in strain EF.A is located in a cysteine-beta synthase (CBS) domain. CBS domains are common and frequently found in two to four tandem copies in both cytosolic- and membrane-associated proteins and are present in proteins from all domains of life (79). CBS domains are important sites for ATP-binding with implications for the regulation of magnesium homeostasis (85). The CBS domains in LP965 are highly conserved and shared among related proteins. This indicates that the domains are important for the function of the CorC protein and could also serve as docking sites for PlnEF peptides. Of all deposited crystal structures for CorC-like proteins, 30C0 shared the highest amino acid sequence identity to LP965 (approximately 50%). It is notable that 30C0 is predicted to form a dimer. After modeling LP965 CorC to this template, it was concluded that there is a strong possibility that LP965 also forms a homodimer structure with two pairs of CBS domains. However, molecular characterization of the LP965 CorC protein as well as direct binding with PlnEF requires more investigation.

[0103] Comparisons of PlnEF-resistance and CorC amino acid composition among L. pentosus strains provide additional supportive evidence that the extracellular, C-terminal end of the CorC protein has a role in conferring sensitivity to the bacteriocin. CorC proteins in nine olive-associated L. pentosus strains were found to be similar to those contained in the LP965 and LP8826 genomes. Although the L. pentosus CorC proteins share high levels of amino acid sequence conservation, two strains that were more resistant to PlnEF also contained unique amino acid substitutions in the predicted, extracellular portion of the protein.

[0104] It was found that the majority of LP965 PlnEF-resistant isolates contained a single mutation resulting in a G to V amino acid change at position 334 in the CorC protein (see Example 2). These findings might indicate the mutation was selected early during the enrichment for PlnEF resistant mutants and these five isolates comprise a clonal population which was selected for in subsequent propagations steps. Although mutations were not found in the corC locus of EF.B and EF.C, the expression of corC was downregulated in EF.B compared to WT LP965 and the growth rates of both isolates were improved in the presence of PlnEF compared to the wild-type strain. Additionally, strain EF.C was able to reach higher final cell numbers (as measured by Oϋboo) than the WT strain in the presence of PlnEF despite having identical MICso values. These results indicate that there are other factors contributing to LP965 PlnEF sensitivity which remain to be identified. However, these other proteins or cell constituents likely only confer modest effects compared to the specific amino acid composition of CorC.

IV. Probiotic Bacteria

[0105] Probiotic bacteria, also referred as probiotics, are live, non-pathogenic bacteria, which can confer health benefits to a host organism ( e.g a human), /. e. by expressing one or more proteins or small molecules that are beneficial to the host organism. In some embodiments, the probiotic bacteria are Gram-negative bacteria. In some embodiments, the probiotic bacteria are Gram-positive bacteria. Some species, strains, and/or subtypes of non- pathogenic bacteria are currently recognized as probiotic bacteria. Examples of probiotic bacteria include, but are not limited to, certain strains belonging to the genus Bifidobacteria (e.g., Bifidobacterium bifldum), Escherichia coli (e.g., Escherichia coli strain Nissle), Enterococcus (e.g., Enterococcus faecium), Lactobacillus (e.g., Lactobacillus acidophilus, and Lactobacillus bulgaricus, Lactobacillus paracasei, and Lactobacillus plantarum). The probiotic may be a variant or a mutant strain of a bacterium. Non-pathogenic bacteria may be genetically engineered to enhance or improve desired biological properties, e.g., survivability. Non-pathogenic bacteria may be genetically engineered to provide probiotic properties. In some embodiments, probiotic bacteria may be genetically engineered to enhance or improve probiotic properties.

[0106] In some embodiments of the methods described herein, a subject may be administered a population of probiotic bacteria (e.g., a population of L. plantarum) that express the PlnE peptide and the PlnF peptide. In some embodiments, the population of probiotic bacteria (e.g., the population of Lactobacillus plantarum probiotic bacteria) may be modified as a population of recombinant probiotic bacteria expressing the PlnE peptide and the PlnF peptide. In some embodiments, the probiotic bacteria (e.g., L. plantarum) may express the PlnE peptide and the PlnF peptide as two separate peptides. In other embodiments, the probiotic bacteria (e.g., L. plantarum) may be modified to express the PlnE peptide and the PlnF peptide as a conjugated polypeptide, i.e., in which the C-terminus of the PlnE peptide is conjugated to the N-terminus of the PlnF peptide either directly or by way of a linker, or the C- terminus of the PlnF peptide is conjugated to the N-terminus of the PlnE peptide either directly or by way of a linker. The probiotic bacteria (e.g., L. plantarum) expressing the PlnE peptide and the PlnF peptide may be engineered to resist protease and/or acid degradation in the gut (e.g., intestines) of the subject. For example, the probiotic bacteria (e.g., L. plantarum) may be genetically modified to also produce one or more protease inhibitors (e.g., aspartic protease inhibitors, cysteine protease inhibitors, metalloprotease inhibitors, serine protease inhibitors, threonine protease inhibitors, and trypsin inhibitors). To increase PlnE peptide and PlnF peptide expression, a population of L. plantarum probiotic bacteria may be modified to increase the plnEFI operon expression, especially when the bacteria is in the gastrointestinal (GI) tract. L. plantarum (e.g., strain NCIMB8826) may be modified to produce a population of L. plantarum probiotic bacteria. In some embodiments, a population of probiotic bacteria may be transformed with high-copy number plasmids encoding the PlnE peptide and/or the PlnF peptide and/or may undergo chromosomal modification to increase the bacterial expression of the PlnE peptide and/or the PlnF peptide.

V. Effects of Plantaricin EF In vitro and In vivo

[0107] Although probiotics are attractive for their potential to maintain and improve human health in numerous ways, only a few effector compounds (e.g., proteins, metabolites, polysaccharides) produced by these microorganisms required for altering host epithelial and immune response pathways have been identified. Herein, the disclosure shows that the L. plantarum bacteriocin PlnEF is a probiotic effector that promotes epithelial barrier integrity in vitro and in vivo. Feeding mice LP with plnEFI biosynthetic capacity was sufficient to induce a reduction in weight gain and food intake on a HFD. The lack of change in intestinal microbiota and metabolome composition in response to L. plantarum supports a mechanism by which the anti-obesogenic effects of this strain were the result of PlnEF-induced improvements to intestinal barrier integrity.

[0108] Application of PlnEF to Caco-2 cells exposed to pro-inflammatory cytokines prevented reductions in macromolecular permeability, transepithelial resistance, and reduced production of IL-8. Although another L. plantarum bacteriocin, PlnA, was previously shown to improve barrier function and cell viability when applied to differentiated Caco-2 cells challenged with INF-ylFN-y [33], a higher concentration of PlnA was applied than used here and the result was not confirmed in vivo. When PlnA was applied to an in vitro model of intestinal inflammation, PlnA prevented cytokine induced reductions in TER 24 h post induction, but only PlnEF sustained this protective effect for an additional 24 h (FIG. 2A). Taken together, there appears to be a broader role for Lactobacillus bacteriocin interactions with the intestinal epithelium. This is reinforced by the previous findings that a L. salivarius mutant unable to synthesize the bacteriocin Abpl l8 exhibited an improved barrier-protective capacity compared to the wild-type strain in a Caco-2 cell model [34]

[0109] The importance of PlnEF biosynthetic capacity of L. plantarum to influence in the GI tract was demonstrated with the significantly higher levels of ZO-l in the ileum following the administration of the wild-type NCIMB 8826-R when comparted to mice fed the D plnEFI mutant. L. plantarum was previously shown to increase the production of ZO-l in the human duodenum [14] and rhesus macaques [35], however the L. plantarum protein or metabolite responsible for conferring these effects were not identified. Plantaricin interactions with the intestine might not be limited to epithelial cells because production of IL-23 was also exclusively limited to the colonic tissues of mice fed the wild-type strain. The majority of intestinal IL-23 acts upon the IL-23 receptor+ type 3 innate lymphoid cell linage (ILC3) to promote intestinal barrier function [36] Promoting the proliferation and maturation of these cells has been noted as essential for epithelial cell protection from chemical [37] and pathogen induced colitis [38] Induction of IL-23 was impaired in obese mice and resulted in a higher mortality following gastrointestinal pathogen infection [32] Prior findings showing that plantaricin biosynthesis-deficient mutants differentially induce cytokine production by human PBMCs [16] and DC [17] are consistent with the possibility that PlnEF might induce conserved signal transduction pathways in both epithelial and immune cells to elicit anti inflammatory outcomes.

[0110] As shown in Example 1, the lack of change in the intestinal microbiota and metabolomes between the LP and MU fed mice is further evidence that plantaricin biosynthetic capacity was directed at the epithelium. Even though plantaricin is effective at killing related Lactobacillus species in vitro [39], there were no differences between the indigenous Lactobacillus populations among mice fed either L. plantarum strain. While it is possible that L. plantarum plantaricin biosynthesis modified metabolic or other functional pathways produced by the intestinal microbiota, the lack of change to the cecal and colonic metabolomes between the WT and MU fed mice indicates otherwise.

[0111] Remarkably, there were also no observable differences between the intestinal microbiota of mice fed either L. plantarum strain or the HF controls. Prior studies have shown that L. plantarum can induce taxonomic shifts among the fecal microbiota over short time scales [19,40] However, co-occurrence analysis revealed that /.. plantarum was not integrated into bacterial networks in the distal gut, and therefore after an initial perturbation caused by the introduction of probiotic [40], the intestinal microbiota might have been more greatly influenced by the regular consumption of a HFD. The only consistent change between L. plantarum-i d and control mice in this study was an increase in Lactobacillus in the ileum, cecum, and stools. These bacteria identified by 16S rRNA marker gene sequencing were confirmed to be L. plantarum by qPCR and corresponded to viable cell recovery on selective laboratory culture medium.

