CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of U.S. Provisional Application No. 62/976,885, filed February 14, 2020, the entire contents of which are incorporated herein by reference.

STATEMENT REGARDING SEQUENCE LISTING The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is 73528_SEQ_fmal_20210211_ST25. The text file is 20 KB; was created on February 11, 2021; and is being submitted via EFS- Web with the filing of the specification.

STATEMENT OF GOVERNMENT LICENSE RIGHTS This invention was made with government support under Grant No. R01 AI080609, awarded by the National Institute of Health Sciences. The Government has certain rights in the invention.

BACKGROUND

The application of traditional antibiotics can have many undesirable consequences. Most notably, their activity can promote the emergence of resistance and disrupt health- beneficial microbial communities. Since these drawbacks are a direct result of off-target effects, the scientific community has sought to develop alternative antimicrobial strategies, with a focus on enhanced and tunable selectivity. Recent examples of such efforts include bacteriophage harboring programmable Cas9 and bacteria that deliver regulated toxins by conjugation. While these and other strategies hold promise, there are many hurdles to overcome prior to their widespread implementation. For instance, phage-based approaches suffer from the rapid evolution of resistance in target populations, the particles can be rendered inactive in transit to the target site (e.g. in the gastrointestinal (GI) tract), and the often narrow host range of phage can lead to challenges in identifying clones active against a particular bacterium. Selective antibacterial methodologies relying on conjugation circumvent some of these limitations. In particular, a donor strain derived from species evolved to transit the GI tract can be utilized. However, a disadvantage of these approaches is that their specificity derives from a detailed knowledge of the target bacterium. It is therefore unclear whether such approaches could be widely adopted or used in situations involving poorly characterized pathogens. Moreover, there is a relatively low efficiency of plasmid transfer.

Accordingly, despite the advances in the art of selective antibacterial methods, there remains a need for a flexible and selective antibacterial platform that is amenable to facile optimization to selectively target bacteria of interest within heterogenous populations for clearance and can be applied in contexts ranging from therapeutic to industrial applications. The present disclosure addresses these and related needs.

SUMMARY

This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

In one aspect, the disclosure provides a transgenic Gram-negative bacterium. The transgenic Gram-negative bacterium comprises a functional type VI secretion system and expressing at least one heterologous affinity reagent that specifically binds a surface antigen on a target Gram-negative bacterium.

In some embodiments, the heterologous affinity reagent is a single domain antibody, or an antigen-binding fragment thereof. In some embodiments, the target Gram negative bacterium is a different species or strain from the transgenic Gram-negative bacterium. In some embodiments, surface expression of the surface antigen is substantially unique to the target Gram-negative bacterium within a heterogeneous population.

In some embodiments, the transgenic Gram-negative bacterium is a probiotic bacterium compatible with the intestinal flora of a subject. In some embodiments, the transgenic Gram-negative bacterium is a species of Enterobacter, Bacteroides, Escherichia, Pseudomonas, Burkholderia, Vibrio, Delftia, and Serratia. In some embodiments, the transgenic Gram-negative bacterium is selected from Enterobacter cloacae, Bacteriodes fragilis, Bacteriodes uniformis, Bacteroides dorei, Bacteroides xylanisolvens, Bacteroides ovatus, Pseudomonas putida, Escherichia coli, and other known probiotic species or strains.

In some embodiments, the target Gram-negative bacterium is E. coli and the at least one surface antigen is BamA, or an antigenic domain thereof. In some embodiments, the heterologous affinity reagent is a single domain antibody that comprises an amino acid sequence with at least 90% identity to SEQ ID NO:3, or an antigen-binding fragment thereof.

In some embodiments, target Gram-negative bacterium includes a pathogenic E. coli and the at least one surface antigen is an extracellular domain of intimin. In some embodiments, the heterologous affinity reagent is a single domain antibody that comprises an amino acid sequence with at least 90% identity to SEQ ID NO:4, or an antigen-binding fragment thereof.

In some embodiments, the transgenic Gram-negative bacterium expresses a plurality of different heterologous affinity reagents, wherein each heterologous affinity reagent specifically binds a different surface antigen on at least one target Gram-negative bacterium. In some embodiments, each different heterologous affinity reagent specifically binds a different surface antigen on a different target Gram-negative bacterium.

In another aspect, the disclosure provides a probiotic composition comprising the transgenic the Gram-negative bacterium as described herein and a therapeutically effective carrier and/or excipient for oral administration, wherein the transgenic Gram-negative bacterium is compatible with the intestinal flora of a subject.

In another aspect, the disclosure provides a method of selectively depleting target Gram-negative bacteria from an environment, comprising contacting the target bacteria with a plurality of the transgenic Gram-negative bacteria recited or the related composition described herein. In some embodiments, the environment is an aqueous environment. In some embodiments, the environment is an intestinal environment. In some embodiments, the environment is on an industrial surface.

In another aspect, the disclosure provides a method of selectively depleting target Gram-negative bacteria and/or preventing colonization by the target Gram-negative bacteria in a subject in need thereof. The method comprises administering to the subject an effective amount of the transgenic Gram-negative bacterium or the related composition described herein.

In some embodiments, the target Gram-negative bacteria is pathogenic. In some embodiments, the target Gram-negative bacteria is pathogenic E. coli and the at least one surface antigen is BamA, or an antigenic domain thereof. In some embodiments, the heterologous affinity reagent is a single domain antibody that comprises an amino acid sequence with at least 90% identity to SEQ ID NO:3, or an antigen-binding fragment thereof. In some embodiments, the target Gram-negative bacteria is pathogenic E. coli and the at least one surface antigen is intimin, or an extracellular domain thereof. In some embodiments, the heterologous affinity reagent is a single domain antibody that comprises an amino acid sequence with at least 90% identity to SEQ ID NO:4, or an antigen-binding fragment thereof. In some embodiments, the effective amount of the transgenic Gram negative bacteria or the composition is administered for delivery to the gastro-intestinal tract of the subject. In some embodiments, the effective amount of the transgenic Gram negative bacterium is administered orally. In some embodiments, the target Gram-negative bacteria is depleted from and/or prevented from colonizing the gastro-intestinal tract environment of the subject. In some embodiments, the transgenic Gram-negative bacterium is compatible with the intestinal flora of the subject.

In another aspect, the disclosure provides a disinfecting composition comprising the transgenic the Gram-negative bacterium described herein and an effective carrier for application to a surface.

In another aspect, the disclosure provides a method of selectively depleting an industrial surface of a target Gram-negative bacterium, comprising contacting the surface with an effective amount of the transgenic Gram-negative bacterium described herein. In some embodiments, the transgenic Gram-negative bacterium is in aqueous solution.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

FIGURES 1A-1C: Cell-cell adhesion mediated by surface-expressed nanobody- antigen pairs promotes targeted killing via the T6SS in liquid medium. (1 A) Schematic of the strategy to achieve programable and selective cell killing. Bacteria with unique surface antigens (shapes) and a programmed T6S+ inhibitor cell (PIC, pink) are represented. (IB) Growth of E. cloacae ArhsA rhsL· rhsB rhsle tae4 tai4 (A ei x3), under contact-promoting (solid) or well-mixed (liquid) growth conditions in co-culture with the indicated donor E. cloacae strain (n=3 technical replicates ± S.D.; * p<0.05, t-test). E. cloacae AicmF is deficient in T6SS activity due to lack of a required structural component. (1C) Impact of E. cloacae PICs expressing the indicated cell surface-displayed nanobody on growth of a T6S-susceptible strain E. cloacae A ei x3, containing deletions of the T6SS effector genes rhsA, rhsB and tae4 and their corresponding immunity genes) expressing cognate or orthogonal antigens during co-culture in liquid medium (n=2 biological replicates with 3 technical replicates each, ± S.D.). See also FIGURES 5A-5E.

FIGURES 2A-2D: Programmed inhibitor cells (PICs) achieve potent and specific killing of a cognate antigen expressing species in liquid culture and under aggregation- promoting conditions. (2A) Growth of E. coli MG1655 expressing the indicated antigens during co-culture in liquid medium with PICs expressing cognate or orthogonal nanobodies. (n=2 biological replicates with 3 technical replicates each, ± S.D.). (2B) Survival of Ag-Y expressing E. coli grown to stationary phase in pure culture, then mixed with the indicated PIC. The control PIC expressed the empty surface-display construct used for nanobody presentation (n=2 biological replicates with 3 technical replicates each, ± S.D.). (2C) Viable CFUs (percentage of control PIC treated cultures incubated without PEG800) of Ag-Y expressing E. coli after 6 hrs growth with the PICs indicated in liquid medium amended with the indicated concentration of PEG 8000 (n=3 technical replicates, representative of 2 biological replicates, ± S.D.). (2D) Quantification of aggregate sizes (number of aggregates counted containing the indicated number of cells) observed by phase contrast microscopy of cultures grown with indicated concentration of PEG 8000 as described in (2C). See also FIGURES 6A-6E.

FIGURES 3A-3E 3: PICs selectively deplete target cells from defined bacterial mixtures. (3 A), Relative fitness of a control E. coli strain (control strain as described in FIGURES 1A-1C) compared to a strain displaying Ag-Y when grown in liquid co-culture with PIC ^{xb Y} with (parental) or without (A icmF) an active T6SS (n=3 technical replicates, representative of 2 biological replicates, ± S.D.; * p<0.05, t-test). The initial ratio (by OD6OO nm) of the E. coli control to Ag-Y expressing strain is indicated below. (3B) Representative fluorescence micrographs indicating relative abundance of E. coli control (red) and Ag-Y expressing (green) cells after growth with parental or T6S-inactive strain (Aicinl _' ) pic ^{Nb Y} . The initial ratio of E. coli control to Ag-Y was 1:1. PIC cells are not fluorescently labeled. Scale bar, 5 pm. Full micrographs in FIGURE 7A. (3C, 3D), Comparison of the change in relative abundance of community members after 8 hr growth with or without PIC addition. The E. coli strain included is indicated at top. Initial abundance (by OD6oo nm): PICs, 20%; target E. coli, 6%. (E), Change in abundance of community members with and without 8 hr treatment with ciprofloxacin at 40 ng/mL (2.5x MIC of E. coli). Initial and final read counts for each species in C-E presented in TABLE 1. See also FIGURES 7A-7C. FIGURES 4A-4E: PICs targeting native a natural surface antigen achieves selective killing in a complex community and resistance to killing is slow to emerge. (4A) Viable CFUs (percentage of control PIC treated cultures) remaining after growing the indicated E. coli strains for 6 hrs with the PICs noted at top in liquid medium amended with 5% PEG8000 to mimic a natural polymer-rich environment. Data represent 2 biological replicates each containing 3 technical replicates, ± S.D, * p<0.05, t-test. (4B) Viable CFUs (percentage of control PIC-treated cultures) remaining after growing E. coli MG1655 expressing full-length intimin from E. coli 0157:H7 for 6hrs with the indicated PICs in liquid medium amended with 5% PEG8000 (Data represent 2 biological replicates, each containing 3 technical replicates ± S.D.; * p<0.05, t-test). Nb-Int targets the carboxyl- terminal 280 amino acids of intimin, which are not found in the surface display construct. (4C) E. coli MG1655 expressing full-length intimin recovered (% of control E. coli in parallel mixes) after incubating for 1 hour with PIC ^{xb l} (50-fold E. coli by ODeoonm) together with a complex bacterial community (200-fold E. coli by ODeoonm) isolated directly from fresh mouse fecal samples. (4D) Stacked bar plot showing the relative abundance of mouse fecal community members before and after incubation with PIC ^{xb l} . as determined by sequencing the V3-V4 region of the 16S rRNA gene. OTUs were clustered at 97% identity, and the 30 most abundant OTUs (by sequence count, E. cloacae excluded) are shown. The E. coli strains added to the communities indicated at top. Normalized OTU counts are presented in TABLE 2. (4E) Competitiveness of PIC ^{xb BamA} toward E. coli DH5a relative to the competitiveness of control PICs after the indicated number of rounds of incubating E. coli in the presence of PIC ^{xb BamA} for 6 hr. Each round of selection was initiated with a fresh population of the parent PIC. n= 3 technical replicates ± SD. See also FIGURES 8A and 8B.

