FIELD OF THE INVENTION

[0001] The present invention relates to lysin polypeptides for prophylaxis and treatment of infections caused by Gram Negative Bacteria including Escherichia coli. Further, the present invention relates to compositions comprising lysin polypeptides for prophylaxis and treatment of infections caused by Gram Negative Bacteria including Escherichia coli. In particular, the present invention relates to lysin polypeptides and compositions comprising the same against the infections caused by Uropathogenic Escherichia coli (UPEC).

BACKGROUND OF THE INVENTION

[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art. [0003] One of the most common and frequent bacterial infections in humans are Urinary Tract Infections (UTIs) and represents an important public health problem with a substantial economic burden. It can be acquired through community as well as health care settings. Every year 150 million people suffer from UTI globally with the Enterobacteriaceae family bacteria contributing 84.3% of all cases. Women, as compared to men, are more prone to UTIs and approximately 50% of them encounter UTI once in their lifespan. Uropathogenic E. coli (UPEC) is the most common pathogen implicated in 80% UTI cases. UPECs are a type of extra-intestinal pathogenic E. coli, and among the most widespread class of extra-intestinal infections found to be the most prevalent cause of UTIs in population of all ages and covering both the genders.

[0004] Antibiotics including P-lactams are used for the treatment of UTIs in several countries. The indiscriminate and widespread use of antibiotics has led to the development of antimicrobial resistance in the pathogens involved at an alarming pace challenging the use of antibiotics. Also, the pipeline for the development of new antibiotics is too small to deter the increasing problem of antimicrobial resistance (AMR). In addition, there has been an increase in the multidrug resistant (MDR) strains of UPEC which thwart the therapy for the treatment of UTIs. Members of Enterobacteriaceae can harbor genes conferring resistance to almost all antibiotics and plasmids harboring these resistance determinants can be transferred between other bacteria, even between species. Therefore, the acquisition of resistance to new antibiotics may only be a matter of time.

[0005] Thus, there is an urgent need of identifying and developing new and alternative antimicrobial agents. Enzybiotics or endolysins are good candidates and have shown promising results against gram positive bacteria. However, the gram negative bacteria have an outer membrane acting as a barrier apart from the peptidoglycan layer, few endolysins with exogenous activity has been observed.

[0006] Given the occurrence of various types of drug resistance such as antimicrobial resistance (AMR), multi drug-resistant (MDR) and extensively drugresistant (XDR) strains of E. coli and other pathogens, alternative solutions are being explored and bacteriophages and the encoded lysin enzymes are gaining attention globally, especially in the past few years. The report identifying lysin PlyF3017 acting against Gram-negative bacteria was the first report on a highly- active therapeutic lysin. It was identified from a group of naturally -produced Acinetobacter baumanii lysins and displayed great activity against planktonic and biofilm A. baumanii cells, both in vitro and in vivo. Therefore, given the steep rise in AMR, development of lysin enzymes as ‘enzybiotics’ can be a game changer in combating AMR when potent antimicrobials are contemplated. However, lysins as therapeutics haven’t yet been much explored in the treatment of UTI.

[0007] Therefore, there remains an urgent need to overcome the scourge of UTIs, a common bacterial infection in the humans, especially caused by UPEC which is responsible for more than 80% of the cases of UTIs in the humans. Accordingly, there remains a need of identifying and developing effective agent, which will not only treat and deter the infections but are also resistant against the gram-negative pathogens, especially UPEC that can provide one or more advantages such as can be easily manufactured, scalable and can overcome deficiencies associated with the known art.

OBJECTS OF THE INVENTION

[0008] The object of the present invention is to provide agent against E. coli causing infections in the urinary tract (UTI).

[0009] Another object of the present invention is to provide agent against drugresistant E. coli causing UTI.

[00010] One of the objects of the present invention is to provide lysin polypeptides for prophylaxis and/or treatment of infection caused by Gram Negative Bacteria including Escherichia coli.

[00011] Still another object of the invention is to provide lysin polypeptides effective against the infections caused by Uropathogenic Escherichia coli (UPEC). [00012] Yet another object of the present invention is to provide a composition comprising lysin polypeptides for prophylaxis and/or treatment of infections caused by Gram Negative Bacteria including Escherichia coli.