[0112] The minimal impact that L. plantarum had on the intestinal environment was confirmed by the metabolomes (Example 1). To this regard, out of >60 metabolites measured, only cecal cholate and fecal threonine concentrations were altered compared to the HF controls. Both WT and MU L. plantarum were associated with significant reductions in cecal cholate compared to the HFD group. Because both strains possess bile salt hydrolases [41], this finding could indicate that L. plantarum was actively altering bile acid metabolism. Consistent with the finding that mice given LP or MU also exhibited improved OGT compared to the controls, altered bile acid metabolism has been consistently associated with homeostatic model assessment (HOMA)-insulin resistance in obese individuals [42] Hence, it is notable that both LP and MU resulted in improved OGT compared to the controls but did not result in changes to serum insulin levels.

[0113] Unlike the Abpl l8 bacteriocin deficient mutant of L. salivarius in DIO mice [26], NCIMB8826-R resulted in persistent, rather than transient, reductions in weight gain as well as reduction in feed intake. Other strains of L. plantarum were shown to reduce weight gain, adipose tissue deposits [43], systemic triglycerides [44], and improve OGT [45] and liver fatty acid oxidation [46] in DIO mice. Although few significant changes were found in adipose tissue and systemic metabolic markers, LP was fed less frequently than in those studies and other factors such as diet formulation could account for these differences. Similarly, L. plantarum ( e.g strain NCIMB8826) was found to be less effective than A. muciniphila in improving gut barrier function in DIO mice [47] However, in that study L. plantarum was only fed for four weeks and the diet contained a higher percentage of fat (60%) than the 43% fat diet used here.

[0114] Although modulation of the gut microbiota is frequently regarded to be the primary mechanism of probiotic-mediated health benefits [48], L. plantarum appears to attenuate weight gain by conferring more direct effects on the host intestinal epithelium. Bacterial cell components that have been evaluated to improve epithelial barrier function (in vitro or in vivo) are currently limited to an outer membrane protein produced in high abundance by A. muciniphila [12], extracellular peptidoglycan hydrolases p40 and p75 from L. rhamnosus GG [49], and /.. brevis- secreted polyphosphate [50] Importantly, each of these molecules induce different mechanisms resulting in barrier function protection. This knowledge informs efforts to elucidate signaling pathways induced by PlnEF and supports the possibility of combining probiotic strains and/or effectors for improving the likelihood of mitigating pathologies caused by the habitual consumption of obesogenic diets. VI. Methods

[0115] The present disclosure provides methods of reducing gut inflammation, improving gut barrier function, treating diseases associated with gut inflammation and/or compromised gut barrier function, and/or reducing body weight in a subject in need thereof. The disclosure also provides methods of treating or preventing an autoimmune disease in a subject in need thereof. In the methods described herein, the methods may reduce food intake and/or increases satiety of the subject. In methods described herein, a subject may be administered directly or indirectly a PlnE peptide (e.g., a PlnE peptide having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99% sequence identity) to the sequence of SEQ ID NO: l) and a PlnF peptide (e.g., a PlnF peptide having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99% sequence identity) to the sequence of SEQ ID NO:2). In some embodiments, the PlnE peptide and the PlnF peptide may be directly administered in the same pharmaceutical composition. In some embodiments, the PlnE peptide and the PlnF peptide may be directly administered substantially simultaneously in two separate pharmaceutical compositions with one pharmaceutical composition comprising the PlnE peptide and the other pharmaceutical composition comprising the PlnF peptide. In other embodiments of the methods described herein, a subject may be administered indirectly a PlnE peptide and a PlnF peptide. For example, the subject may be administered a population of probiotic bacteria (e.g., a population of L. plantarum) that express the PlnE peptide and the PlnF peptide.

[0116] Methods described herein may reduce, ameliorate, and/or prevent inflammation of the gut. The methods may reduce or ameliorate one or more symptoms of gut inflammation in the subject. For example, the methods may reduce or ameliorate abdominal pain, vomiting, diarrhea, constipation, bloating, abdominal distension, rectal bleeding, and internal cramps. Methods described herein may also strengthen, enhance, and/or protect of the gut barrier of the subject, especially strengthen, enhance, and/or protect the epithelial layer of the gut of the subject. As demonstrated in Example 1, production of ileal tight junction protein ZO-l is increased significantly by feeding mice L. plantarum bacteria which express PlnE peptide and PlnF peptide, thus improving intestinal epithelial barrier integrity. Moreover, Example 1 also shows that purified PlnE peptide and PlnF peptide alleviated the effects of pro-inflammatory cytokines on epithelial barrier integrity. Further, IL-23, a cytokine that has been linked to improved epithelial barrier integrity, was increased significantly in mice fed L. plantarum bacteria, while IL-23 was only minimally or not detected in the controls. [0117] Methods described herein may treat or prevent a disease associated with and/or caused by gut inflammation and/or compromised gut barrier function. Examples of diseases associated with and/or caused by gut inflammation and/or compromised gut barrier function include, but are not limited to, inflammatory bowel disease ( e.g Crohn's disease and ulcerative colitis), gastrointestinal infectious viral and bacterial diseases, celiac disease, microscopic colitis, diarrheal disease, bile acid malabsorption, irritable bowel disease, and colon cancer. In some embodiments, diseases associated with and/or caused by gut inflammation and/or compromised gut barrier further include, e.g., graft-versus-host disease, atherosclerosis, cardiovascular disease, irritable Bowel Syndrome (IBS), and necrotizing enterocolitis.

[0118] In some embodiments, the methods described herein may treat or prevent an autoimmune disorder in the subject. In some embodiments, the autoimmune disorder is associated with and/or caused by gut inflammation and/or compromised gut barrier function in the subject. Examples of autoimmune disorders include, but are not limited to, acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis, antiphosphobpid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticarial, axonal & neuronal neuropathies, Balo disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal ostomyelitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid/benign mucosal pemphigoid, Crohn's disease, Cogan's syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST disease, essential mixed cryoglobulinemia, demyelinating neuropathies, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus, Dressier' s syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis (GPA), Graves' disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, herpes gestationis, hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, immunoregulatory lipoproteins, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile idiopathic arthritis, juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus (systemic lupus erythematosus), chronic Lyme disease, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's), neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatry Disorders Associated with Streptococcus), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Tumer syndrome, pars planitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, type I, II, & III autoimmune polyglandular syndromes, polymyalgia rheumatic, polymyositis, postmyocardial infarction syndrome, postpericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm & testicular autoimmunity, stiff person syndrome, subacute bacterial endocarditis (SBE), Susac's syndrome, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis/giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, type 1 diabetes, asthma, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesiculobullous dermatosis, vitiligo, and Wegener's granulomatosis.

[0119] Methods described herein may also reduce body weight of a subject in need thereof. In some embodiments, the subject is on a high-fat diet (HFD) (i.e., a HFD including at least 40% fat, e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% fat). HFDs can result in excessive weight gain, a risk factor for type 2 diabetes, cardiovascular diseases, and other disorders. Symptoms of impaired physiologic function resulting from HFDs may due in part to decreased gut barrier integrity. Translocation of undesirable contents across the gut barrier to other body sites and tissues may result in systemic inflammation. Improving gut barrier function and reducing gut inflammation may improve pathologies associated with HFDs, even without significant dietary restrictions. In some embodiments, the subject may be overweight. In some embodiments, the subject has a body mass index (BMI) of 25 or greater.

VII. Peptide Production and Purification

[0120] Aside from being expressed by a population of probiotic bacteria ( e.g a population of L. plantarum ) in the gut of a subject if the subject is administered the population of probiotic bacteria in methods described herein, PlnE peptide and PlnF peptide may be expressed in vitro by a host cell. A host cell refers to a vehicle that includes the necessary cellular components, e.g., organelles, needed to express the peptides described herein from their corresponding nucleic acids. The nucleic acids may be included in nucleic acid vectors that can be introduced into the host cell by conventional techniques known in the art (transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, etc.). Host cells used for in vitro peptide expression and purification can be of either mammalian or bacterial origin. Host cells can also be chosen that modulate the expression of the peptide or protein constructs, or modify and process the peptide or protein product in the specific fashion desired. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of protein products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the protein expressed.

[0121] For expression and secretion of peptide or protein products from their corresponding DNA plasmid constructs, host cells may be transfected or transformed with DNA controlled by appropriate expression control elements known in the art, including promoter, enhancer, sequences, transcription terminators, polyadenylation sites, and selectable markers. Methods for expression of therapeutic peptides and proteins are known in the art. See, for example, Paulina Balbas, Argelia Lorence (eds.) Recombinant Gene Expression: Reviews and Protocols (Methods in Molecular Biology), Humana Press; 2nd ed. 2004 edition (July 20, 2004); Vladimir Voynov and Justin A. Caravella (eds.) Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology) Humana Press; 2nd ed. 2012 edition (June 28, 2012).