FIGURES 5A-5E: T6SS activity is responsible for PIC-mediated inhibition of susceptible E. cloacae. (5A) Growth of the indicated E. cloacae donor strains grown coculture with E. cloacae ArhsA rhsErksB rhsle tae4 tai4 (A ei x3) in solid or liquid media. (5B) Growth of the indicated PICs grown in liquid coculture with the antigen-expressing E. cloacae A ei x3 strains shown (n=2 biological replicates with 3 technical replicates each, ± S.D.) (5C) Viable CFUs (percentage of control PIC-treated cultures) of E. cloacae A ei x3 expressing Ag-X or Ag-Y remaining after incubation with the indicated PICs for 6 hr. (n=3 technical replicates ± S.D.) (5D) Anti-myc or anti-RpoB (loading control) immunoblot indicating levels of the myc-tagged intimin-nanobody fusion proteins produced by the indicated strains under inducing or non-inducing conditions. (5E) Flow cytometry analysis of the indicated strains treated with anti-myc antibodies followed by Alexa fluor 488-conjugated secondary antibody. Fluorescence intensity at 525/50 nm indicates the degree of surface presentation of myc-tagged intimin-nanobody fusion proteins.

FIGURES 6A-6E: Antigen-nanobody mediated targeting by PICs does not impact PIC cell growth during killing and PEG8000 mediates bacterial aggregation in the absence of antigen-nanobody mediated interactions. (6A), Competitiveness of the indicated PICs grown on a solid surface with E. coli expressing intimin-fused Ag-Y, or truncated intimin alone (Ctrl) (n=3 technical replicates ± S.D.) (6B), Growth of the indicated PICs during liquid coculture with E. coli expressing cognate or orthogonal antigens (n=2 biological replicates with 3 technical replicates each, ± S.D.) (6C) Viable CFUs (percentage of control PIC-treated cultures) of E. coli displaying the indicated antigens after incubation with the PICs noted at bottom (n=3 technical replicates ± S.D.) (6D) Quantification of the indicated PIC populations during incubation with stationary phase E. coli displaying Ag-Y (n=2 biological replicates with 3 technical replicates each, ± S.D.) (6E) Phase contrast micrographs of mixed E. cloacae and E. coli cultures grown for 6 hrs in the indicated concentrations of PEG 8000. Scale bar, 20 pm.

FIGURES 7A-7C: PICs selectively inhibit target cells in a synthetic community. (7 A) Fluorescence micrographs indicating relative abundance of E. coli control (red) and Ag-Y expressing (green) cells after 6 hrs growth with parental or T6S-inactive strain (A icrnF) PICs expressing Nb-Y. The initial ratio of E. coli control to Ag-Y was 1:1. Scale bar, 10 pm. (7B) Competitiveness of T6SS ⁺ or inactive (A icmF) E. cloacae toward the indicated species after growth on solid medium ( S . prot, Serratia proteamaculans ; F. novi, Francisella novicida; S. paki, Sphingobacterium pakistanense). Competitive indices for each species were normalized to those obtained with parental E. cloacae (n=3 technical replicates ± S.D.) (7C) Comparison of the change in relative abundance of community members after growth with or without PIC addition. The E. coli strain included is indicated at top. Initial abundance (by OD6oo nm): PICs, 6.7%; target E. coli, 2%. Initial and final read counts for each species presented in TABLE 1.

FIGURES 8A and 8B: Selective inhibition of intimin-expressing E. coli by PIC ¹ * ^Int in a complex community derived from mouse fecal samples. (8A) Viable CFUs (percentage of control E. coli) of intimin-displaying or control E. coli after 1 h incubation with pic ¹ * ¹ " ¹ in the presence of a complex mouse fecal community. The initial abundance of E. coli relative to community members is indicated at bottom. n=4, ± S.D. (8B) Stacked bar plot showing the relative abundance of mouse fecal community members before and after incubation with PIC ^{xb l} . as determined by sequencing the V3-V4 region of the 16S rRNA gene. OTUs were clustered at 97% identity, and the top 30 most abundant OTUs (by sequence count) are shown. The E. coli strains added to the communities indicated at top. The mouse fecal community was obtained from a different mouse colony than that shown in FIGURE 4D. Normalized OTU counts are presented in TABLE 2.

DETAILED DESCRIPTION

There are a variety of situations in which the selective removal of individual species or strains of bacteria from complex communities is advantageous over traditional, broadly acting antibacterial approaches. However, generalizable strategies that accomplish this with high specificity have been slow to emerge. This disclosure is based on the development of programmed inhibitor cells (PICs) that direct the potent antibacterial activity of the type VI secretion system (T6SS) against specified target cells. As described in more detail below, the PICs express surface-displayed nanobodies that mediate addressable cell-cell adhesion, overcoming the barrier to T6SS activity in fluid conditions. Notably, the PICs were surprisingly demonstrated to efficiently deplete low abundance target cells without significant collateral damage from complex, undefined microbial communities. The data establish that the only requirements for PIC targeting using this platform are a Gram-negative cell envelope and a unique cell surface antigen. Therefore, this approach is generalizable to a wide array of bacteria and will find application in medical (e.g., clinical), research, and environmental settings.

Transgenic Gram-Negative Bacteria

In accordance with the foregoing, in one aspect this disclosure provides a transgenic Gram-negative bacterium, comprising a functional type VI secretion system and expressing at least one heterologous affinity reagent that specifically binds at least one surface antigen on a target Gram-negative bacterium.

In this aspect, the genetically modified Gram-negative bacterium serves as the programmed inhibitor cell (PIC), as they are often referred to herein, that can specifically bind and adhere to unique surface markers on other target Gram-negative bacterium, which provides contact for an appropriate duration for the genetically modified Gram-negative bacterium (i.e., the PIC) to kill the target Gram-negative bacterium via its type VI secretion system.

Gram-negative bacteria have been widely characterized for the disability to retain the crystal violet stain used in Gram-staining. Structurally, the Gram-negative bacteria can be generally characterized by their cell envelopes, which have a thin peptidoglycan cell wall disposed between two cytoplasmic membranes. Unlike Gram-positive bacteria, Gram-negative bacteria lack a thick peptidoglycan wall outside the plasma membrane. In this aspect, the genetically modified Gram-negative bacterium can be any Gram-negative bacterium amenable to genetic modification (i.e., to express the at least one heterologous affinity reagent). The selection of the specific species or strain of Gram-negative bacterium can be appropriately made based on the intended use or application of the Gram-negative bacterium. For example, as described in more detail below, the disclosed transgenic Gram negative bacterium can be used in therapeutic applications and, thus, can be selected for general compatibility and/or appropriateness with the extant flora or microbiota in a subject's gastro-intestinal (GI) tract. Thus, in such embodiments the transgenic Gram negative bacterium is selected for suitability for administration as a pro-biotic to a subject. In exemplary embodiments, the transgenic Gram-negative bacterium is a species of Enterobacter, Bacteroides, Escherichia, Pseudomonas, Burkholderia, Lactobacillus, Vibrio, Delftia, and Serratia. Illustrative, nonlimiting examples include Enterobacter cloacae, Bacteriodes fragilis, Bacteriodes uniformis, Bacteroides dorei, Bacteroides xylanisolvens, Bacteroides ovatus, Pseudomonas putida, Escherichia coli, and the like. Other potential gut-compatible probiotic strains are known and are encompassed by this disclosure. In other exemplary applications, the disclosed transgenic Gram-negative bacterium can be used in targeted applications, such as in industrial surface and food preparation, and can similarly be selected for desirable qualities, such as robustness and durability in the particular environmental conditions (e.g., industrial surface) to which it will be applied and lack of negative effects on the substrate.

The Gram-negative bacteria selected to express at least one heterologous affinity reagent can be distinguished from the target Gram-negative bacterium, which expresses a surface antigen. Typically, the transgenic Gram-negative bacterium is a different species or strain from the target Gram-negative bacterium. In some embodiments, surface expression of the surface antigen is substantially unique to the target Gram-negative bacterium, which can be determined with reference to the expression (or lack thereoi) in other species or strains within a defined environment (e.g., within a heterogenous population). The term, "substantially unique" as used herein, indicates that a substantial majority, for example, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 98%, and substantially all, of the expression of the surface antigen is restricted to the target Gram negative bacterium as compared to other bacteria in the same environment. Thus, the surface antigen can be present on other bacteria strains, but to a lesser extent than as in the target Gram-negative bacterium, either at lower average concentrations on each cell surface or at lower rates on individuals within each subpopulation of the heterogeneous population in the environment.

As indicated above, the disclosed transgenic Gram-negative bacterium has a functional Type VI secretion system (T6SS). The T6SS is a molecular system present in a wide range of Gram-negative bacteria that can transport proteins from the cytosol across the cellular envelope to the outside environment, such as into neighboring cells. While the T6SS was originally discovered in the context of pathogenesis of the bacteria towards higher organisms, it is now understood to play a large role in interbacterial competition and antagonism. The T6SS machinery includes several proteins that assemble into various inter-related structures, including a phage tail-like tube, a phage base plate-like structure, and a cell-envelope spanning membrane complex. The phage tail-like tube structure contains a sheath arranged around a tube and a trimer of a spike-like protein that caps the tube. Upon contraction of the sheath, the tube and spike-like proteins are propelled out of the cell and can potentially penetrate into a neighboring cell. The base plate-like structure and the membrane complex participate within the bacterial cytoplasm to recruit and stabilize the T6SS machinery for deployment. The membrane complex serves as a closable channel through the membrane through which the phage tail like tubule can move. Various substrates can be delivered through the T6SS machinery that are toxic to neighboring bacterial cells. Species deploying such toxins via the T6SS prevent self-intoxication through the production of immunity proteins that specifically inactivate particular toxins.

In some embodiments, the transgenic Gram-negative bacterium comprises a functional T6SS that can deliver one, two, three, four, or more antibacterial toxins to the exterior of the bacterial cell. Bacterial toxin molecules that are delivered by the T6SS are known. In some embodiments, one or more, including all, of the T6SS components, such as those are described above, are constitutively expressed. For example, the transgenic Gram-negative bacterium can further comprise additional genetic alterations, whether spontaneous mutations or engineered alterations, to implement in constitutive expression of T6SS components. Alternatively, the one or more components of the T6SS can be inducible, such as under desired conditions. Such embodiments can include inducible promoter sequence(s) operatively linked to one or more genes encoding components of the T6SS (including genes encoding the bacterial toxins).

While the T6SS in its natural setting is generally indiscriminate toward any Gram negative bacterial cell in its delivery of antibacterial toxins, selectivity of the antibacterial effect is imposed within the disclosed Gram-negative bacterium by inclusion of an expressed heterologous affinity reagent that specifically binds to a surface antigen on a target Gram-negative bacterium. Through this specific binding, delivery of the powerful antibacterial activity is restricted to the prolonged cell-to-cell interactions conferred by the affinity reagent-surface antigen binding. For example, in liquid environments, the typical cell-to-cell interactions are insufficient to initiate effective T6SS-mediated killing. However, the presence of the affinity reagent with specific binding capacity for a surface feature of the target cell provides the necessary stability to the cell-to-cell interaction (e.g., in maintaining duration and proximity of contact) to allow the T6SS to function to selectively kill the target cell without detectable detriment to other "off-target" bacteria in the environment.

As used herein, an "affinity reagent" refers to any moiety, construct, or molecule (typically proteinaceous) that can specifically bind to a target antigen. As used herein, the term "specifically binds" refers to an association or union of a binding domain, or a molecule containing the binding domain, (i.e., the affinity reagent) to a target molecule (i.e., the target antigen) without significantly binding or associating with distinct antigens. In some embodiments, specific binding is manifested in a binding affinity or K _a (i.e., an equilibrium association constant of a particular binding interaction with units of 1/M) equal to or greater than 10 ⁵ M ^{- 1} for the antigen, while not significantly associating with any other distinct antigen. Affinity reagents can be classified as "high affinity" or "low affinity". "High affinity" refers to affinity reagents with a K _a of at least 10 ⁷ M ¹ , at least 10 ⁸ M ¹ , at least 10 ⁹ M ^-1 , at least 10 ¹⁰ M ^-1 , at least 10 ¹¹ M ^-1 , at least 10 ¹² M ^-1 , or at least 10 ¹³ M ^-1 . "Low affinity" refers to those antibodies or antibody derivatives with a K _a of up to 10 ⁷ M

^! , up to 10 ⁶ M ¹ , up to 10 ⁵ M ¹ . Alternatively, affinity can be defined as an equilibrium dissociation constant (K^) of a particular binding interaction with units of M. For example, the affinity reagent can have a binding affinity within a range characterized by a K _d from about 50nM (lower binding affinity) to about 0.001 nM (higher binding affinity). For example, the binding domain has a binding affinity for the GLUT protein characterized by C¾) of about 50nM, 40nM, 30nM, 20nM, lOnM, 5nM, InM, 0.75nM, 0.5nM, O.lnM, 0.05nM, O.OlnM, 0.005nM, and O.OOlnM, or even smaller. Typical ( ¾ ranges characterizing the binding affinity of the cell -targeting domain for the antigen characteristic of the cell -type of interest include from about 30nM to about lOnM, from about 20nM to about InM, from about lOnM to about O.lnM, from about 0.5nM to about 0.05nM, and from about 0. InM to about .OOlnM, or even lower, or any subrange therein.