[00013] One more object of the invention is to provide a composition comprising lysin polypeptides for prophylaxis and/or treatment of infections caused by Uropathogenic Escherichia coli (UPEC).

SUMMARY

[00014] In an aspect, the present invention provides lysin polypeptides active against Gram Negative Bacteria including Escherichia coli.

[00015] In one specific aspect the present disclosure provides a lysin polypeptide comprising an amino acid sequence having at least 80% or at least 85% or at least 90% or at least 95% identity to a sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 16, or a fragment thereof having lysin activity, wherein the lysin polypeptide inhibits the growth, or reduces the population, or kills Gram Negative Bacteria including Escherichia coli.

[00016] In one aspect the present invention provides lysin polypeptides effective against the infections caused by an Escherichia coli selected from the group consisting of but not limited to, Uropathogenic Escherichia coli (UPEC), Shiga toxin-producing Escherichia coli (STEC), Avian Pathogenic Escherichia coli (APEC) and Mammary pathogenic Escherichia coli (MPEC).

[00017] In another aspect, the present invention provides a composition comprising a lysin polypeptide for prophylaxis and/or treatment of infections caused by Gram Negative Bacteria including Escherichia coli.

[00018] In one more specific aspect the present disclosure provides a composition for prophylaxis and/or treatment of infections caused by an Escherichia coli selected from the group consisting of but not limited to, Uropathogenic Escherichia coli (UPEC), Shiga toxin-producing Escherichia coli (STEC), Avian Pathogenic Escherichia coli (APEC) and Mammary pathogenic Escherichia coli (MPEC)), the composition comprises a lysin polypeptide comprising an amino acid sequence having at least 80% or at least 85% or at least 90% or at least 95% identity to a sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 16, or a fragment thereof having lysin activity.

[00019] In some aspects, the composition further comprises one or more antibiotics suitable for the treatment of Gram- negative bacteria.

[00020] In another aspect, the present invention provides a method of inhibiting the growth, or reducing the population, or killing of at least one species of Gramnegative bacteria, the method comprising contacting the bacteria with a composition containing an effective amount of a lysin polypeptide comprising an amino acid sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 16, or active fragments thereof, wherein the lysin polypeptide has the property of inhibiting the growth, or reducing the population, or killing Gram Negative Bacteria including Escherichia coli.

[00021] In a related aspect, the disclosure provides a method of treating a bacterial infection caused by an Escherichia coli selected from the group consisting of but not limited to, Uropathogenic Escherichia coli (UPEC), Shiga toxin-producing Escherichia coli (STEC), Avian Pathogenic Escherichia coli (APEC) and Mammary pathogenic Escherichia coli (MPEC), comprising administering to a subject diagnosed with, at risk for, or exhibiting symptoms of a bacterial infection, a composition containing an effective amount of a lysin polypeptide comprising an amino acid sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 16, or an active fragment thereof, wherein the lysin polypeptide has the property of inhibiting the growth, or reducing the population, or killing Uropathogenic Escherichia coli (UPEC).

DRAWINGS

[00022] The following drawings form part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[00023] Figure Idepicts representative In-silico strategy followed to identify prophage-encoded lysin polypeptides.

[00024] Figure 2 depicts optical images of SDS-PAGE of 7 recombinant lysin polypeptides namely those having SEQ ID NOs: 1, 10, 11, 7, 15, 2 and 9.

[00025] Figure 3 depicts optical images of Western Blot of 7 recombinant lysin polypeptides namely those having SEQ ID NOs: 1, 10, 11, 7, 15, 2 and 9.

[00026] Figure 4 depicts SDS-PAGE Image of lysin polypetide comprising amino acid having SEQ ID NO: 10 (Induced Fraction) in duplicate.

[00027] Figure 5 depicts SDS-PAGE Image of NiNTA purified lysin polypetide comprising amino acid having SEQ ID NO: 10 in duplicate.

[00028] Figure 6 depicts Western Blot Image of purified lysin polypetide comprising amino acid having SEQ ID NO: 10.

[00029] Figure 7 is a graph depicting lysis of E. coli (BL21 DE3) cells showing drop in ODeoonm cells in the treated with lysin polypetide comprising amino acid having SEQ ID NO: 10.