[0122] The PlnE peptide and PlnF peptide as described herein may be purified by any method known in the art of peptide and protein purification, for example, by chromatography (e.g., ion exchange, affinity (e.g., Protein A affinity), and size-exclusion column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. For example, PlnE peptide and PlnF peptide can be isolated and purified by appropriately selecting and combining affinity columns such as Protein A column with chromatography columns, filtration, ultra filtration, salting-out and dialysis procedures (see, e.g., Process Scale Purification of Antibodies, Uwe Gottschalk (ed.) John Wiley & Sons, Inc., 2009; and Subramanian (ed.) Antibodies-Volume I-Production and Purification, Kluwer Academic/Plenum Publishers, New York (2004)). In some instances, PlnE peptide and/or PlnF peptide may be conjugated to marker sequences, such as a purification peptide to facilitate purification. An example of a purification peptide is a hexa-histidine peptide, which binds to nickel -functionalized agarose affinity column with micromolar affinity. Other purification peptides include, but are not limited to, the hemagglutinin“HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al, 1984, Cell 37:767).

VIII. Pharmaceutical Compositions

[0123] As described herein, the PlnE peptide and the PlnF peptide may be directly administered to the subject in the methods of the disclosure, or may be administered to the subject indirectly as a population of probiotic bacteria (e.g., a population of L. plantar um) that express the PlnE peptide and the PlnF peptide in the gut of the subject. In some embodiments, a pharmaceutical composition of the disclosure may include a PlnE peptide comprising a sequence that is substantially identical to a sequence of SEQ ID NO: l and a PlnF peptide comprising a sequence that is substantially identical to a sequence of SEQ ID NO:2, and one or more pharmaceutically acceptable carriers or excipients. In other embodiments, if the subject is directly administered the PlnE peptide and the PlnF peptide, each peptide may be included in its own pharmaceutical composition. The subject may be administered two pharmaceutical compositions substantially simultaneously, with one pharmaceutical composition comprising the PlnE peptide and the other pharmaceutical composition comprising the PlnF peptide.

[0124] In some embodiments, a pharmaceutical composition of the disclosure may include a population of probiotic bacteria (e.g., a population of L. plantarum) expressing a PlnE peptide comprising a sequence that is substantially identical to a sequence of SEQ ID NO: l and a PlnF peptide comprising a sequence that is substantially identical to a sequence of SEQ ID NO:2, and one or more pharmaceutically acceptable carriers or excipients. In other embodiments, the subject may be administered two pharmaceutical compositions substantially simultaneously, with each pharmaceutical composition comprising a population of probiotic bacteria ( e.g ., a population of /.. plantarum) that express only one of the two peptides.

[0125] In any of the pharmaceutical compositions described herein, the pharmaceutical composition may further comprise one or more protease inhibitors, which may prevent the degradation of the PlnE peptide and the PlnF peptide in the gut of the subject and increase the half-life of the peptides. Examples of protease inhibitors include, but are not limited to, aspartic protease inhibitors, cysteine protease inhibitors, metalloprotease inhibitors, serine protease inhibitors, threonine protease inhibitors, and trypsin inhibitors.

[0126] In the methods described herein, the PlnE peptide and the PlnF peptide may be administered directly or indirectly to the subject. In either route, the peptides are delivered to the gut (e.g., intestines) of the subject. In some embodiments, the peptides may be formulated and delivered to the gut of the subject as a foodstuff or a suppository. In other embodiments, the peptides may be formulated and delivered to the gut of the subject as a fecal transplant.

[0127] Pharmaceutical compositions including either therapeutic peptides or probiotic bacteria may be formulated by methods know to those skilled in the art. Pharmaceutical compositions, especially those including therapeutic peptides, may be administered parenterally in the form of an injectable formulation including a sterile solution or suspension in water or another pharmaceutically acceptable liquid. For example, the pharmaceutical composition can be formulated by suitably combining the PlnE peptide and/or the PlnF peptide with pharmaceutically acceptable vehicles or media, such as sterile water for injection (WFI), physiological saline, emulsifier, suspension agent, surfactant, stabilizer, diluent, binder, excipient, followed by mixing in a unit dose form required for generally accepted pharmaceutical practices. The amount of active ingredient included in the pharmaceutical preparations is such that a suitable dose within the designated range is provided. The sterile composition for injection can be formulated in accordance with conventional pharmaceutical practices using distilled water for injection as a vehicle. For example, physiological saline or an isotonic solution containing glucose and other supplements such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride may be used as an aqueous solution for injection, optionally in combination with a suitable solubilizing agent, for example, alcohol such as ethanol and polyalcohol such as propylene glycol or polyethylene glycol, and a nonionic surfactant such as polysorbate 80TM, HCO-50, and the like commonly known in the art. In some embodiments, pharmaceutical compositions including either therapeutic peptides (e.g., a PlnE peptide comprising a sequence that is substantially identical to a sequence of SEQ ID NO: l and a PlnF peptide comprising a sequence that is substantially identical to a sequence of SEQ ID NO:2) or probiotic bacteria may be formulated as encapsulated tablets. Formulation methods for therapeutic peptide or protein products are known in the art, see e.g., Banga (ed.) Therapeutic Peptides and Proteins: Formulation, Processing and Delivery Systems (2d ed.) Taylor & Francis Group, CRC Press (2006).

[0128] Pharmaceutical compositions including a population of probiotic bacteria and one or more pharmaceutically acceptable carriers or excipients may be formulated as dietary adjuncts. In some embodiments, the pharmaceutical compositions including a population of probiotic bacteria and one or more pharmaceutically acceptable carriers or excipients may be formulated as encapsulated tablets. In some embodiments, the pharmaceutical compositions may be formulated for oral administration. Exemplary carriers and excipients do not adversely affect the viability of the probiotic bacteria. In one example, the pharmaceutical composition may include a carrier to facilitate the probiotics being delivered to the gastro-intestinal tract in a viable and metabolically-active condition. In one example, the probiotic bacterial cells are also delivered in a condition capable of colonizing and/or metabolizing and/or proliferating in the gastrointestinal tract of the subject. For example, a pharmaceutical composition including a population of probiotic bacteria may be a foodstuff. A foodstuff may be liquids (e.g., drinks), semi-solids (e.g., gels, jellies, yogurt, etc) and solids. Exemplary foodstuffs include dairy products, such as fermented milk products, unfermented mild products, yogurt, cheese, fermented cream, milk-based desserts, milk powder, or milk concentrate. The pharmaceutical composition including a population of probiotic bacteria may also be presented in the form of a capsule, tablet, syrup, etc. The pharmaceutical composition may include additional components, such as vitamins, one or more minerals, such as calcium or magnesium, one or more carbohydrates, such as lactose, maltodextrin, inulin, dextrose, mannitol, maltose, dextrin, sorbitol, fructose, and a mixture thereof.

EXAMPLES

Example 1 - Effects of Plantaricin EF (PlnEF) In vitro and In vivo

Materials and methods

Mice and experimental protocol

[0129] Mouse weight and food intake were monitored every other day over the course of the nine-week study. Freshly expelled mouse stools were collected once a week (including before the HFD started) for plating and 16S rRNA sequencing. At the time of sacrifice, the mice were anesthetized under 2% isoflurane (VetOne, Boise, Idaho, USA) and blood was collected by cardiac puncture following diaphragotomy. Termination was then ensured by cervical dislocation. Epididymal and mesenteric fat depots and ileal, cecal, and colonic tissues and contents were collected, weighed, snap-frozen in liquid nitrogen, and stored at -80 °C until further analysis. Some ileal tissue was preserved in O.C.T. cryopreserving medium (Sakura Finetek, Torrence, CA, USA) and stored at -80 °C for tissue sectioning.

Immunofluorescence staining

[0130] Ileal tissues were sectioned on a Frigocut 2800n Cryostat (Leica, Buffalo Grove, IL, USA) into 5 mM sections at the University of California Davis Center for Comparative Medicine and applied to Superfrost Plus microscope slides (Thermo-Fisher, San Diego, CA, USA) to be stored at -20 °C until staining. Tissues were fixed to slides by incubation at 15 min room temperature (RT) in 4% paraformaldehyde- (Thermo-Fisher). Slides were washed for 5 min three times in Tris-buffered saline (TBS) with 0.1% triton X-100 (Sigma-Aldrich) (TBS- T). Non-specific binding was blocked by addition of 10% bovine serum albumin (Thermo- Fisher) in TBS-T for 30 min with a parafilm coverslip. After blocking, 100 pL of primary antibody (ZO-l, 61-7300, Thermo-Fisher) diluted 1:50 in TBS was applied to each slide and incubated in a moist chamber overnight at 4 °C with a parafilm cover slip. Slides were then washed three times in TBS-T for 10 min. Secondary antibody (Alexa-488, A11008, Thermo- Fisher) diluted 1:50 in TBS was applied to each slide in 100 pL aliquots and incubated at RT in the dark for 1 h. Slides were washed three times in TBS-T for 5 min and incubated with 4',6-Diamidino-2-Phenylindole, dihydrochloride (DAPI, Thermo-Fisher) at 1 : 1000 dilution in TBS-T for 2 min. Slides were washed in TBS-T for 5 min, followed by two washes in TBS for 5 min. Slides were mounted with 20 pL of Prolong Diamond anti-fade mounting media (Thermo-Fisher) and a glass cover slip. Slides were dried overnight in the dark at RT.

[0131] Slides were imaged on a TCS SP8 STED 3X (Leica) with a 63X/1.4 oil immersion objective at the University of California Davis Center for Advanced Imaging. Two slides per mouse taken from non-serial ileal sections were imaged. As controls, some slides were stained with secondary antibody only, or serially diluted primary antibody. Z-stacking was conducted with 16 frames per sec line averaging, and 1248 x 1248 formatting. 3D-rendering and quantification of images was completed with Imaris software version 8.4 (Bitplane, Concord, MA, USA). For ZO-l, identical threshold parameters were applied to all images, and the minimum voxel number for surfaces was set to 500. For DAPI, the minimum voxel number for surfaces was set to 1000. Objects in images were quantified as volume in cubic microns and expressed as total volume of ZO-l relative to total volume of DAPI.