Exemplary affinity reagents encompassed by the disclosure include aptamers and antibodies, and antigen-binding fragments or derivatives thereof. As used herein, the term "antibody" encompasses immunoglobulin molecules and antigen binding antibody derivatives and fragments thereof, derived from any antibody-producing animals (e.g., cartilaginous fish, mouse, rat, rabbit, camelids, and primate including human), that specifically bind to an antigen of interest. Exemplary antibodies include monoclonal antibodies, chimeric antibodies (e.g., mouse-rabbit, mouse-human, mouse-primate, primate-human monoclonal antibodies), and humanized antibodies. An antibody "derivative" encompasses fragments, modifications, fusions, or other antibody-related constructs that incorporate structure of at least part of an antibody molecule, typically at least the antigen-binding domain of the antibody molecule. An antigen-binding antibody derivative or fragment will typically contain at least a portion of the complementarity determining regions (CDRs) of the original antibody sufficient to bind to the antigen of interest. Illustrative examples of antibody fragments and derivatives encompassed by the present disclosure include Fab, Fab', F(ab)2, F(ab')2 and Fv fragments, linear antibodies, single-chain antibody molecules, multi-specific antibodies formed from antibody fragments, and the like. Single-chain antibodies include single-chain variable fragments (scFv) and single-chain Fab fragments (scFab). A "single-chain Fv" or "scFv" antibody fragment, for example, comprises the VJJ and Vp domains of an antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide can further comprise a polypeptide linker between the VJJ and Vp domains, which enables the scFv to form the desired structure for antigen. Single-chain antibodies can also include diabodies, triabodies, and the like. In certain embodiments, heterologous affinity reagent is a "single-domain" antibody (sdAb), also referred to herein as a "nanobody", or an antigen-binding fragment or derivative thereof. Single-domain antibodies are antibody fragments that contain a single monomeric variable domain that can bind to target antigens with high specificity. Single-domain antibodies are characteristic of camelids and cartilaginous fishes. For example, camelid sdAbs are called V _h H fragments and have a single variable domain (V _H ) of a heavy chain antibody molecule. Exemplary design and production of affinity reagents based on camelid sdAbs are described in Pain, C., et al., "Camelid single-domain antibody fragments: Uses and prospects to investigate protein misfolding and aggregation, and to treat diseases associated with these phenomena," Biochiinie 1 1 (2015 ): 82- 1 16. incorporated herein by reference in its entirety. Fish sdAbs are called V _AAR ("new antigen receptor" variable) fragments, that similarly have a single variable domain joined with a number of constant domains. The sdAbs of the present disclosure can be VHH or V _AAR fragments or be derived therefrom. These sdAbs are able to bind to antigens without associated light chain variable domains. Alternatively, sdAbs can be engineered from more conventional or canonical antibody structures, such as by monomerizing the traditional antibody domains by removing or replacing lipophilic residues or by introducing premature stop codons, and screening for binding affinity of the single domain chains.

In some embodiments the affinity reagent comprises at least the antigen binding domain of the antibody constructs described above, such as at least the variable domain comprising the minimal CDRs to permit specific antigen binding. Furthermore, the affinity reagents of the present disclosure will also typically have a domain to anchor the affinity reagent in the outer membrane of the Gram-negative bacteria that expresses it. In some embodiments, the affinity reagent (e.g., sdAbs and other antibody fragments and derivatives) comprise the variable domain or domains) and do not include constant domains, whether in the exterior or interior of the cell. In some embodiment, the affinity reagent (e.g., sdAbs and other antibody fragments and derivatives) comprise the variable domain or domains) comprises a single VHH an no constant domain. In some embodiment, the affinity reagent (e.g., sdAbs and other antibody fragments and derivatives) comprise the variable domain or domains) comprises a single V _h H and no constant domain.

Due to the smaller size of sdAbs and other antibody fragments and derivatives (e.g., Fab, Fab', F(ab)2, F(ab')2 and Fv fragments, linear antibodies, single-chain antibody molecules (e.g., scFv)), they are more amenable to heterologous expression in bacterial cells using coding sequence operably linked to an appropriate promoter. In some embodiments, the affinity reagent (e.g., sdAbs and other antibody fragments and derivatives) comprise the variable domain or domains) are displayed in monomeric form on the surface of the bacterium.

It will be apparent to the skilled practitioner that the affinity reagent can comprise antigen binding molecules other than antibody-based affinity reagents, such as peptidobodies, antigen-binding scaffolds (e.g., DARPins, HEAT repeat proteins, ARM repeat proteins, tetratricopeptide repeat proteins, and other scaffolds based on naturally occurring repeat proteins, etc. [see, e.g., Boersma and Pluckthun, Curr. Opin. Biotechnol. 22:849-857, 2011, and references cited therein, incorporated herein by reference]), which include a functional binding domain or antigen-binding fragment thereof.

In some embodiments, the affinity reagent is an aptamer. As used herein, the term "aptamer" refers to peptide molecules that can bind to specific target molecules, such as a bacterial surface antigen protein. Peptide aptamers are protein molecules that often contain a peptide loop attached at both ends to a protamersein scaffold. The loop typically has between 10 and 20 amino acids long, and the scaffold is typically any protein that is soluble and compact. One example of the protein scaffold is Thioredoxin-A, wherein the loop structure can be inserted within the reducing active site. Peptide aptamers can be generated/selected from various types of libraries, such as phage display, mRNA display, ribosome display, bacterial display, and yeast display libraries.

In addition to the above affinity reagents, the disclosure also encompasses other peptide or protein-based binding reagents that can be engineered to selectively bind target antigens and are amenable for expression in Gram-negative bacteria. See, e.g., Chevalier, A., et ak, "Massively parallel de novo protein design for targeted therapeutics," Nature _, 550 (2017):74-79, incorporated herein by reference in its entirety.

The disclosure encompasses heterologous affinity reagent that can specifically bind to any surface antigen of interest. As indicated above, the selection of surface antigen is without limitation except that the surface antigen is ideally unique or substantially unique to the target Gram-negative bacterium, for example as defined relative to a heterogeneous population in an environment. In some embodiments, the surface antigen is not expressed by the transgenic Gram-negative bacterium.

Simply for purposes of illustration, in one embodiment the surface antigen to which the heterologous affinity agent specifically binds can be BamA, which can be expressed by E. coli. As described in more detail below, BamA was used as a target surface antigen to show proof of concept for the presently disclosed PIC platform technology. BamA is a widely conserved, essential and cell surface-accessible outer-membrane protein that is required for the biogenesis of transmembrane b-barrel proteins. Accordingly, in some embodiments, the heterologous affinity reagent specifically binds an extracellular domain of BamA. An exemplary full-length sequence for BamA is set forth in SEQ ID NO:34. In further embodiments, the heterologous affinity reagent contains the CDRs included in the sequence set forth as SEQ ID NO: 3, or an antigen-binding variant with at least 90% sequence identity thereof. In some embodiments, the heterologous affinity reagent comprises the variable domain included in the sequence set forth as SEQ ID NO:3, or an antigen-binding variant with at least 90% sequence identity thereof (e.g., with at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% thereof). In some embodiments, the heterologous affinity reagent comprises the sequence set forth as SEQ ID NO:3, or an antigen-binding variant with at least 90% sequence identity thereof (e.g., with at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% thereof).

Furthermore, as described in more detail below, it was demonstrated that the functionality of the PIC platform is not limited to transgenic Gram-negative bacteria expressing affinity reagents specific for BamA, but rather can be used as a platform to target Gram-negative bacteria that express any other surface antigen of interest. Specifically, it is demonstrated that sdAbs that specifically binding intimin, which is alternative surface protein expressed on E. coli cells. Intimin is a useful target surface antigen, considering the critical role the protein plays in adherence to epithelia by certain pathogenic strains of E. coli. Accordingly, in some embodiments, the heterologous affinity reagent specifically binds an extracellular domain of intimin. An exemplary sequence for the extracellular domain of intimin is set forth in SEQ ID NO:35. In some embodiments, the heterologous affinity reagent specifically binds the C-terminus of the protein, which localizes distal to the cell surface. In further embodiments, the heterologous affinity reagent contains the CDRs included in the sequence set forth as SEQ ID NO:4, or an antigen-binding variant with at least 90% sequence identity thereof (e.g., with at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% thereol). In some embodiments, the heterologous affinity reagent comprises the variable domain included in the sequence set forth as SEQ ID NO:4, or an antigen-binding variant with at least 90% sequence identity thereof. In some embodiments, the heterologous affinity reagent comprises the sequence set forth as SEQ ID NO:4, or an antigen-binding variant with at least 90% sequence identity thereof (e.g., with at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% thereol).

Accordingly, it is demonstrated that appropriate affinity reagents can be generated or obtained to specifically bind to any surface antigen appearing on a target Gram-negative bacterium of interest. As indicated above, the surface antigen can be substantially unique to the target Gram-negative bacterium and thus serve as a functional marker to differentiate the target Gram-negative bacterium from other species in the heterogeneous population or environment for selective targeting and removal. In the clinical setting context, the designated surface marker can be substantially unique to a pathogenic species or strain of Gram-negative bacteria targeted for removal or prophylaxis. Given the vast sequence and characterization data for pathogens and other Gram-negative species and strains of interest, a person of ordinary skill in the art can reasonably designate and characterize a desired surface antigen for targeting with the disclosed transgenic Gram-negative bacteria PIC platform.

Furthermore, discovery and production of affinity reagents such as single-domain antibodies, other antibody-like molecules, and aptamers that specifically bind to antigens of interest can be accomplished using any technique commonly known in the art. See, e.g., in Harlow et ak, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); and Hammerling et ak, in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981), incorporated herein by reference in their entireties.

Genetic manipulation of a Gram-negative bacterium to achieve the disclosed transgenic a Gram-negative bacterium expressing the desired heterologous affinity reagent can be performed according to standard procedures in the art. For example, once the affinity reagent, whether antibody-based or otherwise, is identified, the nucleic acid sequence encoding the affinity reagent can be introduced to the desired Gram-negative bacteria (see above) through known transformational techniques. Typically, encoding nucleic acid is integrated into an expression cassette configured to promote expression of the affinity reagent in bacterial cell. The expression cassette typically comprises at least an appropriate promoter sequence operatively linked to the encoding sequence to facilitate transcription in the bacterial cell. The promoter can be inducible or constitutive. "Inducible promoter" refers to a promoter capable of facilitating transcription of a coding sequence upon certain environmental conditions or presence of specific inducing agents, and typically results in a transient expression pattern. Many inducible promoters are known. "Constitutive promoter" refers to a promoter that is capable of facilitating continuous transcription of a coding sequence or gene under its control and/or to which it is operably linked. Constitutive promoters and functional variants are well known in the art and include, but are not limited to, BBa_J23100, a constitutive E. coli os promoter (e.g., an osmY promoter (International Genetically Engineered Machine (iGEM) Registry of Standard Biological Parts Name BBa_J45992; BBa_J45993)), a constitutive E. coli s32 promoter (e.g., htpG heat shock promoter (BBa_J45504)), a constitutive E. coli s70 promoter (e.g., lacq promoter (BBa_J54200; BBa_J56015), E. coli CreABCD phosphate sensing operon promoter (BBa_J64951), GlnRS promoter (BBa_K088007), lacZ promoter (BBa Kl 19000; BBa Kl 19001); M13K07 gene I promoter (BBa_M13101); M13K07 gene II promoter (BBa_M13102), M13K07 gene I II promoter (BBa_M13103), M13K07 gene IV promoter (BBa_M13104), M13K07 gene V promoter (BBa_M13105), M13K07 gene VI promoter (BBa_M13106), M13K07 gene VII I promoter (BBa_M13108), Ml 3110 (BBa_M13110)), a constitutive Bacillus subtilis s ^A promoter (e.g., promoter veg (BBa_K143013), promoter 43 (BBa_K143013), Pl _iaG (BBa_K823000), Pl _epA

(BBa_K823002), P _veg (BBa_K823003)), a constitutive Bacillus subtilis s ^B promoter (e.g., promoter etc (BBa_K143010), promoter gsiB (BBa_K143011)), a Salmonella promoter (e.g., Pspv2 from Salmonella (BBa_Kl 12706), Pspv from Salmonella (BBa_Kl 12707)), a bacteriophage T7 promoter (e.g., T7 promoter (BBa_1712074; BBa_1719005; BBa_J34814; BBa_J64997; BBa Kl 13010; BBa Kl 13011; BBa Kl 13012; BBa_R0085; BBa_R0180; BBa_R0181; BBa_R0182; BBa_R0183; BBa_Z0251; BBa_Z0252; BBa_Z0253)), and a bacteriophage SP6 promoter (e.g., SP6 promoter (BBa_J64998))

As described above, in some embodiments, the affinity reagent comprises minimal components that result in an expressed monomeric protein domain, e.g., a single V _H domain, that can bind to the antigen and no constant domains from typical antibody constructs are included. However, the expressed affinity reagent requires a transmembrane domain that will anchor the antigen binding domain on the cell surface. Thus, the affinity reagent can be a fusion of an extracellular antigen binding domain and a transmembrane domain. The expression cassette comprises a fusion nucleic acid that encodes both the transmembrane domain and the extracellular antigen binding domain in frame and operatively linked to the same promoter sequence. For example, as described in more detail below, an affinity reagent expressed in Gram-negative bacteria contained a single variable domain fused to the transmembrane domain of intimin protein (and excluding any extracellular domain of intimin). Any appropriate transmembrane domain and encoding sequence appropriate for expression in a Gram-negative bacteria is encompassed by the disclosure and can be implemented by a person of ordinary skill in the art.