Gene expression analysis

[0132] Total RNA was isolated from ileal and adipose tissue using the phenol-chloroform extraction method. Approximately 20-30 mg of the tissue was cut on dry ice and immediately placed into a pre-chilled 2 mL microfuge tube containing 1.4 mm Matrix D ceramic beads (MP Biomedicals, Solon, OH, USA) with 500 pL 10% SDS and 1 M Tris-EDTA. The tissues were then disrupted and homogenized in a Fast Prep 24 instrument (MP Biomedicals) at speed setting of 6 m/s for 40 seconds (or 2x30 s for fat pads with 1 min cooling rest on ice between the two runs). Purified RNA was treated with DNase using TURBO DNA-free™ Kit (Ambion, Austin, TX, USA) following the manufacturer's instructions. The quantity and quality of RNA was determined using the NanoDrop 2000c (Thermo-Fisher) and the Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany), respectively. RNA integrity numbers (RIN) were between 7.1 and 9.9. The RNA was immediately stored at -80 °C until use.

[0133] First-strand complementary DNA (cDNA) was generated using the RETROscript kit (Thermo-Fisher) according to the manufacturer’s instructions. Real-time quantitative PCR (qPCR) was performed using a 7500 Fast Real-time PCR system (Applied Biosystems, Carlsbad, CA, USA). Each reaction mix (20 pL) consisted of 2.5 ng cDNA template, 10 pL Fast SYBR ^® Green Master Mix (Applied Biosystems), 200 nmol of each forward and reverse primer (Table 1) and nuclease free water (Ambion). All reactions were performed in duplicate. Amplification was initiated at 95 °C for 20 s, followed by 40 cycles of 95 °C for 3 s (denaturation), and 60 °C for 30 s (annealing extension). Primer specificity was assessed by adding a melting curve step at the end of amplification. Data were analysed using the 2 ^DDa method corrected for primer efficiencies according to [52] using HF group mean as the reference condition b-actin was used for transcript normalization.

Table 1 - RT-PCR primers used for intestinal tissue gene expression in this study

Protein measurements in blood, ileum, colon, and adipose tissue

[0134] Blood was incubated at room temperature for 30 min, centrifuged at 2,350 xg for 10 min, and the supernatant (serum) was collected in lithium-heparin tubes (BD Biosciences, San Jose, CA) and frozen at -80 °C until further analysis. Metabolic markers insulin and leptin were measured in the sera using the Meso Scale Discovery platform (Rockville, MD, USA) according to the manufacturer’s instructions with a duplex Mouse metabolic kit (Meso Scale).

[0135] Approximately 200 mg of epididymal adipose tissue, or 50 mg of proximal colon, and ileum tissue from each mouse was cut on dry ice, weighed, and placed into a pre-chilled 2 mL microfuge tube containing 1.4 mm Matrix D ceramic beads (MP Biomedicals). Tissues were suspended in five volumes of cold PBS containing a protease inhibitor cocktail (Sigma Aldrich) and 1% Triton X-100 (Thermo-Fisher). The tissues were then homogenized twice in a Fast Prep 24 instrument (MP Biomedicals) for 30 s at speed setting 6.5 with 1 min cooling on ice between the two runs. The extracts were centrifuged at 14,000 xg for 10 min at 4 °C, the supernatants were collected and stored at -80 °C until analysis.

[0136] Adipose tissue concentrations of adiponectin, IL-6, leptin, MCP-l, PAI-l, resistin, and TNFa were quantified using a Mouse Adipocyte Magnetic Bead kit (EMD Millipore, Temecula CA, USA). The colon and ileum tissue concentrations of IL-22 and IL-23 were quantified using a Mouse Thl7 Magnetic Bead kit (EMD Millipore). All assays were run using a Bio-plex Magpix multiplex reader (Bio-Rad, Hercules, CA, USA), and data were analysed with the Bio-Plex Manager software version 6.1 (Bio-Rad).

Bacterial composition analysis

[0137] DNA was extracted using the QIAamp fast DNA stool mini kit (Qiagen, Hilden, Germany) with modifications. Cecal, ileal content or feces were suspended in 100 pL lysis buffer consisting of 200 mM NaCl, 100 mM Tris-HCl, 20 mM EDTA and (100 mg/ml) lysozyme, and incubated at 37 °C for 30 min. Prior to purification, the suspension was mechanically disrupted in InhibitEX buffer (Qiagen) and 300 mg of 0.1 mm zirconium beads (BioSpec Products, Inc. Bartlesville, OK, USA) in a FastPrep-24 instrument (MP Biomedicals) run twice at speed setting 6.5 for 1 min. DNA was stored at -20 °C. [0138] The V4 region of the 16S rRNA gene was amplified using barcoded F515 primers and the R806 primer [3,4] Each reaction (50 pL) consisted of 5 ng DNA template, 0.8 U Ex Taq DNA polymerase (Takara), IX Ex Taq buffer, 2.5 mM MgCh. 200 mM dNTPs (Takara) and 0.1 pM of each primer. Amplification was initiated at 94 °C for 3 min, followed by 25 cycles of 94 °C for 45 s, 50 °C for 60 s, and, 72 °C for 40 s, prior to a final extension at 72 °C for 10 min. A negative control was included in each PCR run to confirm the absence of contamination. The concentration of each PCR product was measured by Quant-iT PicoGreen dsDNA (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. Approximately 50 ng of each amplicon was pooled and purified using Wizard® SV Gel and PCR Clean-Up System (Promega). The purified amplicon mixture was used for DNA library preparation and paired-end Illumina Mi-Seq (PE250) sequencing (Illumina Inc., San Diego, CA) at the University of California Davis Genome Center.

[0139] DNA sequence analysis was performed using the pipeline Quantitative Insights Into Microbial Ecology (QIIME) version 1.8.0 [55] The forward and reverse Illumina reads were assembled using the fastq-join script with a minimum overlap of 175 bp allowing 1 % differences within the overlap region. Only the joined-end reads were used for further analysis. The barcode region was extracted following by trimming off the primer regions, and trimmed reads were passed quality control while demultiplexing. Only high-quality reads that achieved following criteria were retained; 1) no errors in barcode, 2) no ambiguous bases in sequence, and 3) possess at least the minimal acceptable Phred quality score at 30. Chimeric sequences were filtered by performing chimera detection using USEARCH 6 [56] The retained reads with a median sequence length in 294 bp were clustered into Operational Taxonomic Units (OTUs) based on 97% sequence similarity against the Greengenes reference database version 13 8 [56] A representative sequence from each OTU was used to build phylogenetic trees for subsequent UniFrac distance measurements, and the representative sequence was aligned by the uclust-based consensus taxonomy assigner for taxonomic assignment. The sum of the count number of each OTU which was less than 0.005% of total observed counts were removed. PD whole tree rarefaction analysis was conducted to determine feeding group a-diversity. The b diversity between treatment groups was determined using principal coordinates analysis (PCoA) plot on weighted UniFrac distances [57] These were visualized with XLSTAT ver. 2017.5 (Addinsoft, Paris, France). Lactobacillus species-specific, quantitative PCR

[0140] Genomic DNA of L. plantarum NCIMB8826-R was extracted using the DNeasy Blood & Tissue Kit (Qiagen Inc., Valencia, CA, USA) and quantified with the Quant-iT™ PicoGreen® dsDNA Assay Kit (Life Technologies, Carlsbad, CA, USA). The DNA was then serially diluted and used to construct standard curves ranging from 10 to 10 ⁷ of 16S rRNA gene copies per reaction for absolute quantification. A total of five primer sets for 16S rRNA genes based on the L. plantarum (e.g., strain NCIMB8826) genome were used to estimate 16S rRNA gene copy numbers in the intestinal contents as previously described [58]

[0141] Real-time, quantitative PCR (qPCR) was performed in an ABI 7500 Fast Real-time PCR system (Applied Biosystems, Carlsbad, CA, USA). Each reaction contained SsoFast EvaGreen supermix with low ROX (2X) (Bio-Rad, Hercules, CA, USA), 250 nM of each primer (Life Technologies, Carlsbad, CA, USA) and approximately 30 ng of fecal/cecal bacterial genomic DNA. PCR amplification was initiated at 98 °C for 3 min, followed by 40 cycles of 98 °C for 5 sec and 60 °C for 30 sec. Each reaction was performed in duplicate. A melting curve was added at the final stage to confirm the amplification specificity. PCR amplification efficiency was calculated each time based on the standard curve and data were only considered to be valid when the amplification efficiency was within 90% -100%.

Intestinal metabolome

[0142] Cecal and colon samples were processed for Nuclear Magnetic Resonance (NMR) analysis as previously described [59] In the cecum, the following metabolites were measured: cholate, choline, creatine, formate, glucose, glutamate, glycerol, glycine, hypoxanthine, inosine , isobutyrate, isoleucine, lactate, leucine lysine, maltose, methanol, methionine, phenylalanine, propionate, serine, succinate, taurine, threonine, tyrosine, uracil, uridine, valerate, and valine. In the colon, the following metabolites were reliably measured: 4-hydroxyphenylacetate, acetate, alanine, aspartate, butyrate, cholate, choline, creatine, formate, glucose, glutamate, glycerol, glycine, hypoxanthine, inosine, isoleucine, lactate, leucine, lysine, maltose, methanol, methionine, ornithine, propionate, sarcosine, succinate, taurine, threonine, tyrosine, uridine, valerate, and valine. One mouse in the MU group was removed from analysis, because it had consistently higher values in both the cecum and colon and was identified as an outlier in ROUTS analysis with a false discovery rate of 1%. The b diversity between treatment groups was determined using principal coordinates analysis (PCoA) plot on Bray-Curtis distances. These were visualized with XLSTAT ver. 2017.5 (Addinsoft).