The expression cassette can be further incorporated into vector configured to promote gene expression in bacterial host cells. The vector can be any construct that facilitates the delivery of the nucleic acid to the bacterial cell and/or expression of the nucleic acid within the bacterial cell. The vectors can be viral vectors, circular nucleic acid constructs (e.g., plasmids), or nanoparticles. In some embodiments, the vector comprises sequences to promote integration of the encoding nucleic acid into the bacterial genome to promote more permanent expression of the encoded affinity reagent. The vectors can contain other components, such as origin of replications sequences, selection marker (e.g., antibiotic resistance genes), endonuclease restriction sites, and the like.

In some embodiments, the disclosed transgenic Gram-negative bacterium expresses a plurality of different heterologous affinity reagents. In these embodiments, each heterologous affinity reagent specifically binds a different surface antigen on a target Gram-negative bacterium. The different surface antigens can be present on the same target Gram-negative bacterium or on different Gram-negative bacteria. For example, the transgenic Gram-negative bacterium can comprise a first heterologous affinity reagent that specifically binds a first surface antigen present on a first target Gram-negative bacterium and a second heterologous affinity reagent that specifically binds a second surface antigen present on a second target Gram-negative bacterium, wherein the first target Gram-negative bacterium and second target Gram-negative bacterium are different species or strains of target Gram-negative bacteria.

Probiotic compositions and related formulations

In another aspect, the disclosure provides a probiotic composition and related formulations comprising the transgenic the Gram-negative bacterium, as described above. Probiotics are generally considered live non-toxic microbial supplements (e.g., appropriate for ingestion or application) that can beneficially affect a host by improving the host's microbial balance (e.g., intestinal microbial balance) without causing disease. In the present case, the improvement to the host's microbial balance results from the specific targeting of a Gram-negative bacterium (e.g., a pathogenic strain) for depletion or for the prevention of its colonization. Thus, this aspect encompasses formulations of the probiotic appropriate for methods of administration for application to in vivo therapeutic settings in subj ects (e. g. , subj ects with dysbiosis in the gut, or other infection with an undesired Gram negative bacteria as described above).

The probiotic and related pharmaceutical compositions described herein can be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the Transgenic Gram negative bacteria into compositions for therapeutic or prophylactic use. Methods of formulating pharmaceutical compositions are known in the art (see, e.g., A. Adejare (ed.), (2020) "Remington: The Science and Practice of Pharmacy" Academic Press; incorporated herein by reference in its entirety). In some embodiments, the probiotic and related pharmaceutical compositions are subjected to tabletting, lyophilizing, direct compression, conventional mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping, or spray drying to form tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enterically coated or uncoated according to skill and knowledge common in the art. Thus, the transgenic Gram negative bacteria described herein can be formulated into the probiotic or related pharmaceutical compositions in any suitable dosage form (e.g., liquids, capsules, sachet, hard capsules, soft capsules, tablets, enteric coated tablets, suspension powders, granules, or matrix sustained release formations for oral administration) and for any suitable type of administration (e.g., oral, topical, injectable, immediate-release, pulsatile-release, delay ed- release, or sustained release). The dosage form can be configured for any suitable route of administration, including oral, nasal, intra-ocular, intra-otic, anal, vaginal, and the like.

In some embodiments, the transgenic Gram-negative bacteria in the probiotic composition are present in a dried form that preserves viability and administration. Drying of micro-organisms after production by fermentation is known to the skilled person. For example, EP0818529 describes a representative drying process of pulverization. See also, WO 0144440, each of which is incorporated herein by reference in its entirety. Bacteria can be concentrated from a medium and dried by spray drying, fluidized bed drying, lyophilization (freeze drying) or other known drying processes. For example, micro organisms can be mixed with a carrier material such as a carbohydrate, e.g., sucrose, lactose or maltodextrin, a lipid, or a protein, during or before the drying process.

In some embodiments, the dried probiotic composition is further formulated for oral administration and can be in the form of tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. Pharmacological compositions for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients include, but are not limited to, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose compositions such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP) or polyethylene glycol (PEG). Disintegrating agents can also be added, such as cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate.

In some embodiments, formulated forms including tablets, capsules, pills, etc. comprise one or more layers of coating (e.g., enteric coating) that dissolves upon entering the desired environment (e.g., a specific region of the GI tract) but otherwise protects the transgenic Gram-negative bacteria from degradation due to moisture, heat, pH, etc., when being stored or before arrival at the desired site. Many such coating materials are known and are encompassed by the present disclosure. For examples of enteric coatings, see Ghube K., et al. "A Review on Enteric Coating." Curr. Pharm. Res. (2020) 10(4):3848- 3862; and Hussan, S. D., et al., "A Review on Recent Advances of Enteric Coating." IOSR J. of Pharmacy (2012) 2(6) 5-11; WO2011108826 A2 each of which is incorporated herein by reference in its entirety. Exemplary coatings for purposes of microencapsulation of the live transgenic bacteria include biodegradable synthetic "polymers" such as polylactide, polyglycolic acid, and polyanhydride. Other known "polymers" for live encapsulation include alginate-polylysine-alginate (APA), alginate-polymethylene-co-guanidine- alginate (A-PMCG-A), hydroymethylacrylate-methyl methacrylate (HEMA-MMA), Multilayered HEMA-MMA-MAA, polyacrylonitrilevinylchloride (PAN-PVC), acrylonitrile/sodium methallylsulfonate (AN-69), polyethylene glycol/poly pentamethylcyclopentasiloxane/polydimethylsiloxane (PEG/PD/PDMS), poly N,N dimethyl acrylamide (PDMAAm), Siliceous encapsulates and cellulose sulphate/sodium alginate/polymethylene-co-guanidine (CS/A/PMCG). Other materials that are useful include, without limitation, cellulose acetate phthalate, calcium alginate and k- carrageenan-Locust bean gum gel beads, gellan-xanthan beads, poly(lactide-co- glycolides), carrageenan, starch poly-anhydrides, starch polymethacrylates, and polyamino acids. In some embodiments, the formulation is liquid preparation, e.g., for oral administration. Liquid preparations for oral administration can take the form of solutions, syrups, suspensions, or a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable agents such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate. Preparations for oral administration can be suitably formulated for slow release, controlled release, or sustained release of the transgenic Gram-negative bacteria described herein

In some embodiments, the Gram-negative bacteria are formulated as a comestible product, such as a food product, according to ordinary skill in the art.

The probiotic composition can further comprise additional optional ingredients, as appropriate for the desired mode of administration, including pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, thickeners, buffers, buffering agents, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or agents that are available in the art. Examples of such substances include normal saline solutions such as physiologically buffered saline solutions and water. Specific non-limiting examples of the carriers and/or diluents that are useful in the formulations of the disclosed probiotic composition include water and physiologically acceptable buffered saline solutions such as phosphate buffered saline solutions pH 7.0-8.0. Suitable carriers include, but are not limited to sterile water, salt solutions (such as Ringer's solution), alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, and the like. The pharmaceutical preparations can be mixed with auxiliary agents, e.g., lubricants, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like which do not deleteriously react with the active compounds. They can also be combined where desired with other active substances, such as antibiotics, drugs, foods, nutrients, vitamins, other beneficial substances, prebiotics, anti-inflammatory agents, anesthetics, and other therapeutic agents such as pH encapsulated glucose, lipids or proteins.

Formulations of the probiotic composition can also include topical formulations for local administration according to established practice. See, e.g., Malik, D. S., Expert Opin Ther Pat. (2016), 26(2):213-28, incorporated herein by reference in its entirety including references cited therein. Exemplary topical formulations include the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well known to one of skill in the art. For example, viscous to semisolid or solid formulations can comprise a carrier or one or more excipients compatible with topical application and having a dynamic viscosity greater than water. Suitable formulations can incorporate solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, etc., which may be sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, e.g., osmotic pressure.

Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as freon) or in a squeezable container. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms. Examples of such additional ingredients are well known in the art.

The transgenic Gram-negative bacteria described herein can be formulated into the probiotic or related pharmaceutical compositions in any suitable dosage amount. Suitable dosage amounts for the transgenic Gram-negative bacteria can range from about 10 ⁵ to 10 ¹² bacteria, e.g., approximately 10 ⁵ bacteria, approximately 10 ⁶ bacteria, approximately 10 ⁷ bacteria, approximately 10 ⁸ bacteria, approximately 10 ⁹ bacteria, approximately 10 ¹⁰ bacteria, approximately 10 ¹¹ bacteria, or approximately 10 ¹¹ bacteria. The composition may be administered once or more daily, weekly, or monthly. The composition can be administered before, during, or following a meal. In one embodiment, the pharmaceutical composition is administered before the subject eats a meal. In one embodiment, the pharmaceutical composition is administered currently with a meal. In one embodiment, the pharmaceutical composition is administered after the subject eats a meal.

In an alternative aspect, the transgenic Gram-negative bacteria are not used as a probiotic or therapeutic composition, but rather are applied in other environments, e.g., in industrial surfaces, and thus are not necessarily formulated for application to a subject. Instead the transgenic Gram-negative bacteria are formulated for ease of application to a surface, such as in a spray, aerosol, solution, emulsion, etc. However, in some embodiments the formulation is still configured for potential consumption, such as formulations added to food preparations or used in surfaces related to food preparation and processing. Formulation ingredients and parameters, such as those described above, are applicable in this aspect and are encompassed herein.

Methods

In another aspect, the disclosure provides a method of selectively depleting target Gram-negative bacteria from an environment. The method comprises contacting the target bacteria with a plurality of the transgenic Gram-negative bacteria as described above or the probiotic composition as described above.

The environment can be an aqueous environment. In some embodiments, the environment is an intestinal environment (i.e., in vivo in the GI tract) or in other internal environments of the body that may maintain a natural microbiota (e.g., nasal passages or intra-ear passages). Alternatively, the environment can be surfaces or solutions that comprise heterologous populations of bacteria where at least one potential member of the population is deleterious, and its removal or prevention of colonization is desired. Such instances can be in industrial or food preparation settings.

For example, in a related aspect, the disclosure provides a method of selectively depleting target Gram-negative bacteria and/or preventing colonization by the target Gram negative bacteria in a subject in need thereof. The method comprises administering to the subject an effective amount of the transgenic Gram-negative bacterium as described above or the probiotic composition as described above. For example, the method can be characterized as a method to treat a dysbiosis in the GI tract or elsewhere in the body, or a method to treat or prevent an infection by a particular Gram-negative species or strain that can lead to a dysbiosis. Accordingly, the transgenic Gram-negative bacteria being administered is typically a species/strain of bacteria that is compatible with the gut flora of the subject. Exemplary species and strains are described elsewhere above and below and are incorporated into this aspect of the disclosure.

Thus, in one embodiment the method comprises administering the effective amount of the transgenic Gram-negative bacterium as described herein or the composition as described herein to the subject to selectively deplete target Gram-negative bacteria and/or prevent colonization by the target Gram-negative bacteria from the GI tract of the subject. The transgenic Gram-negative bacterium as described herein or the composition as described herein can be formulated for oral or rectal administration, as described above. The effective amount of the composition and relevant dosing regimens to treat the subject (including prophylactic treatment) can be determined by a person of ordinary skill in the art.

As described above, the disclosed transgenic Gram-negative bacterium and probiotic formulations thereof can be used to target any Gram-negative bacterium for which a surface antigen is known. In some embodiments, the target Gram-negative bacteria is pathogenic. In some embodiments, the target Gram-negative bacteria is a pathogenic strain of E. coli. In further embodiments, the surface antigen expressed by the target Gram negative bacteria (and specifically bound by the expressed heterologous affinity reagent) is BamA, described above. Exemplary affinity reagents that specifically bind BamA are described elsewhere above and below. In another illustrative embodiment, the surface antigen expressed by the target Gram-negative bacteria (and specifically bound by the expressed heterologous affinity reagent) is intimin. Exemplary affinity reagents that specifically bind intimin are described elsewhere above and below.

In another related aspect, the disclosure provides a method of selectively depleting target Gram-negative bacteria and/or preventing colonization by the target Gram-negative bacteria in a defined environment. The environment can be a surface (e.g., industrial or clinical surface; a surface of a device or hardware), an agricultural or food preparation, or surface that will contact such a preparation, and the like. In this aspect the disclosed transgenic Gram-negative bacterium can be considered a disinfectant composition where the transgenic Gram-negative bacterium is an innocuous species or strain that expresses a heterologous affinity reagent that specifically binds a surface antigen of a target Gram negative bacterium, which is undesirable for that environment. In some embodiments, the target Gram-negative bacterium can be considered pathogenic. In other embodiments, the target Gram-negative bacterium can lead to spoiling of food ingredients or other preparations.

The method can comprises deploying a preparation of the disclosed transgenic Gram-negative bacteria and allowing the transgenic Gram-negative bacteria to contact the target Gram-negative bacteria present in the environment. In some embodiments, the disclosed transgenic Gram-negative bacteria is deployed in a liquid, e.g., aqueous, solution. The solution can be poured into or over, sprayed onto, or wiped over the environment or surface. Alternatively, the solution can be mixed into an existing liquid that defines the environment, such as a liquid beverage or food preparation, industrial production of microorganisms, and the like. Appropriate dosing to deliver an effective amount of the transgenic Gram-negative bacteria into a particular environment can be readily determined based on a variety of factors.

Additional Definitions

Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook J., et al. (eds.), Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Plainsview, New York (2001); Ausubel, F.M., et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, New York (2010); and Coligan, J.E., et al. (eds.), Current Protocols in Immunology, John Wiley & Sons, New York (2010) Mirzaei, H. and Carrasco, M. (eds.), Modem Proteomics - Sample Preparation, Analysis and Practical Applications in Advances in Experimental Medicine and Biology, Springer International Publishing, 2016, and Comai, L, et al., (eds.), Proteomic: Methods and Protocols in Methods in Molecular Biology, Springer International Publishing, 2017, for definitions and terms of art.

For convenience, certain terms employed herein, in the specification, examples and appended claims are provided here. The definitions are provided to aid in describing particular embodiments and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims.

As used herein, the term "nucleic acid" refers to any polymer molecule that comprises multiple nucleotide subunits (i.e., a polynucleotide). Nucleic acids encompassed by the present disclosure can include deoxyribonucleotide polymer (DNA), ribonucleotide polymer (RNA), cDNA or a synthetic nucleic acid known in the art.

As used herein, the term "polypeptide" or "protein" refers to a polymer in which the monomers are amino acid residues that are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used, the L-isomers being preferred. The term polypeptide or protein as used herein encompasses any amino acid sequence and includes modified sequences such as glycoproteins. The term polypeptide is specifically intended to cover naturally occurring proteins, as well as those that are recombinantly or synthetically produced. One of skill will recognize that individual substitutions, deletions or additions to a peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a percentage of amino acids in the sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:

(1) Alanine (A), Serine (S), Threonine (T),

(2) Aspartic acid (D), Glutamic acid (E),

(3) Asparagine (N), Glutamine (Q),

(4) Arginine (R), Lysine (K),

(5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V), and

(6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Reference to sequence identity addresses the degree of similarity of two polymeric sequences, such as protein or nucleic acid sequences. Determination of sequence identity can be readily accomplished by persons of ordinary skill in the art using accepted algorithms and/or techniques. Sequence identity is typically determined by comparing two optimally aligned sequences over a comparison window, where the portion of the peptide or polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical amino-acid residue or nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Various software driven algorithms are readily available, such as BLAST N or BLAST P to perform such comparisons.

The terms "subject," "individual," and "patient" are used interchangeably herein to refer to an individual being assessed for treatment and/or being treated. In certain embodiments, the subject is a human. While subjects may be human, the term also encompasses other mammals, particularly those mammals useful as laboratory models for human disease, e.g., mouse, rat, dog, non-human primate, and the like. The term "treating" and grammatical variants thereof may refer to any indicia of success in the treatment or amelioration or prevention of a disease or condition (e.g., infection with a bacterial strain, dysbiosis, etc.), including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms can be based on objective or subjective parameters, including quantification of infectious species (i.e., target Gram-negative bacteria) or the results of an examination by a physician to assess symptoms. Accordingly, the term "treating" includes the administration of the compounds or agents of the present disclosure to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with disease or condition (e.g., the presence of the infectious or disfavored target bacteria, dysbiosis, etc.). The term "therapeutic effect" refers to the reduction, elimination, or prevention of the disease or condition, symptoms of the disease or condition, side effects of the disease or condition in the subject, or amount of infectious agent (i.e., target bacteria) itself.

The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or."

Following long-standing patent law, the words "a" and "an," when used in conjunction with the word "comprising" in the claims or specification, denotes one or more, unless specifically noted.

Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise," "comprising," and the like, are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to indicate, in the sense of "including, but not limited to." Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words "herein," "above," and "below," and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of the application. The terms "about" or "approximately" indicate a number within range of minor variation above or below the stated reference number. For example, "about" can refer to a number within a range of 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% above or below the indicated reference number. Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. It is understood that, when combinations, subsets, interactions, groups, etc., of these materials are disclosed, each of various individual and collective combinations is specifically contemplated, even though specific reference to each and every single combination and permutation of these compounds may not be explicitly disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in the described methods. Thus, specific elements of any foregoing embodiments can be combined or substituted for elements in other embodiments. For example, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed. Additionally, it is understood that the embodiments described herein can be implemented using any suitable material such as those described elsewhere herein or as known in the art.

Publications cited herein and the subject matter for which they are cited are hereby specifically incorporated by reference in their entireties.

EXAMPLES

The following examples are provided for the purpose of illustrating, not limiting, embodiments of the disclosure.

Example 1

This example describes the design and implementation of an illustrative embodiment of a programmable cell-to-cell recognition system that can selectively deplete target bacteria from mixed populations. Results of this study were published in Ting, See- Yeun, et al. (2020) "Targeted depletion of bacteria from mixed populations by programmable adhesion with antagonistic competitor cells." Cell Host & Microbe 28.2: 313-321, which is incorporated herein by reference in its entirety.

Introduction

A generalizable platform was designed and implemented for selectively depleting target bacteria within mixed communities. Recent findings by the inventors and others have revealed potent mechanisms of contact-dependent antagonism between bacteria. Among these, the type VI secretion system (T6SS) is a widespread pathway that catalyzes the delivery of antibacterial toxins between neighboring Gram-negative cells. A hallmark of this system is its capacity to target cells indiscriminately, a behavior attributable to its promiscuous delivery mechanism and toxins that disrupt broadly conserved cellular processes. In this investigation, it was reasoned that if the antibacterial activity of the T6SS could be specifically directed toward target cell populations, these properties of the system could facilitate its development into a flexible, alternative antibacterial platform.

Beyond the requirement for a Gram-negative target cell, the only known barriers to T6S-based intoxication are close (< 200 nm) and long-term (> minute) cell-cell association. The stringency of these requirements is illustrated by the observation that cells susceptible to the T6SS of a strain under conditions of dense co-cultivation on solid media, where intimate cell-cell contacts are enforced, are fully protected from the system in liquid media, where cell-cell contacts are transient. Therefore, it was reasoned that selective T6S-based depletion of bacteria could be achieved by promoting the specific adhesion of a T6S ⁺ inhibitor cell to the targeted population. The antigens expressed on the surface of a bacterial cell are variable and can resolve the identity of these organisms at multiple taxonomic levels. In this study, it is demonstrated that this principle can be exploited to achieve selective and programmable T6S-based killing by generating strains expressing surface-displayed antibodies directed at unique cell surface epitopes of target cell populations.

Results

Nanobodv-antigen pairs enable programmable T6S-mediated killing in liquid medium

To test the hypothesis that specific adhesion of a T6S ⁺ cell to a defined target population can promote selective killing under fluid conditions, the ability of bacteria to present functional camelid-derived single-domain antibodies (nanobodies) on their cell surface was leveraged (FIGURE 1A). For proof-of-concept studies, characterized nanobody (Nb)-antigen (Ag) pairs were utilized in conjunction with a previously developed autotransporter display system (Pinero-Lambea, C., et al. (2015). Programming controlled adhesion of E. coli to target surfaces, cells, and tumors with synthetic adhesins. ACS Synth Biol 4, 463-473, incorporated herein by reference in its entirety). Elegant work by Glass et al. demonstrated that cognate Nb-Ag pairs displayed by this system facilitate specific cell-cell adhesion, allowing bacterial cell patterning (Glass, D.S., and Riedel- Kruse, I.H. (2018). A Synthetic Bacterial Cell-Cell Adhesion Toolbox for Programming Multicellular Morphologies and Patterns. Cell 174, 649-658 e616). As a first step, the feasibility of this approach to promote intraspecific killing by the g-proteobacterium Enterobacter cloacae was examined. This bacterium is genetically tractable, its T6SS is active under standard laboratory conditions, and three toxic substrates of its T6SS, RhsA, RhsB, and Tae4 have been described. Although it is a member of the normal human gut microbiota, E. cloacae is also considered an opportunistic pathogen and an emerging antibiotic resistant threat.

In wild-type bacteria, self-intoxication by the T6SS is prohibited by immunity proteins encoded by genes adjacent to those encoding the effectors they inactivate. Therefore, a susceptible target strain was generated for these studies by deleting rhsA, rhsB, tae4 and their adjacent cognate immunity genes, rhsL i, rhsle, and tai4 ( Aei x3). As expected, this strain was susceptible to T6S-based intoxication by the parent strain when cell-cell contacts are enforced by growth on solid media, but not in liquid media, where contacts are transient (FIGURES 1A and 5A).

Next, nanobody and antigen expression systems were introduced into the parent and T6S-susceptible strains, respectively. To facilitate mismatched control experiments, two characterized Nb-Ag pairs were utilized: Nb-X-Ag-X and Nb-Y-Ag-Y (Glass and Riedel - Kruse, 2018, supra). The amino acid sequence for the nanobody X ("Nb-X") is set forth in SEQ ID NO: 1 and the amino acid sequence for the nanobody Y ("Nb-Y") is set forth in SEQ ID NO:2. It was found that in liquid cultures, the growth of antigen-expressing T6S- susceptible strains was specifically inhibited by parental strains expressing cognate, but not non-cognate nanobodies (FIGURES 1C and 5B). This inhibition was not observed when the T6SS of the nanobody-expressing strain was inactivated (FIGURE 5C). Immunoblotting analysis and cell surface accessibility assays ruled out the possibility that T6S inactivation interfered with nanobody expression or localization (FIGURES 5D and 5E). Together, these data demonstrate that cell adhesion via Nb-Ag interactions can direct the antibacterial activity of the T6SS at target cells. The bacteria (e.g., E. cloacae strains) expressing nanobodies that promote adhesion to target cells are henceforth referred to as PICs (programmed inhibitor cells).

To determine whether this approach could be utilized more broadly, the capacity of PICs to deplete E. coli was tested. E. coli does not possess immunity factors against E. cloacae T6S effectors. Therefore, the strain is likely to be inherently susceptible to RhsA, RhsB, Tae4, as well as other, yet undescribed effectors that E. cloacae may deliver. Using co-culture on solid media, E. coli susceptibility to T6S-mediated intoxication by E. cloacae was confirmed (FIGURE 6A). Congruent with the intraspecies competition assays described above, it was found in this interspecies mixture that PICs inhibited target cell growth in liquid media only when they possessed both an active T6SS and expressed a matched nanobody (Figure 2A, and Figure S2A-C). The magnitude of the growth inhibitory effect of PICs targeting E. coli (PIC ^{xb x} . 570-fold; PIC ^{xb Y} . 220-fold) was significantly higher than that observed for a self-derived strain sensitized only to RhsA, RhsB, andTae4 (PIC ^{XI X} . 6.4-fold; PIC ^{xb Y} . 3.3-fold), suggesting A. cloacae indeed utilizes T6S effectors beyond those that identified here. PICs could be inhibiting the proliferation of E. coli by inducing bacteriostasis, killing, or by a combination of these mechanisms. It was found that within 30 minutes of addition, PICs reduced the colony forming units (c.f.u.) of stationary phase non-growing target cells by 16.1 -fold (FIGURES 2D and 6D). This experiment indicates PICs kill E. coli, though it does not rule-out a mixed mechanism that also involves bacteriostasis. These data demonstrate the capacity of programmable cell cell adhesion to promote efficient interspecies killing by the T6SS.