Bacterial strains and growth conditions

[0143] Isolation of the rifampicin-resistant mutant L. plantarum NCIMB8826-R [19] and construction of strain LM0419, a plnEFI deletion mutant of L. plantarum NCIMB8826-R [18] were described previously. L. plantarum strains were routinely grown statically at 37 °C in de Man Rogosa and Sharpe (MRS) medium (BD, Franklin Lakes, NJ, USA). For selective enrichment of L. plantarum NCIMB8826-R and LM0419 from mouse stools, rifampicin (Thermo Fisher Scientific, Waltham, MA, USA) was included in MRS agar at a concentration of 50 pg/mL.

Mice study design

[0144] The mouse study was conducted under approval of the UC Davis Animal Care and Use Committee (protocol #17500). A total of 30 C57B16/J male mice at six weeks old were obtained from the Jackson Laboratory (Jackson Laboratory, Sacramento, CA, USA). Mice were housed and acclimated to the facility as previously described [30] The mice were then fed a high-fat diet (HFD) (Harlan) formulated to contain 43% kcal from fat (mainly animal lard) for the duration of nine weeks (Table 2). The mice were divided into two groups and were given either 2 ^c 10 ⁹ cells of L. plantarum NICMB8826-R (HF-LP) in 20 pL PBS, 2 ^c 10 ⁹ cells of LM0419 (HF-MU), or 20 pL PBS (HF), orally, on alternate days, for the duration of nine weeks.

Table 2 - Dietary Composition

a HF, high-fat diet; b Teklad Custom Research Diet (Harlan Laboratories, Inc. Madison, WI); c Amioca™ from Ingredion Incorporated (Bridgewater, NJ) consists primarily of amylopectin

(100% glycemic starch).

Plantaricin peptides

[0145] The leaderless forms of PlnE (FNRGGYNF GKS VRHV VD AIGS V AGIRGILKS IR (SEQ ID NO: l)) and PlnF (VFHAY S ARGVRNNYKS AV GPADWVIS AVRGFIHG (SEQ ID NO:2)) were chemically synthesized by GenScript (Piscataway, NJ, USA) and Thermo-Fisher (Waltham, MA, USA), respectively. The processed form of plantaricin A (PlnA) (KSSAYSLQMGATAIKQVKKLFKKWGW (SEQ ID NO: 3)) was synthesized by GenScript.

Cell culture

[0146] The human intestinal Caco-2 cell line ATCC HTB-37 was cultured as previously described with minor modifications [31] Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM), 20% fetal bovine serum, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids and 2 mM GlutaMax at 37 °C in 10% CO2 (all reagents from Gibco, Life Technologies, Carlsbad, USA). To form monolayers of intestinal epithelial cells, approximately 3.3 ^c 10 ³ cells were seeded on polycarbonate permeable membrane supports (Transwell #3413; Coming, Acton, USA), and cultured for at least 21 days. Transepithelial resistance (TER) was measured with an epithelial volt-ohm-meter (EVOM, WPI, Sarasota, USA) equipped with STX-2“chopstick” electrodes.

[0147] Caco-2 monolayers with an initial TER more than 250 ohms _' cm ² were used for the assays. TER was measured in each monolayer before adding 200 pL of culture medium containing 500 mM PlnEF onto the apical surface for 3 h prior to treatment of the basolateral medium with IFN-g (100 ng/ml; R&D Systems, Minneapolis, USA) overnight at 37 °C. The media of the basal compartment wells were changed, and the wells were supplemented with TNF-a (10 ng/ml; R&D Systems). TER measurements were taken every 24 h after the TNF-a treatment.

[0148] Basolateral supernatants were harvested after 2 hr of TNF-a treatment and stored at -80 °C until quantification of IL-8 with the IL-8 Human ELISA kit according to manufacturer’s instructions (Thermo-Fisher). Thereafter, paracellular permeability was determined by the flux of fluorescein isothiocyanate-dextran (FITC-dextran; 4 kDa; Sigma, St Louis, Missouri, USA). A total of 2 mg/mL FITC-dextran was added to the apical compartment of each insert. Every 30 min, aliquots were taken from the basolateral side and the concentration of FITC-dextran was calculated based on a standard curve.

Statistical Analysis

[0149] Data were analyzed using GraphPad Prism 6 software for Windows (GraphPad Software, Inc., La Jolla, CA, USA). Unless otherwise stated, data are presented as individual mice as data points with mean ± standard error of the mean (sem) represented as bars. Group means were compared by one-way ANOVA or in the case of non-parametric data a Kruskall- Wallis test was used followed by an appropriate post-hoc test. For analyzing average body weight over time, a repeated measure ANOVA with the Greenhouse-Geisser correction was used followed by Tukey’s multiple comparisons test to compute P values. A P value <0.05 was considered significant (*, P<0.05; **, P<0.0l; ***, P<0.00l; and ****, P<0.000l).

Results

Purified PlnEF alleviates the effects of pro-inflammatory cytokines on epithelial barrier integrity in vitro

[0150] Exposure of the apical surface of Caco-2 monolayers to PlnEF peptides prevented TNF-a and INF-g induced translocation of FITC-dextran and maintained transepithelial resistance similar to non-inflamed, control cells (FIGS. 1A, 2A, and 2B). Neither of the individual peptides, PlnE or PlnF, was capable of sustained alleviation of cytokine-induced barrier disruption (FIGS. 1A, 2A, and 2B). Furthermore, concentrations of the monocyte chemokine IL-8 were significantly reduced in basolateral supernatants from Caco-2 cells treated with PlnEF (FIG. 1B). There was no depreciation of barrier integrity (as measured by FITC-dextran translocation or TER reduction) when PlnEF were applied in the absence of TNF-a and IFN-g (data not shown). These findings show that PlnEF can induce a protective state in colonic epithelial cells and that both peptides are required for biological activity.

Production of ileal tight junction protein ZO-l is increased by feeding LP to mice on a HFD

[0151] To determine whether L. plantar um plantaricin biosynthesis contributes to the anti-obesogenic effects of this species through a mechanism that involves modulation of the intestinal barrier, mice were administered L. plantarum NCIMB8826-R (HF-LP) or a plnEFI- deficient mutant (HF-MU) (10 ⁹ cells every 48 h) while being maintained on a HFD for 9 weeks. Thin sections of ileal tissue were immuno-fluorescently labeled for the tight junction protein zonula occludin-l (ZO-l) and imaged with high-resolution Z-stack microscopy. The production of ZO-l protein was significantly increased in mice fed LP (HF-LP) when compared to mice fed the MU strain (P =¾ 0.05) as well as the HF controls (P U 0.01) (FIGS. 3A and 3C). There was also an increase in transcript levels of ZO-l mRNA in whole ileal tissues in response to LP-feeding, however this change was not significant (FIG. 3B). Additionally, transcript levels of occludin mRNA were quantified, but were unchanged between feeding groups (data not shown). plnEFI is required for LP-induced reductions in weight gain of mice on a

HFD

[0152] Mice administered either LP or MU had an improved oral glucose tolerance when tested 9 weeks after the start of L. plantarum feeding (FIG. 4A). While all mice had similar serum insulin levels (data not shown), mice given either L. plantarum strain contained significantly reduced concentrations of sera glucose two hours after glucose administration when compared with HF controls (P =¾ 0.05, FIG. 4A). However, only LP-fed mice gained significantly less weight compared to HF controls (P =¾ 0.05, FIG. 4B), amounting to a 10% difference in weight change (FIG. 5A). By comparison, mice fed the DrIhEIΊ mutant (MU) had weights similar to the controls (FIG. 4B) and exhibited only a 3% total reduction in weight gain (FIG. 5A). The differences in weight gain for mice given the wild-type L. plantarum strain were observable approximately 7 weeks (47 days) after the start of the study (HF vs HF- LP, P =¾ 0.05, FIG. 4C). LP administration also resulted in significantly less food intake compared to the HF control mice (P =¾ 0.001, FIG. 4D). These changes occurred independently from alterations in feed efficiency (weight gained/kcal consumed) (FIG. 5B) or fat pad weight (FIGS. 5C and 5D).

LP reduced PAI-1 in adipose tissue and increased IL-23 in the intestine

[0153] In HF-LP but not HF-MU mice, a significant decrease in plasminogen activator inhibitor-l (PAI-l) was observed in adipose tissue compared to the HF controls (P % 0.05, FIG 6A). There was also a trend for reduced leptin in adipose tissue of mice given HF-LP compared to HF-MU (P = 0.19) or HF alone (P = 0.19, FIG. 5B). Other inflammatory cytokines in adipose tissue (TNF-a, MCP-l, IL-6) and markers of metabolic dysfunction (adiponectin, resistin, data not shown) were unchanged between the mice.

[0154] Concentrations of IL-23pl9 and IL-22, cytokines that have been linked to improved epithelial barrier integrity in obese mice [32] were also investigated. Approximately half of the mice fed LP (HF-LP) contained elevated levels of IL-23 in the ileum (4/10 mice) and colon (5/10 mice). This cytokine was only minimally or not detected in the HF controls and mice given the MU strain (HF-MU) (FIGS. 6A-6D). Although IL-23 can lead to the production of IL-22 and regenerating islet-derived protein III-g (Regllly) [32], no significant changes in IL- 22 protein quantities (FIGS. 6A-6D) and Regllly transcripts were found (data not shown).

LP did not alter the gut microbiota of DIO mice

[0155] Diet-induced alterations to the gut microbiota were confirmed at appeared both 6 and 9 weeks after the start of the HFD. Bacterial species richness (a-diversity) and community composition (b-diversity) in the feces was significantly changed compared to the baseline when a chow diet was consumed (FIGS. 7A, 8A, and 8B). Among the predominant taxa, Turicibacterales was significantly enriched by HFD whereas proportions of Bacteroidales were reduced (FIG. 8C). Notably, because there were no feeding-group specific differences, the impact of switching from a chow to HFD on the fecal microbiota (FIG. 7A) occurred independently of whether L. plantarum was consumed (FIGS. 7D, 8A, and 8B).

[0156] Administration of either WT or MU L. plantarum also did not result in significant alterations to the intestinal microbiota compared to the HF controls at other intestinal sites (FIGS. 7B, 7C, 9A-9E, 10A-10E, and 11A-11E). At each intestinal site, there were no significant differences between groups in OTU richness (data not shown), PD whole tree rarefaction curves, or b-diversity (FIGS. 9A-9E, 10A-10E, and 11A-11E). Although the ileal microbiota was enriched in Lactobacillales from L. plantarum feeding (17.8% ± 4.3, 34.7% ± 6.7, 34.2% ± 7.6; as a percentage of total reads, HF, HF-LP, and HF-MU, respectively) (FIG. 7C), this increase was not significant (FIG. 10C). Lactobacillales comprised a smaller proportion percentage of the microbiota in the cecum (FIG. 11C) and feces (FIG. 12C) and were was also unchanged between feeding groups: mice fed either L. plantarum strain or the controls.

[0157] At the genus level, Lactobacillus was the only taxon that was significantly altered in all intestinal sites (FIG. 7E). Mice fed LP or MU were significantly enriched with OTUs corresponding to L. plantarum in the ileum, cecum, and feces. Other Lactobacillus species indigenous to the murine GI tract were unaffected (FIGS. 12A-12C). Viable cell enumeration of L. plantarum from feces were consistent with an enrichment of L. plantarum in both LP and MU fed mice, and no rifampicin-resistant lactobacilli were recovered from HF control mice. There were no statistically significant differences in L. plantarum numbers between mice given LP and MU as quantified by 16S rRNA sequencing (FIG. 7E) and L. plantarum enumeration by qPCR (FIGS. 13A-13C).

LP feeding did not alter the intestinal metabolome

[0158] In agreement with the lack of interaction between the gut microbiota and PlnEF biosynthetic capacity of L. plantarum, there were no significant changes between the LP and MU fed mice among metabolites measured in the cecum or colon contents. PCoA of Bray- Curtis distances between cecal (FIG. 14A) and fecal (FIG. 14B) metabolomes of HF, LP, and MU-fed mice did not reveal any significant clustering by feeding group. Instead, both LP and MU fed mice had reduced concentrations of cholate in the cecum (P =¾ 0.05, FIG. 14C) and threonine in the colon (P =¾ 0.05, FIG. 14D), respectively, compared to the HF controls.

Example 2 - Sensitivity to Plantaricin EF (PlnEF) is Dependent on CorC

Materials and methods

Bacterial strains and growth conditions

[0159] Strains and plasmids used in this study are listed in Table 3. Lactobacillus strains were grown in de Manne Rogosa Sharpe (MRS, BD Biosystems) laboratory culture broth. L. pentosus and LP965 strains were grown without aeration at 30 °C. L. pentosus strains were isolated from olive fermentations (86), and strain-level variation was confirmed by discrepant sensitivities to PlnEF and comparisons between nucleotide sequences of corC genes (phylogenetic tree: FIG. 15). L. casei BL23 and L. plantarum NCIMB 8826 (LP8826) were grown without aeration at 37 °C. Escherichia coli DH5a was grown in Lennox lysogeny broth (Teknova) with aeration (250 rpm) at 37 °C. When appropriate, 5 or 100 pg/ml chloramphenicol (Cm ^R ) and 5 or 300 pg/ml erythromycin (Ery ^R ) (Sigma-Aldrich) was included in the media for Lactobacillus and E. coli, respectively. To measure sensitivity to Mg ²⁺ and Co ²⁺ , a sterile solution of anhydrous MgSCri or CoSCri was added to MRS to reach a final concentration of 500 mM or 1 pM, respectively.

Table 3

Bacteriocin peptide synthesis

[0160] The full-length peptide sequences of plantaricins taken from the published genome of L. plantarum WCFS1 (Refseq: NC_004567.2) were downloaded fromNCBI and trimmed to delete export-signal peptide sequences. Leaderless forms of plantaricin E

(FNRGGYNFGKSVRHVVDAIGSVAGIRGILKSiR (SEQ ID NO: l)), and plantaricin A (PlnA) (KS S AYSLQMGATAIKQVKKLFKKWGW (SEQ ID NO: 3)) were chemically synthesized by Genscript. Plantaricin F

(VFHAYSARGVRNNYKSAVGPADWVISAVRGFIHG (SEQ ID NO 2)) was synthesized by Thermo-Fisher. Peptides were 98-99% pure and diluted in ultra-pure, molecular grade water (Ambion) prior to being stored at -20 ^° C until use.

Bacteriocin activity assays

[0161] The antimicrobial activity of PlnEF against target cells was tested using a 96-well microtiter plate-based activity assay. Overnight (stationary phase) cultures were diluted 1:50 in MRS broth to an Oϋboo of 0.1. Five pl of this cell suspension was added to 200 pl of MRS per well together with two-fold dilutions of PlnEF or PlnA. The microtiter plates were then measured every 30 min at Oϋboo nm (Synergy 2, Biotek instruments). The minimum inhibitory concentration (MlCrio) was defined as the peptide concentration (the sum of both peptides [in a 1 : 1 ratio]) that inhibited growth by 50% after 6 h incubation.

Isolation of plantaricin EF-resistant mutants

[0162] LP965 was incubated for 24 h on MRS plates containing PlnEF at 5X of the calculated MIC (62.5 nM). A single colony isolate from this plate was used to inoculate MRS broth with 70X MIC (875 nM) PlnEF and incubated overnight. The resulting cell suspension was spread onto MRS agar with and without PlnEF supplemented at a concentration of 5X MIC. A single colony from the PlnEF -containing MRS agar was suspended in PBS prior to plating serial dilutions onto MRS agar containing a 15X MIC (187.5 nM) PlnEF. After a 48 h incubation at 30 ^° C, a total of six individual colonies were recovered. Three colonies and a naive LP965 strain were grown in MRS without bacteriocin overnight. Serial dilutions of the resulting cell suspensions were then plated onto MRS agar containing a 15X MIC

concentration of PlnEF and incubated overnight. Out of a total of 420 colonies, seven PlnEF resistant colonies were picked from separate 15X MIC MRS plates and grown in MRS broth for approximately 36 generations (four subcultures). PlnEF resistance was confirmed for each of the isolates prior to preparation of glycerol stocks and storage at -80 ^° C.

Soft-agar growth inhibition assay

[0163] Single colonies of naive and PlnEF -resistant LP965 strains isolated from -80 ^° C glycerol stocks were grown in MRS broth overnight. This culture was diluted in PBS and inoculated into agar at a final concentration of 10 ⁵ cells/ml. To expose putative mutants to PlnEF, a PlnEF-overexpression strain of L. plantarum NCIMB 8826 was constructed. The genes for plnEFI ( WCFS1 lp_04l9, lp_042l, lp_0422) were amplified with primers V and Z using genomic DNA as a template (Table 4).

Table 4. Primers used for cloning in this study

a Restriction enzyme sites are underlined; linker DNA sequences are italicized

[0164] The product was digested with Sall-HF and SacII-HF (New England Biolabs) and ligated into the multiple cloning site of pJIM2246 (87) resulting in pJIMBGl. Escherichia coli DH5a was transformed with the ligation mixture and transformants were selected for Cm ^R resistance. Plasmids were isolated from E. coli using the Qiaprep Spin Mini-prep kit (Qiagen) and checked by PCR using primers V and Z (Table 4) and DNA sequencing for verification. LP8826 was transformed as previously described (77) with pJIMBGl (containing plnEFI), before inoculation onto MRS agar plates at a cell density of 1 x 10 ¹⁰ cells/ml in phosphate buffered saline (PBS). Upon drying, molten soft agar (1.5% w/v agar) was then overlaid on top. A resistant LP965 phenotype was detected by a reduced zone of inhibition compared to the naive strain after overnight incubation at 37 ^° C.

Isolation of genomic DNA and whole genome sequencing

[0165] DNA was isolated from stationary phase cultures by incubating cell pellets with lysozyme (Sigma, 10 mg/ml) for 1 h at 37 ^° C followed by a 1 h incubation with proteinase K (Qiagen, 1 mg/ml) at 56 ^° C. DNA was then recovered by phenol-chloroform extractions, followed by ethanol precipitation (88). High molecular weight DNA was confirmed on a 1% agarose gel and quality confirmed by measuring spectrophotometric absorbance at 260 and 230 nm (Nanodrop 200c, Thermo-Fisher). DNA Sequencing was performed using a PacBio RSII instrument with desired insert size of 10 kb and P6C4 chemistry according to manufacturer’s instructions at the University of California, Davis, DNA Technologies Core. DNA sequence analysis

[0166] Sequence SMRTcell files were downloaded from the UC Davis Sequencing core and imported to the PacBio SMRT portal graphical interface unit. The genome of LP965 was assembled de novo using the hierarchical genome assembly protocol 2 (RS_HGAP_assembly.2) with default parameters, except for the following adjustments: minimum subread length 1 kb, genome size 3.4 Mb, target coverage 15X, and minimum seed read 6 kb. This resulted in one chromosome and five plasmids, with a 154-fold minimum coverage. The chromosome and plasmids were then checked for circularity with Gepard vl.4 and overlapping ends were trimmed using Seqbuilder Version 14.0. (DNASTAR. Madison, WI).