Polymer-mediated aggregation can enhance PIC activity

The specificity of PICs is dependent on fluidity; cell-cell contacts that occur independently of Nb-Ag interactions lead to indiscriminate killing (FIGURE 6A). However, in many natural environments, high molecular weight linear polymers are present that can affect the degree to which cells aggregate by both chemical and physical mechanisms. To quantitatively describe the sensitivity of the system to polymer-mediated aggregation, a high molecular weight polymer (PEG 8K) was used to systematically vary the degree of cell aggregation through a process termed depletion aggregation. With this method, a concentration of the polymer was identified that enhances the efficiency of PICs without observably impacting their specificity (5.0%) (FIGURE 2C). As expected, at high concentrations of the polymer (>10% w/v), cellular aggregation is enhanced independently of Nb-Ag interaction and under these conditions PIC targeting is indiscriminate (FIGURES 2C, 2D, and 6E). These findings suggest that PIC efficacy could benefit from natural polymer-mediated aggregation. However, a threshold effect exists, wherein conditions that strongly promote aggregation could generate off-target effects.

PICs function efficaciously in multi-strain and multi-species mixtures

A critical feature of the disclosed approach is its potential to distinguish target from non-target cells. As a first step toward addressing the specificity of the system, PICs were exposed to fluorescently-labeled E. coli cells expressing matched antigens diluted to varying degrees within differentially fluorescently-labeled cells expressing control antigens. In this scenario, no evidence of off-target activity was found, including when control cells outnumbered target cells by 1000-fold (FIGURES 3 A, 3B, and 7A). Moreover, the efficiency of PICs was unaffected by the degree of target cell dilution. The detection limit of the assay prohibited testing beyond this dilution of target cells; however, these data clearly demonstrate that PICs can exhibit high specificity for target cells found within mixed populations.

Next, the PIC system was challenged by substantially increasing the diversity of bacteria present with the target cell. Specifically, PICs and the target cells were introduced, initially at 20% and 6% abundance (by Oϋboo _hih ), respectively, to a synthetic community consisting of 12 species derived from four phyla, including both Gram-positive and Gram negative representatives. Quantification of community constituents by 16S rRNA gene sequencing before and after cultivation revealed no detectable PIC off-target activity, despite the susceptibility of several members to the E. cloacae T6SS (FIGURES 3C and 7B, and TABLE 1). In contrast, the level of target cells within the community was substantially reduced (96%) by PICs. Reduction of the initial abundance of target and PIC cells to 3% and 9%, respectively, did not decrease PIC efficacy (98%) (FIGURE S3C and TABLE 1). As a reference, the synthetic community was also exposed to ciprofloxacin, an antibiotic used to treat serious E. coli infections, among many other indications. In the experimental regime, ciprofloxacin displayed similar potency toward E. coli as PICs; however, the antibiotic caused substantial collateral effects within the community (FIGURE 3E). These findings demonstrate the feasibility of using PICs to deplete target cells from multispecies environments. Moreover, they highlight the improved specificity achievable by PICs in this context relative to conventional antibiotics.

TABLE 1. 16S V3-V4 amplicon sequencing results from examining PIC and antibiotic

PICs selectively target via natural surface antigens

The antigens employed to this point are not naturally found on the cell surface of bacteria. The utility of PICs hinges on their ability to kill target cells via the recognition of native antigens. BamA is a widely conserved, essential and cell surface-accessible outer- membrane protein that is required for the biogenesis of transmembrane b-barrel proteins (Konovalova, A., et al. (2017). Outer Membrane Biogenesis. Annu Rev Microbiol 71, 539- 556, incorporated herein by reference in its entirety). Naturally occurring molecules targeting BamA have validated the protein as an antibacterial target and, most pertinent to this study, nanobodies have been reported that recognize the E. coli protein in its membrane-integrated state. Thus, it was assessed whether display of these BamA-targeting nanobodies (Nb-BamA) could facilitate PIC-mediated killing of E. coli. An exemplary amino acid sequence for Nb-BamA is set forth in SEQ ID NO:3. Indeed, it was found that PIC ^Nb - ^BamA effectively suppressed the proliferation of the E. coli laboratory strains DH5a and DH10B in a Nb-BamA- and T6SS -dependent manner (FIGURE 4A). Interestingly, the parent of these strains, MG1655, which possesses an identical bamA sequence, was not inhibited by the PICs despite its susceptibility to the T6SS of E. cloacae (FIGURE 2A). BamA is an integral membrane protein lacking a significant ectodomain; therefore, it is hypothesized that structures protruding from the cell surface of MG1655 could reduce PIC efficacy by blocking access to their target antigen. This hypothesis was tested by evaluating PIC susceptibility of an MG1655 derivative bearing a truncated LPS core (ArfaD) (Coleman, W.G., Jr. (1983). The rfaD gene codes for ADP-L-glycero-D- mannoheptose-6-epimerase. An enzyme required for lipopolysaccharide core biosynthesis. J Biol Chem 258, 1985-1990). This mutation rendered MG1655 vulnerable to PIC ^{Nb BamA} - mediated inhibition, suggesting that the PIC platform will be most efficacious when targeting antigens that protrude from the cell surface.

The finding that bacterial surface structures can interfere with PIC targeting led to the examination of suitability of another native E. coli target protein, intimin. Owing to the critical role intimin plays in adherence to epithelia by certain pathogenic strains of E. coli, the protein has been the subject of considerable study (see, e.g., Celli, J., Deng, W., and Finlay, B.B. (2000). Enteropathogenic Escherichia coli (EPEC) attachment to epithelial cells: exploiting the host cell cytoskeleton from the outside. Cellular Microbiology 2, 1-9; McWilliams, B.D., and Torres, A.G. (2014). Enterohemorrhagic Escherichia coli Adhesins. Microbiol Spectr 2, each of which is incorporated herein by reference in its entirety). Unlike BamA, intimin adopts an extended structure and protrudes significantly (14 nm) from the cell surface. Indeed, the intimin-derived display system utilized in this study - despite containing a significantly truncated form of the protein - effectively presents synthetic antigens for PIC targeting (FIGURES 1A-1C). To target intimin, a nanobody that recognizes the C-terminus of the protein (Nb-Int) was used, which localizes distal to the cell surface and is not present in the disclosed display system (Ruano- Gallego, D., et al. (2019). Screening and purification of nanobodies from E. coli culture supernatants using the hemolysin secretion system. Microb Cell Fact 18, 47). An exemplary amino acid sequence for Nb-Int is set forth in SEQ ID NO:4. It was found that piC ^Nb - ^int _e fn _cien tiy and specifically depletes E. coli producing the cell surface adhesin (FIGURE 4B). It is worth noting that the degree of E. coli targeting via interaction with intimin exceeds that of Ag-X, Ag-Y or BamA. A multitude of factors could contribute to the magnitude of targeting achievable by different Nb-Ag interactions, but taken together with the variability in BamA-targeting between E. coli strains, these results are consistent with distance of target antigens from the cell surface, and thus their accessibility for nanobody binding, being an important variable influencing PIC activity.

PICs selectively deplete target cells from gut-derived communities

Intimin expression is a characteristic trait of enteropathogenic E. coli strains (Celli et al., 2000, supra,· McWilliams and Torres, 2014, supra). Thus, the next step was to determine whether robust and selective intimin-mediated PIC targeting could be achieved in the context of a complex and undefined community derived from the mammalian GI tract. Toward this end, intimin-producing E. coli was introduced into freshly isolated total fecal bacteria from conventionally reared mice, and the capacity of PIC ¹ * ¹ " ¹ to deplete these strains from the mixture was measured. To mimic conditions in vivo, these experiments were conducted under a regime that limits proliferation (e.g. high cell density and short timescale). One hour after the addition of PIC ^{xb lnl} . E. coli c.f.u. levels dropped by approximately 90%, whereas in separate experiments using E. coli expressing only the truncated version of intimin not recognized by Nb-Int, E. coli levels were unaltered (FIGURE 4C). This degree of targeting was maintained across a wide range of target cell abundance (1-0.05%) and in fecal microbiota deriving from two independently reared mouse colonies (FIGURE 8A). Additionally, the effects of PICs on the fecal community was examined using 16S rRNA gene sequencing. Among OTUs with >10 counts (the cutoff for analysis), those corresponding to E. coli underwent the greatest extent of PIC- mediated depletion in both fecal microbiome samples (FIGURES 4D and 8B, and TABLE 2). The magnitude of this depletion was less than that determined by c.f.u. enumeration, which is attributed to the persistence of DNA following cell lysis. The overall effect of PICs on the community composition was minor, and mostly limited to low abundance OTUs. The current data does not indicate whether the small changes that were observed derived from indirect consequences of E. coli depletion or bona fide off-target killing. The community profiling also revealed that the two murine microbiomes that were tested diverge greatly in the phylogenetic distribution of their constituents, suggesting that PIC- mediated targeting in this environment may be largely insensitive to the specific bacteria present.

TABLE 2. 16S V3-V4 amplicon sequencing analysis of the bacterial community of fresh mouse fecal samples amended with the indicated initial concentration of . coli, before and after 1 hr incubation with PIC ^{xb lm} . Fraction change represents the final divided by initial proportion of the community containing E. coli Ag-Int divided by that of the community containing control E. coli.

39 21 40 0.683482871

31 22 24 1.053984801

24 22 22 0.6162708333

18 26 23 0.9262331397

26 29 25 0.8163090145

21 31 19 1.225446443

36 31 29 1.204318056

38 33 26 1.207505664

51 35 34 0.891111957

60 36 47 0.7800615307

76 50 61 0.7896809287

49 51 41 1.130351771

80 53 50 0.9874665212

69 58 59 0.808668946

114 69 86 0.8560451922

90 83 93 0.9575982562

127 89 101 0.691802805

100 91 79 1.227188924

125 103 86 1.484404944

139 132 132 1.054546776

244 133 149 0.8724492418

174 144 151 0.8982310094

165 145 136 1.006699768

244 151 141 1.090338629

253 187 192 0.8509183326

290 199 216 0.7915996658

270 223 243 0.9293430845

262 244 237 0.7807134335

304 249 228 1.065634982

481 315 331 0.8832532779

322 321 295 1.025977322

530 329 303 1.067235735

436 349 305 1.346676081 Bacteroides sp. 16 15 10 17 0.5628006773 74 66 81 59 1.249618596

37 52 21 24 1.254994808

5250 4859 5373 5458 0.9298291588

59 32 58 14 2.293137946

36 48 34 40 1.156617925 90 108 88 79 1.364171849

31 22 15 17 0.6390510916

23 27 14 13 1.290187613

37 23 21 36 0.3700625714 26 26 20 21 0.9719478363 137 135 238 217 1.102967412 347 340 383 326 1.174797118 299 354 351 338 1.254742898 359 414 350 347 1.187071063 34 40 23 22 1.255216056 58 42 46 57 0.5963984818 11 28 23 32 1.867133883 30 24 23 15 1.251868813 10 11 10 18 0.6236665283 16 25 25 19 2.09816042 25 18 20 21 0.6998024421

24 20 34 31 0.9327563913 30 30 37 35 1.078862098 66 58 58 44 1.182201841 45 44 51 45 1.130915305 56 53 44 48 0.8853837321 50 38 46 53 0.6731747391 63 61 58 55 1.042045892 63 75 76 76 1.214934795 87 90 103 80 1.35926067 79 89 107 119 1.033789193 119 120 215 170 1.301535684

Resistance to PICs that target BamA is slow to emerge

A concern with any antimicrobial approach is the emergence of resistance. In the disclosed PIC approach, two potential broad routes by which target cells could acquire resistance were considered: target cell mutations diminishing susceptibility to PIC toxins or mutations that influence nanobody recognition of target cell surface antigens. In general, the T6SS delivers a payload of biochemically diverse toxins that act simultaneously on a range of essential cellular structures, and the PICs employed are not an exception. Based on this, it is hypothesized that target modification represents an unlikely physiological route to high level T6SS resistance. Rather, toxin neutralization via the horizontal acquisition of genes encoding toxin-specific resistance determinants appears to be widespread in microbial communities. Since this resistance mechanism cannot be accurately modeled in an in vitro setting, the resistance study was designed to capture mutations that could impact cell surface antigen recognition. Specifically, PICs that recognize BamA were employed, as this protein has been shown to accumulate antibiotic-resistance conferring substitutions within the b-barrel region of the protein that is recognized by Nb-BamA. To evaluate the potential for PIC resistance to emerge, E. coli was passaged serially in the presence of PIC ^Nb - ^BamA After each passage, a population derived from all surviving colonies was used as inoculum for the subsequent passage and the sensitivity of the population to PIC ^{xb BamA} - based targeting was quantitatively assessed. Remarkably, after 10 passages, no change in the sensitivity of E. coli to PIC ^{xl BamA} was found, suggesting that resistance to PICs - at least those targeting BamA - can be slow to emerge (FIGURE 4E). Based on the structure of the BamA-Nb-BamA complex, which shows that Nb-BamA makes extensive contacts with the inner surface of the b-barrel, it is believed that the substitutions necessary to break this interaction are incompatible with BamA function.