[0167] All sequences were then compiled into a single FASTA file and used as a reference sequence for iterative re-aligning of PacBio reads using the algorithm (RS Resequencing. l). After three rounds of re-alignments, the final assembly resulted in an average 31 OX coverage (chromosome of 3,015,426 bp, and plasmids with the following sizes, plasmid 1: 66,439 bp, plasmid 2: 52, 109 bp, plasmid 3: 41, 818 bp, plasmid 4: 23, 484 bp, and plasmid 5: 16,940 bp). To ensure a high-quality reference genome, short read archive data generated from LP965 (accession number: SRR1553345) was downloaded from NCBI. A total of 4.7 million 75 bp (349.3 Mb) single end Illumina reads were assembled into 307 contigs (Cellera assembler, default parameters). These contigs were then aligned to the PacBio assembly data (RS AHA Scaffolding. l) to generate a single high confidence FASTA file with one chromosome and five plasmids (LP965). The LP965 genome sequence was then uploaded to RAST (89) for annotation and further analyzed with Seqbuilder.

[0168] The LP965.EF.A strain genome was assembled using the high-quality, wild-type genome of LP965 as a reference and the program (RS Resequencing. l) with default parameters except for the following adjustments: minimum subread length of 8 kb, minimum read quality 75, minimum polymerase read length of 12 kb. This resulted in one chromosome and five plasmids with an average coverage of 193X. To identify variants between the wild- type and EF.A strain, each genome was compiled into single FASTA files and aligned with MAUVE 2.4.0 (90). This resulted in five indels and three single nucleotide polymorphisms found on the chromosome.

[0169] Each region with conflicting base calls was used to design PCR primers to amplify a 250 bp segment of DNA (Table 5). PCR was conducted with Takara ex-Taq according to manufacturer’s instructions. The PCR products were purified with the Wizard SV gel and PCR Clean-Up system (Promega) before being submitted for bi-directional DNA sequencing at the University of California Davis Sequencing core.

Table 5. DNA sequencing primers used in this study

CorC and plnEF deletion mutagenesis

[0170] LP965 was not amenable to genetic manipulation with techniques described to transform this strain previously (76) or by adjusting parameters (glycine concentrations, recovery times, voltage) described therein. Repeated attempts to delete the corC open reading frame (ORF) in LP8826 (WCFS1 lp_267l) using methods previously established for clean deletions in this strain (77) were equally unsuccessful. Primers used for these attempts are listed in Table 4 (A/B and C/D).

[0171] To construct a plnEF deletion mutant of L. plantarum NCIMB8826, the pRV300 system was used as previously described (77). Primer sets used were designated as W/X and Y/Z (Table 4). The loss of plnEF was confirmed with PCR using primer set W/Z. Heterologous expression of L. plantarum corC in L. casei BL23

[0172] The corC genes from LP965 and EF.A were amplified with primers A and D using genomic DNA as a template (Table 4). The product was digested with EcoRl-HF and Sacl- HF (New England Biolabs) and ligated into the multiple cloning site of pJIM2246 (87) resulting in pJIMDHl (WT LP965 CorC) and pJIMDH2 (EF.A CorC). E. coli DH5a was transformed and selected for as previously described. Plasmids were isolated from E. coli and checked by PCR using primers A and D (Table 4) and DNA sequencing for verification. L. casei BL23 electrocompetent cells were prepared and transformed as previously described (91). Briefly, freshly prepared BL23 cells were electroporated with 400 ohms, 2 kV, and 25 pF and then immediately transferred in MRS supplemented with 0.5 M sucrose. The cells were then incubated for 4 h and transformants were selected by plating serial dilutions on selective medium. PCR and DNA sequencing confirmed the presence of the plasmids in L. casei BL23.

Quantitative PCR

[0173] Total RNA was harvested from late exponential-phase cultures (88). DNA was digested with the Turbo-DNAse kit (Ambion) according to manufacturer’s instructions. Quality was confirmed (16S rRNA / 23S rRNA ratio >1.7) with the Agilent RNA 6000 Nano kit and analyzed on an Agilent 2100 bioanalyzer. cDNA was retro-transcribed from 1 pg of total RNA with the RETRO-script kit (Ambion). Quantitative qPCR was performed on an Applied Biosystems 7500 using gene-specific primers (corCFor: 5’-

GGTCTCGTTAGCTGGTATCATT-3’, corCRev: 5’ -CGGATAGGTTGAAGAGGGATAAG- 3’) and SYBR green PCR mastermix (Applied biosystems). The following qPCR parameters were used: 95 ^° C for 10 min, followed by 40 cycles of denaturation at 95 ^° C for 15 s, annealing and extend at 60 ^° C for 1 min. The 3 -step amplification was followed by a melting cycle (95 ^° C for 10 s, 65 ^° C for 60 s and 97 ^° C for 1 s). All measurements were made in duplicate. As an internal control, rpoB (rpoBF or: 5’-CGATGACTCTAACCGTGC-3’, rpoBRev: 5’-CAAGGCAATCCCTGAGTC-3’) was used and relative transcript quantification was calculated through the delta delta CT method (92) using the wild type strain transcript levels as the reference condition.

CorC amplicon sequencing and analysis of L. plantarum and L. pentosus

[0174] The corC gene from /.. plantarum and environmental L. pentosus isolates (Table 3) was amplified with primers designed using genomic DNA of LP965 and L. pentosus BGM48 as the template, respectively (LplantcorCF or: 5’-CCCGCTGACTACGATAGACC- 3’, LplantcorCRev: 5’ -TGCCATTTTAAGTAGTGCGTGT-3’ , LpentcorCFor : 5’- GAGTAGGCC AGACCTGC AAC-3’ , LpentcorCRsr. 5’ GGCCTGACTTTTCCCTTTTC-3’ ). Purified PCR amplicons were submitted for DNA sequencing, and forward and reverse DNA sequencing reads were conjoined and trimmed with SeqMan Pro 14 (DNASTAR). DNA sequences were aligned with MegAlign Pro 14 (DNASTAR) using the MUSCLE aligner (93). To identify conserved regions of the CorC protein, the LP965 CorC amino acid sequence was submitted to pBLAST with default parameters. The top 250 sequences from this search were downloaded and aligned. Amino acid sequences are colored with the Zappo scheme according to their physico-chemical properties (94).

[0175] For phylogenetic analysis and tree-building, the evolutionary history was inferred by using the Maximum Likelihood method based on Tamura-Nei distances for nucleotides (95) or the JTT matrix-based model for amino acids (96). The bootstrap consensus tree inferred from 1000 replicates was computed (97) and only bootstrap values above 50 were considered of value. Evolutionary analyses were conducted in MEGA7 (95).

Structural modeling of CorC protein

[0176] A 3D structure model of the CorC protein from LP965 was performed using relax application (98-101) in Rosetta structural modeling software (102-104) using the x-ray structure of a hemolysin-like protein containing a CBS domain of O. oeni PSU (PDB ID: 30CO) as a template. Sequence identity between the CorC protein region from Y216 to G351 and 30CO (A211 to G346) was -50% (FIG. 16). All structural modeling figures were generated using the UCSF Chimera package (105). The highest ranked model built by Rosetta was used to investigate the mutational hotspot region in the CorC protein. Further domain annotation was accomplished by submitting the LP965 CorC protein to prosite domain scanner and the SWISS-MODEL online server.

Statistics

[0177] Growth assays were conducted with technical replicates in triplicate and data are representative of at least two independent experiments. Data are presented as average values ± SD. Analysis of covariance (ANCOVA) was used to determine whether the growth rates of cultures in logarithmic phase were statistically different from one-another. One-way ANOVA was used to determine significant differences between final optical densities of cultures and relative expression of corC transcripts. Accession numbers

[0178] LP965 WT sequence data was deposited into the National Center for Biotechnology

Information with accession numbers: CP023490 to CP023495, the EF.A PlnEF-resistant strain was deposited in NCBI with accession numbers: CP026505-CP026510.

Results

Selection of PlnEF-resistant LP965 mutants

[0179] Plantaricin E (PlnE) and F (PlnF) were synthesized and combined in equal molar ratios prior to measuring for their inhibitory activity against LP965. The PlnEF MIC50 of LP965 in MRS broth was 12.5 nM, and LP965 did not grow in the presence of PlnEF at concentrations above 50 nM when measured over a 24 h period (data not shown). Spontaneous, PlnEF-resistant mutants of LP965 were enriched by exposing the strain to PlnEF at doses above 50 nM on MRS agar. A total of seven (out of >400) putative PlnEF-resistant isolates were randomly selected and confirmed for PlnEF resistance using soft agar inhibition (data not shown) and growth assays (FIG. 17). Five of the seven isolates (designated EF.A, and EF.D to G) exhibited a four-fold increase in PlnEF MIC50 values (MIC5o>50 nM) compared to LP965. The remaining two isolates (EF.B and EF.C) were less resistant (MIC50 = 25 nM and MIC50 = 12.5 nM, respectively). However, growth rates and maximum cell numbers (as measured by Oϋboo) of all seven isolates were increased relative to the wild-type LP965 strain in the presence of 25 nM PlnEF (FIG. 17). This effect was not the result of a change in the capacity to grow in MRS laboratory culture medium lacking PlnEF because the growth rates of PlnEF-resistant mutants did not significantly deviate from LP965 when incubated under standard conditions (static, 37 ^° C for 24 h) (ANCOVA of growth rates F(3, 148) = 0.0555, P = 0.9827, data not shown). Moreover, PlnEF resistance was specific and not the result of general, stress-related responses to antibacterial peptides because all strains grew equally well in the presence of 1000 nM of the unrelated bacteriocin plantaricin A (PlnA) (ANCOVA of growth rates F(3, 44) = 1.752 P = 0.17, FIG. 17).