Discussion

These results represent a unique platform that harnesses the potent antibacterial activity of the T6SS to eradicate specific bacteria from polymicrobial assemblages. This system - when appropriately tailored for individual targets - is believed to be applicable in basic research, biotechnology, and the clinic for therapeutic purposes. Optimization or customizations might include i) tailoring the effector repertoires of PICs to achieve maximal killing efficiency and specificity toward the target, ii) defining the features of cell surface antigens that best promote PIC adherence, Hi) increasing the expression of the T6SS within PICs, iv) identifying or engineering alternative PIC strains (e.g. inherently avirulent or engineered attenuation, adapted to particular environments of interest!, and v) engineering PICs to express immunity determinants that provide protection from antibacterial toxins produced by target cells encoding their own T6SS or another antagonistic pathway.

No resistance of target cells sensitized to PIC-mediated growth suppression via nanobody-BamA interactions was detected. While this is likely to continue in natural (e.g., in vivo ) systems, it is difficult to know how well the present in vitro experiments will predict resistance in a natural system. Clearly, the function and essentiality of the antigen targeted, and the nature of the interaction of the antigen with the nanobody, will impact the rate by which resistance mutations arise. Proteins and other non-proteinaceous antigens present within or immediate to the bacterial outer membrane likely have a higher probability of being essential than those that extend from the surface, yet these data suggest that those protruding from the surface best support PIC targeting. An effective means of circumventing this tradeoff could be to develop PICs that recognize multiple antigens on target cells. In addition to stemming the emergence of resistance, the initial binding of a PIC cell to an antigen distal to the cell surface may facilitate interactions with more proximal antigens. The identity of the target cell will also exert significant influence on the rate of resistance to PICs. Recent work suggests that cell surface features such as exopolysaccharides can interfere with T6S-based intoxication. If target bacteria produce such structures constitutively or if readily acquired mutations can activate their expression, the efficacy of PICs could be impaired through both reduced efficacy of T6S-mediated targeting and occlusion of surface antigens.

It is likely that no single alternative antimicrobial technology will prove most useful in all, or even a majority of scenarios. Nevertheless, PICs have several potential advantages that are worth noting. First and foremost among the advantages is their generality. Owing to the promiscuity of the T6SS, the single specialized reagent required to target a new Gram-negative bacterium by the approach is a nanobody that recognizes a unique epitope on the cell surface of that bacterium. PICs targeting Gram-positive bacteria could also be developed. In this case, target cell killing could be achieved using the Esx pathway. Though divergent in sequence and mechanism from the T6SS, the Esx pathway - present widely in Gram-positive bacteria - can also catalyze indiscriminate cell contact-dependent toxin delivery into neighboring cells (see, e.g., Cao, Z., et al. (2016). The type VII secretion system of Staphylococcus aureus secretes a nuclease toxin that targets competitor bacteria. Nat Microbiol 2, 16183; Whitney, J.C., et al. (2017). A broadly distributed toxin family mediates contact-dependent antagonism between Gram-positive bacteria. Elife 6, e26938, each of which is incorporated herein by reference in its entirety.

Other notable advantages of PICs are the limited biological information needed for targeting and the short timescale theoretically required for PIC preparation and deployment. Surface-exposed candidate antigens on target bacteria can be defined with high confidence from most bacteria using commonplace bioinformatic tools that require only genomic (or metagenomic) sequence data. A set of candidates could then be applied to currently available pipelines for the in vitro selection of specific nanobodies from highly diverse libraries (~10 ¹³ ), allowing functional screening of these to begin within a period of three weeks. Altogether, these advantages bode well for the development of PICs into viable alternatives to traditional antimicrobials in myriad applications.

Experimental Model and Subject Details

Bacterial strains and culture conditions

The bacterial strains and plasmids used in this study are listed in the TABLE 3. PICs were derived from Enter obacter cloacae ATCC-13047. Escherichia coli strains used in this study included MG1655, DH5a, and DH10B for targeting by PICs, DH5a and EC 100 l pir for plasmid maintenance, and SI 7-1 l pir for conjugal transfer of plasmids into E. cloacae. Other bacterial strains used in the synthetic community competition include Serratia proteamaculans 568, Agrobacterium tumefaciens FACH, Paracoccus denitrificans ATCC-177441, Flavobacterium johnsoniae UW101, Sphingobacterium pakistanense, Chromobacterium violaceum, Mycobacterium smegmatis MC2 155, Enterococcus faecalis OG1RF, Listeria monocytogenes 10403S, Francisella novicida U112, Xanthomonas maltophilia ATCC-13637, and Aeromonas hydrophila An65A68. Bacteria were cultured routinely at 37°C in Luria Bertani (LB) medium supplemented with 0.5% (w/v) glucose, unless noted otherwise. Antibiotics and chemicals were used at the following concentrations: 50 pg ml ¹ streptomycin; 50 pg mT ¹ spectinomycin; 150 pg ml ¹ carbenicillin; 50 pg ml ¹ kanamycin; 25 pg ml ¹ chloramphenicol; 40 ng ml ¹ ciprofloxacin; 400 ng ml ¹ anhydrotetracycline.

Mouse studies

Murine fecal samples employed in this study were obtained from two separately reared colonies of C57BL/6J mice maintained in specific pathogen free (SPF) conditions. Mice used to establish the colonies were originally purchased from Jackson Laboratories. Daily care of the colonies was provided and SPF conditions were ensured through the rodent health monitoring program overseen by the Department of Comparative Medicine at the University of Washington.

TABLE 3 Key Resource Table.

Methods

Plasmid construction

All primers used in plasmid construction and generation of mutant strains are listed in TABLE 4. Tet expression plasmids used for nanobody and antigen presentation in E. cloacae and E. coli in this study were generated from pDSG323-derived expression plasmids previously described (Glass, D.S., and Riedel-Kruse, I.H. (2018). A Synthetic Bacterial Cell-Cell Adhesion Toolbox for Programming Multicellular Morphologies and Patterns. Cell 174, 649-658 e616). The expression plasmids for synthetic antigens and cognate nanobodies described by Glass et al. were modified to introduce a 3' myc tag to the expressed proteins by cloning of the amplified tag into the Ndel and Pstl restriction sites. Nbl/Agl and Nb3/Ag3 were renamed Nb-X/Ag-X and Nb-Y/Ag-Y, respectively for the purposes of our study. The Nb-Int and Nb-BamA expression plasmids display the previously characterized nanobodies IB10 and Nb_B12, respectively Ruano-Gallego, D., et al. (2019). Screening and purification of nanobodies from E. coli culture supernatants using the hemolysin secretion system. Microb Cell Fact 18, 47) (Kaur et al., (2019). Identification of conformation-selective nanobodies against the membrane protein insertase BamA by an integrated structural biology approach. J Biomol NMR 73, 375-384). Each was constructed by synthesizing the nanobody-encoding sequence as a Gblock (IDT) and subsequently inserting it (by Gibson assembly) downstream of the gene encoding the truncated version of intimin in pDSG323. To generate the intimin expression plasmid, the full-length intimin gene ( eae , EDL933 4947) was PCR amplified from genomic DNA of EHEC 0157:H7 strain EDL933 and substituted for the truncated version of intimin used for nanobody or antigen display in pDSG323 (Glass and Riedel-Kruse, 2018, supra). For the production of E. cloacae in-frame deletion constructs, 750 bp regions flanking the deletion were amplified, joined using splicing by overlap extension (SOE) PCR, and subsequently cloned into the E. cloacae suicide vector pRE118-pheS using the Sad and Xbal restriction sites (pRE118-pheS was a gift from Christopher Hayes of UC Santa Barbara).

TABLE 4 Oligonucleotides used in this study.

Generation of mutant bacterial strains

To generate mutations in E. cloacae, deletion constructions in pRE118-pheS were transformed into E. coli SI 7-1 l pir. E. coli SI 7-1 l pir donor cells carrying the deletion constructs and E. cloacae recipient strains to be mutated were grown overnight on LB plates containing antibiotics as appropriate, then scraped together to create a 2:1 mixture of each donor-recipient pair that was spread on an LB agar plate and incubated at 37°C for 6 hours to facilitate plasmid transfer via conjugation. Cell mixtures were then scraped into PBS and plated on LB medium agar plates supplemented with kanamycin and streptomycin to select for E. cloacae containing the deletion construct inserted into the chromosome. E. cloacae merodiploid strains were then grown overnight in non-selective LB medium at 37°C, followed by counter selection on M9 minimal medium agar plates with 0.4% glucose and 0.1% (w/v) / chlorophenylalanine. Kanamycin sensitive colonies were screened for allelic replacement by colony PCR and mutations were confirmed by Sanger sequencing of PCR products.

E. coli bearing a deletion of rfaD was generated by the lambda red recombinase system ( Datsenko, K.A., and Wanner, B.L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences of the United States of America 97, 6640-6645). In brief, PCR products containing a chloramphenicol resistance cassette flanked by 50 bp of homology to the 5' and 3' termini of the rfaD gene were electroporated into E. coli MG1655 carrying pKD46 induced to expressed the recombinase with 0.2 % (w/v) L-arabinose for 5 hours at 30°C. E. coli was then incubated in LB for 1 hour, plated on chloramphenicol containing LB agar and incubated overnight at 37°C.

Bacterial competition assays

Competitions between two bacterial strains

Bacterial competitions on solid media were performed as previously described (LeRoux, M., et al. (2015). Kin cell lysis is a danger signal that activates antibacterial pathways of Pseudomonas aeruginosa. Elife 4). Briefly, overnight cultures were spun for 1 min at 17,900 x g to pellet cells and culture supernatant was removed. Cell pellets were washed once with LB medium, spun again, and finally resuspended in LB medium. Cell suspensions were then normalized to Oϋboo = 2.0. Mixtures of E. cloacae and target cells were then established at 20:1 (E. cloacae vs. E. cloacae Aei x3) or 3:1 ( . cloacae vs. E. coli) v/v ratios. Starting ratios were established by performing 10-fold serial dilutions and plating on appropriate selective media. Competitions were initiated by spotting 3 x 5 ul of each mixture on nitrocellulose filters placed on 3% (w/v) agar LB plates and incubated at 37°C for 3 or 6 hours. Cells were then harvested by scraping individual spots from excised sections of the nitrocellulose filter into LB medium. Suspensions were serially diluted and plated on selective media for quantification of CFUs. For intraspecies competitions, E. cloacae Aei x3 was marked by chromosomal insertion of a spectinomycin resistance gene, while donor E. cloacae strains were unmarked; E. cloacae Aei x3 populations were quantified by enumerating CFU obtained on LB with spectinomycin, and donor populations quantified by subtracting this number from the total CFU enumerated on non- selective LB plates. For interspecies competitions, E. coli was marked by chromosomal insertion of a chloramphenicol resistance gene; S. proteamaculans, F. novicida, and S. pakistanense were marked by a kanamycin resistance gene; E. cloacae strains were unmarked as in the intraspecies competition experiments; E. cloacae was distinguished by plating on LB containing streptomycin (intrinsic resistance). Competitive indices for each experiment were determined by dividing the final donor to recipient ratio by the initial donor to recipient ratio.

For bacterial competitions in liquid media, overnight cultures were pelleted, washed, and resuspended in LB as described above. For both intra- and interspecies competitions, E. cloacae donor and target cells were diluted to Oϋboo = 0.03 and 0.0003, respectively. Competitions were performed in LB with 0.5% glucose and 400 ng/mL of anhydrotetracycline to induce nanobody or antigen expression. PEG 8000 was added to the medium when indicated in the figure legends. Starting ratios of donor and recipient strains were established as described above. Competitions were incubated at 37°C with shaking at 200 rpm for 6 hours. Cells were collected at indicated time points (2, 4, 6, 8, or 24 hours), serially diluted, and plated on selective media for quantification of CFUs.