PlnEF-resistant isolates contain mutations in a gene coding for a putative membrane-bound, magnesium/cobalt efflux protein

[0180] Whole genome sequencing was conducted on the WT LP965 strain and a single L. plantarum isolate displaying a high-level PlnEF resistance (EF.A). A high-confidence reference genome for LP965 was constructed using long (PacBio) and short (Illumina) read sequence data followed by alignment against the EF.A genome. These alignments revealed three chromosomal point mutations and five gaps (a result of putative indels) between the two genomes. To rule out sequencing errors, the eight genomic regions with sequence variations were amplified by PCR and subjected to DNA sequencing (Table 5). Only one of the eight putative mutations was verified and this mutation was found to be localized in corC, a putative membrane-bound, magnesium/cobalt efflux protein. Compared to LP965, a guanine was changed to a thymine in EF.A at position 846,396. This single nucleotide change is predicted to result in the substitution of a valine (V) instead of a glycine (G) at amino acid residue 334 (G334V) of CorC.

[0181] The corC gene and 100 bp flanking DNA from the six remaining PlnEF-resistant clones was amplified and sequenced. This showed the presence of the single point mutation in the corC gene (translated to G334V) of the five highly-PlnEF-resistant mutants (EF.A, and EF.D to EF.G). The other two PlnEF-resistant isolates (EF.B and EF.C) did not contain mutations in corC or the upstream intergenic region. Gene transcript quantification showed that expression of corC in EF.B was 2.5-fold down regulated compared to LP965 during late exponential-phase growth (p < 0.01), indicating that EF.B PlnEF-resistance was the result of reduced CorC expression, but no changes in relative transcript levels could be attributed to EF.C.

Heterologous expression of wild-type LP965 corC increases Lactobacillus casei BL23 sensitivity to plantaricin EF

[0182] Numerous attempts to truncate or delete LP965 corC were unsuccessful using methods commonly used for genetic modification of other L. plantarum strains (76). Additionally, attempts to delete or truncate corC in L. plantarum NCIMB 8826 (LP8826) were also unsuccessful. Because LP8826 is amenable to genetic manipulation (77) it was concluded that CorC is likely essential for L. plantarum during growth in MRS, however, it is possible that use of other mutagenesis approaches besides the suicide vector pRV300 used here could be successful for constructing a corC deletion mutant.

[0183] Therefore, to confirm that CorC is required for PlnEF sensitivity, corC from wild- type LP965 and the EF.A mutant were introduced into L. casei strain BL23. This strain is more resistant to PlnEF than LP965 (MIC50 = 25 nM) and contains a CorC homolog with only 61% amino acid identity to LP965 CorC. Introduction of wild-type LP965 corC into L. casei BL23 on a stably -maintained plasmid, decreased the MIC50 of PlnEF from 25 nM to 12.5 nM (FIG. 18). Conversely, BL23 harboring the G334V mutant from EF.A was as equally -resistant as strain BL23 containing the expression plasmid pJIM2246 (MIC50 = 25 nM) (FIG. 18). The growth rates of BL23 harboring either corC variant were not impaired in the absence of PlnEF (data not shown). These results confirm that wild-type LP965 corC increases sensitivity to PlnEF and that the single G334V amino acid substitution is sufficient to prevent this increased sensitivity from occurring.

Mutations in the EF.A CorC protein reside in a highly conserved functional domain

[0184] CorC is predicted to be a membrane-bound, magnesium and cobalt efflux protein containing four transmembrane domains as well as two extracellular cysteine-beta-synthase domains (CBS, also known as Bateman domains (79)) and two extracellular, transporter- associated domains (PFAM03471). To capture the diversity of CorC -like proteins that have been published, the LP965 CorC protein was aligned to the closest 250 related proteins identified in Genbank. This alignment showed that the EF.A mutation is located in a highly conserved CBS domain of the CorC protein (FIG. 20, black star).

[0185] To elucidate the molecular basis for CorC interactions with PlnEF, structural modeling was performed for the LP965 WT CorC protein region from Y216 to G351, encompassing the extracellular CBS domain mutated in EF.A. This was performed using a CorC -like protein from Oenococcus oeni (PDB ID:30CO) because it is highly related to CorC in LP965 and a crystal structure for this protein is available. Notably, 30CO forms a dimer and the Y216 to G351 model of LP965 CorC indicated that this region may also be involved in a dimerization interface (FIG. 20A). The template-based model revealed that the G334V mutation resides in a coil located between two beta-sheets (FIGS. 20B and 20C). Because these domains are predicted to be extracellular, the mutations likely result in steric inhibition or alteration in overall charge of the protein, with the valine residue occupying a potential binding site for PlnEF.

Sensitivity of L. pentosus to PlnEF is associated with CorC

[0186] Because L. pentosus is highly related to L. plantarum but lacks genes for the synthesis of PlnE, PlnF, and Plnl, it was hypothesized that L. pentosus sensitivity to PlnEF would vary in a manner consistent with variations in the amino acid sequence of CorC. Assessment of nine L. pentosus strains for PlnEF sensitivity showed that the MIC50 values for seven of those strains were equal to or less than found for LP965 (MIC50 < 12.5 nM PlnEF). The remaining two L. pentosus strains EL8 and EL24 were more resistant to PlnEF (MIC50 > 50 nM and MIC50 = 25 nM, respectively).

[0187] Alignments of the predicted proteins based on corC gene sequences indicated that L. pentosus CorC proteins are highly conserved (FIG. 21). Those CorC proteins shared between 96 to 97% amino acid sequence identity to the CorC proteins in LP965 and LP8826. Among the nine L. pentosus strains, the transmembrane and intracellular domains were distinguished by only two amino acid substitutions (amino acid 62 and 155). For the extracellular portion, a single amino acid substitution at amino acid 445 distinguished four of the L. pentosus strains from the other five. However, both EL8 and EL24 were found to contain additional variations. EL8 was predicted to contain a valine (V) instead of a glutamic acid (E) at position 366 in the transporter-associated domain (PFAM03471). Strain EL24 was predicted to contain a histidine at position 190 instead of an aspartic acid (D190H) in an extracellular region of the protein lacking domain characterization.

PlnEF increases the effects of Mg ²⁺ stress

[0188] Growth rates and maximum cell densities of the L. plantar um PlnEF -resistant mutants were equivalent to the LP965 parental strain when incubated in commercial MRS containing added 500 mM MgSOr or 1 mM C0SO4 (data not shown). However, because sensitivity to these cations might depend on the presence of PlnEF, the effects of elevated Mg ²⁺ and Co ²⁺ concentrations on L. plantarum strain NCIMB 8826 were also tested. This strain (LP8826) has the capacity to synthesize PlnEF, the cognate immunity protein, Plnl, and contains a CorC protein which is 99% identical to CorC in LP965. For comparison, a LP8826 plnEF deletion mutant (LM0420) was also constructed. In MRS containing 500 mM MgSOr. LP8826 exhibited a severely inhibited growth rate and reached lower cell numbers compared to LM0420 (FIG. 22). Both strains grew similarly in MRS lacking additional MgSOr (data not shown). Specificity for Mg ²⁺ as opposed to Co ²⁺ was indicated by the lack of growth impairment for either strain in MRS containing excess C0SO4 (1 mM added to MRS, data not shown). These findings reveal that PlnEF likely increases Mg ²⁺ stress by limiting the efflux functionality of the CorC protein. Because the gene encoding the PlnEF immunity protein Plnl was retained in LM0420, this outcome also suggests that the PlnEF immunity protein Plnl is insufficient to prevent PlnEF-associated impairments to Mg ²⁺ homeostasis.

Example 3 - Effect of PlnEF on ATP Concentrations

[0189] Because bacteriocins can disrupt energy metabolism in sensitive cells, intracellular ATP levels were measured to verify PlnEF resistance among the LP965 mutants. In the absence of PlnEF, incubation in 10 mM glucose resulted in increased intracellular ATP levels of all strains by five-fold within 10 min (FIGS. 23 and 24). As expected, intracellular ATP concentrations of the WT strain declined upon exposure to 25 nM PlnEF (FIG. 23), confirming the negative impact of PlnEF on cell viability. Conversely, intracellular ATP quantities of strains EF.A to EF.E increased until 20 min incubation (FIGS. 23 and 24), and only slightly declined thereafter. When compared to WT LP965, ATP levels of EF-resistant strains were approximately 4-fold higher after 40 min (P < 0.0001, FIGS. 23 and 24). The effect of PlnEF was specific to intracellular ATP quantities (FIGS. 23 and 25). There was no change in extracellular ATP concentrations after PlnEF challenge; whereas, all strains rapidly released ATP (approximately 20 nM) when challenged with a growth-inhibiting concentration (25 mM) of nisin (P < 0.0001, FIGS. 23 and 25).

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[0190] One or more features from any embodiments described herein or in the figures may be combined with one or more features of any other embodiment described herein in the figures without departing from the scope of the disclosure.

[0191] All publications, patents and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.