Competitions between three bacterial strains

E. cloacae expressing Nb-Y was cocultured with two E. coli strains, those displaying Ag-Y or a null control expressing only the intimin display construct (pDSG323) (Glass and Riedel-Kruse, 2018, supra). Suspensions of E. cloacae and each E. coli strain were prepared as described above for interspecies liquid media competitions. Competitions were then initiated in which the starting concentration of E. cloacae and the null control strain were held constant at Oϋboo = 0.03 and 0.01, respectively, while the starting concentration of E. coli expressing Ag-Y was varied by 10 fold dilutions from 0.01 to 0.00001, to establish starting ratios for the two A. coli strains of 1:1, 10:1, 100:1 and 1000:1. The two E. coli populations were distinguished by chromosomal insertion at galK of genes encoding chloramphenicol resistance and dTomato (control strain) or mTagBFP (Ag-Y expressing strain) under the control of the constitutive promoter pLlacO (strains were a gift from Erik Gullberg of Uppsala University). Anhydrotetracycline and glucose were supplemented as describe above. Competitions were incubated at 37°C with shaking at 200 rpm for 6 hours. Initial and after competition samples were collected, plated on LB containing chloramphenicol, and visualized using an Azure Biosystems c600.

Bacterial survival experiments

To assess survival of bacteria targeted by nanobody -producing E. cloacae, strains were grown overnight then diluted to Oϋboo = 0.1 and grown for 4 hours (until stationary phase) in LB with anhydrotetracyline to induce nanobody or antigen production. Cells were then pelleted, washed and resuspended as described above, then normalized to OD ₆ OO = 5.0. Mixtures of E. cloacae and E. coli were then established at 100:1 v/v ratios in LB with glucose and anhydrotetracycline. Starting ratios of cells were established as described above. Competitions were incubated at 37°C with shaking at 200 rpm, and samples were collected at 30, 60, and 90 minutes. Cells were harvested at each time point and then serially diluted and plated on selective media for quantification of CFUs as described above.

Synthetic community competition experiment Synthetic community competition experiments contained the following 12 species in addition to E. cloacae and E. coli : Serratia proteamaculans , Agrobacterium tumefaciens, Paracoccus denitrificans, Flavobacterium johnsoniae, Sphingobacterium pakistanense, Chromobacterium violaceum, Mycobacterium smegmatis, Enterococcus faecalis, Listeria monocytogenes, Francisella novicida, Xanthomonas maltophilia, and Aeromonas hydrophila. Overnight cultures of each of the species were pooled together at equal concentration (normalized to Oϋboo = 0.01) and mixed with E. cloacae expressing Nb-Y (OD6OO = 0.03 or 0.01) and E. coli expressing Ag-Y or the null control described above (OD6OO = 0.01 or 0.003). The mixtures were grown in LB medium with glucose at 30°C (to permit growth of organisms included in the mixture that are unable to grow at 37°C) with shaking at 200 rpm for 8 hours. After the incubation period, competitions were harvested and washed with fresh LB medium. Cells were then incubated with 2mg mL ¹ DNase for 30 minutes at 37°C to remove extracellular DNA and washed a final time with LB containing 10 mM EDTA to inactivate DNase. Total DNA was extracted using the InstaGene Matrix (Bio-Rad). To calculate the abundance ratio of bacterial strains in the mixed population, the V3 and V4 regions of the 16S rRNA gene were amplified using proprietary primers (Genewiz) and sequenced with an Illumina MiSeq. 40,000 to 200,000 paired end reads were generated from each sample; paired end reads were merged using VSEARCH ( Rognes, T., et al. (2016). VSEARCH: a versatile open source tool for metagenomics. PeerJ 4, e2584). Reads were then trimmed and filtered with MICCA 1.7.2, using a minimum length of 250 and an expected error rate of 0.5. MICCA also was used to de novo cluster the sequences, as well as for taxonomic classification with the Greengenes core set database, downloaded from the MICCA FTP site 2019-06-28, micca.org (Albanese, D., et al. (2015). MICCA: a complete and accurate software for taxonomic profiling of metagenomic data. Sci Rep 5, 9743). Species for which <10 read counts were obtained across the majority of samples post cultivation (M smegmatis, L. monocytogenes) were excluded from downstream analyses. The fraction change in relative read abundance for each community member from before to after competition was calculated by dividing the final read proportion deriving from a given community member by the initial proportion.

Complex, undefined community competition experiment

A complex, undefined microbial community was derived from fresh mouse fecal samples by homogenization followed by Nycodenz density gradient centrifugation as described previously, with the modifications indicated below (Hevia, A., er al. (2015). Application of density gradient for the isolation of the fecal microbial stool component and the potential use thereof. Sci Rep 5, 16807). Fecal samples for the two replicates of the experiment were each collected from a distinct, separately reared colony of C57BL/6J mice maintained in specific pathogen free (SPF) conditions. Mice used to establish the colonies were originally purchased from Jackson Laboratories. Daily care of the colonies was provided and SPF conditions were ensured through the rodent health monitoring program overseen by the Department of Comparative Medicine at the University of Washington. Upon collection, mouse fecal samples were resuspended in Phosphate-buffered saline (PBS) buffer and homogenized by Tissue-Tearor homogenizer (BioSpec). Samples were then gently added to the top of 80 % (w/v) Nycodenz, followed by ultra-centrifugation at 10,000 g for 40 minutes. The top layer of PBS buffer was carefully removed, and the high density fecal bacterial community from the middle layer was collected and normalized to ODeoo = 20 (FIGURES 4C, 4D, 8A, E. coli at 0.25% or 0.05%, FIGURE 8B) or 5 (FIGURE 8 A, E. coli at 1%) in PBS buffer. The resulting fecal bacterial community was mixed with equal volumes of E. cloacae expressing Nb-Int and normalized to ODeoo = 5 (FIGURES 4C, 4D, 8 A, E. coli at 1% or 0.25%, FIGURE 8B) or 1 (FIGURES 8A and 8B, E. coli at 0.05%), and E. coli marked with a chromosomally encoded chloramphenicol resistance gene and expressing intimin or the null control (ODeoo = 0.05, FIGURES 4C, 4D, 8A, E. coli at 1% or 0.25%, FIGURE 8B or 0.01, FIGURE 8A, E. coli at 0.05%) in LB amended with glucose and anhydrotetracyline. The mixtures were grown at 37°C with shaking at 200 rpm for 1 hour. Initial and post-incubation samples of the mixtures were plated on LB with chloramphenicol and incubated aerobically to selectively quantify E. coli populations. After the incubation period, cells were pelleted and washed with fresh LB medium. Total DNA was extracted using the DNeasy PowerSoil Kit (Qiagen). The V3-V4 region of the 16S rRNA gene was amplified from the samples using proprietary primers (Genewiz) and sequenced with an Illumina MiSeq as described above. OTU counts were determined using the Genewiz 16S-EZ analysis pipeline.

Assessing conventional antibiotic specificity

The specificity and potency of conventional antibiotics was compared to that of PICs using the 12-member synthetic microbial community described above. Overnight cultures of each of the species were pooled together at equal concentration (normalized to ODeoo = 0.01) and E. coli (ODeoo = 0.01). The mixtures were grown in LB with glucose and ciprofloxacin at 30°C with shaking at 200 rpm for 8 hours. After the incubation period, cells were harvested and washed with fresh LB medium. Extracellular DNA was removed as described above. Total DNA was extracted using the InstaGene Matrix (Bio-Rad) and V3-V4 region of the 16S rRNA genes were amplified and sequenced as described above. Sequence data was analyzed as described for the synthetic community competition experiments.

Assessing potential for the emergence of resistance to ^A

E. cloacae expressing Nb-BamA or the null control and E. coli DH5a containing pBAD33 and pBAD18 (providing resistance to chloramphenicol and carbenicillin for separating E. coli from E. cloacae) were grown overnight in medium amended with appropriate antibiotics. The cultures were then washed, pelleted, and resuspended as described above. Three replicate competitions were then initiated in LB medium amended with glucose, anhydrotetracycline and PEG8000, in which the starting concentration of E. cloacae and E. coli DH5a at ODc.oo were 0.03 and 0.0003, respectively. Competitions were incubated at 37°C with shaking at 200 rpm for 6 hours. Post-competition samples were pooled and plated densely on 150 mm petri plates containing LB with chloramphenicol and carbenicillin to remove E. cloacae. All E. coli DH5a colonies obtained (~10,000/sample) were collected, diluted, and used to initiate the next round of competition along with fresh cultures of E. cloacae expressing Nb-BamA or the null control, prepared as above. This regime was repeated for ten passages. The populations of E. coli and E. cloacae were quantified before and after each round of competition by plating on selective media. The relative competitiveness of PIC ^{xb BamA} at each round was calculated by dividing the competitive index (final E. cloacae/ final E. coli divided by initial E. cloacae/ initial E. coli) for this strain by that of the null control.

Phase contrast and fluorescence microscopy

Imaging was performed on a Nikon Eclipse T/-E wide-field epi-fluorescence microscope, equipped with a sCMOS camera (NEO, Andor Technology) and X-cite LED for fluorescence imaging. Images were obtained through 60X 1.4 NA oil-immersion objective, with a constant focal plane maintained a using a Nikon Perfect Focus system. The microscope is controlled by NIS-Elements.

Samples from bacterial competition experiments were imaged by phase contrast and fluorescence microscopy as previously described (Ting, S.Y., et al. (2018). Bifunctional Immunity Proteins Protect Bacteria against FtsZ-Targeting ADP-Ribosylating Toxins. Cell, 175(5), 1380-1392). Briefly, mixtures of . cloacae and . coli were spotted on a PBS with 2% agarose pad placed on a microscope slide. Still images of the cells were acquired before and after competition, including a phase-contrast image (to visualize cell morphology) and fluorescence image (to distinguish E. coli, which expressed dTomato or mTagBFP, from unlabeled E. cloacae) for each field of view. To visualize bacterial aggregates from cultures amended with varying concentrations of PEG8000, liquid cultures of E. cloacae normalized to Oϋboo 0.03 and E. coli normalized to Oϋboo 0.01 were mixed, grown in LB with glucose and PEG8000 at the concentrations indicated for 6 hrs, diluted, spotted onto an agarose pad, and imaged via phase microscopy. Aggregate sizes were quantified by a masking algorithm in MATLAB, the main stages of which are local nominalization, gradient thresholding, and hole-filling.

Protein expression level analyses

To analyze the expression of nanobodies, E. cloacae strains expressing the proteins from pDSG323-derivatives were grown in LB medium supplemented with or without anhydrotetracycline at 37°C for 6 hours and harvested at an Oϋboo of 1.0. For each quantification assay, cell pellets were resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 0.1% (v/v) Triton X100, 1 mM EDTA, and 1 mM DTT) and with 2X SDS-PAGE sample loading buffer. Samples were boiled at 95°C for 10 minutes and loaded at equal volumes to resolve using SDS-PAGE, then transferred to nitrocellulose membranes. Membranes were blocked in TBST (10 mM Tris-HCl pH 7.5, 150 mM NaCh. and 0.1% w/v Tween-20) with 5% (w/v) non-fat milk for 30 min at room temperature, followed by incubation with primary antibodies (anti-myc or anti-RpoB) diluted 1:1000 in TBST for 1 hour at room temperature. Blots were then washed by TBST, followed by incubation with secondary antibody (Goat anti-mouse HRP conjugated) diluted 1:5000 in TBST for 30 minutes at room temperature. Finally, blots were washed by TBST again and were developed using the Radiance HRP substrate (Azure Biosystems) and visualized using the Azure Biosystems c600.

Cell surface accessibility assays (Flow cytometry and immunofluorescence assay)

Overnight grown cells were diluted to an Oϋboo of 0.03 in LB and incubated either with or without anhydrotetracycline for 6 hours at 37°C. Cells were then harvested at an OD6OO of 1.0 and washed with filtered phosphate buffered saline (PBS; 8 mM of Na2HP04, 1.5 mM of KH2P04, 3 mM of KC1 and 137 mM ofNaCl, pH7). An aliquot of 200 pi of cells was incubated for 1 hour on ice with anti-myc antibodies diluted 1:1000 in PBS and 10% (v/v) goat serum in a final volume of 500 mΐ. Cells were followed by washed with filtered PBS and resuspended in 500 mΐ of filtered PBS containing 10% (v/v) goat serum. Then, bacteria were incubated for 30 minutes at 4°C in the dark with goat Alexa fluor 488- conjugated anti-mouse IgG (1:250; ThermoFisher Scientific). Finally, cells were washed once and resuspended in a final volume of 1 ml of filtered PBS and fluorescence analyzed in a MACSQuantTM VYB flow cytometer (Miltenyi Biotec; Bergisch Gladbach, Germany). Fluorescence was excited at 488 nm and recorded with a 525/50 nm band-pass filter. The results were processed using FlowJo (FlowJo LLC; Ashland OR, USA). Two biological replicates were performed and 100,000 events acquired for each experiment. Quantification and Statistical Analysis

Statistical significance in bacterial competition experiments was assessed by unpaired t-tests between relevant samples. Details of statistical significance is provided in the figure legends. While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.