[00030] Figure 8 depicts optical images of Untreated (UT) and Treated (Tr) colonies. Top rows show untreated colonies and bottom rows show colonies treated with 5pl of lysin polypetide comprising amino acid having SEQ ID NO: 10 at different dilutions ranging from 10- 10’ ⁶ . [00031] Figure 9 is a graph showing Log killing of logarithmic growing E. coli cells by lysin polypetide comprising amino acid having SEQ ID NO: 10.

[00032] Figure 10 is a graph showing lysozyme activity of lysin polypetide comprising amino acid having SEQ ID NO: 10.

[00033] Figure 11 depicts optical image showing lytic activity of lysin polypetide comprising amino acid having SEQ ID NO: 10 (in duplicate) against ATCC 700928 strain.

[00034] Figure 12 depicts optical image showing lytic activity of lysin polypetide comprising amino acid having SEQ ID NO: 10 (in duplicate) against ATCC 25922 strain.

DETAILED DESCRIPTION OF THE INVENTION

[00035] The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.

[00036] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[00037] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[00038] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term "about." Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[00039] As used in the description herein and throughout the claims that follow, the meaning of "a," "an," and "the" includes plural reference unless the context clearly dictates otherwise.

[00040] Also, as used in the description herein, the meaning of "in" includes "in" and "on" unless the context clearly dictates otherwise.

[00041] Unless the context requires otherwise, throughout the specification which follow, the word "comprise" and variations thereof, such as, "comprises" and "comprising" are to be construed in an open, inclusive sense that is as "including, but not limited to."

[00042] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any unclaimed element essential to the practice of the invention.

[00043] The description that follows, and the embodiments described therein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present disclosure. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.

[00044] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

[00045] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.

[00046] The terms “Polypeptide” or "protein” and any variants not specifically listed as used herein interchangeably, according to the present invention means, but not limited to, large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. The chain of amino acid residue may be of any length and may be linear or branched. This term encompasses all the naturally occurring or modified/ recombinant proteins.

[00047] The terms “lysin” or “Endolysin”, used herein interchangeably according to the present invention means, but not limited to peptidoglycan hydrolases produced at the end of the bacteriophage (phage) replication cycle to lyse the host cell. These enzymes are remarkably efficient in hydrolyzing the peptidoglycan layer, resulting in a sudden drop in turgor pressure and osmotic lysis to cause bacterial cell death

[00048] The term “therapeutically effective amount” as used herein according to the present invention means, but not limited to an amount of the active ingredient (i.e., therapeutic protein or antibody) sufficient to produce the desired therapeutic effect in a human or animal, e.g., the amount necessary to treat, cure, prevent, or inhibit development and progression of disease or the symptoms thereof and/or the amount necessary to ameliorate symptoms or cause regression of disease. Such a therapeutically effective amount may vary depending on the structure and potency of the active ingredient and the contemplated mode of administration.

[00049] The term “treatment” as used herein according to the present invention means, but not limited to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those individuals, such as humans and animals, already with the disorder or condition to be treated as well as those prone to have the disorder or those in which the disorder is to be prevented. As used herein, “treatment” also includes reduction of the likelihood of obtaining the disorder, reduction of the severity of the disorder in those already afflicted, and the induction of regression of the disorder or symptoms thereof.

[00050] The term “pharmaceutically-acceptable carrier” as used herein according to the present invention means but not limited to a filler, diluent or encapsulating substance that may be safely used in systemic administration. Depending upon the particular route of administration, a variety of pharmaceutically acceptable carriers, well known in the art may be used. These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions including phosphate buffered saline, emulsifiers, isotonic saline, and pyrogen free water. In particular, pharmaceutically acceptable carriers may contain different components such as a buffer, sterile water for injection, normal saline or phosphate buffered saline, sucrose, histidine, salts and polysorbate. Terms such as “physiologically acceptable”, “diluent” or “excipient” can be used interchangeably.

[00051] The present invention relates to lysin polypeptides active against Gram Negative Bacteria including Escherichia coli.

[00052] The present invention provides lysin polypeptides also known as endolysin polypeptides to act as an enzybiotic for prophylaxis and/or treatment of infections caused by Gram Negative Bacteria including Escherichia coli, preferably UPEC infections.

[00053] In one embodiment, the present disclosure provides a lysin polypeptide comprising an amino acid sequence having at least 80% or at least 85% or at least 90% or at least 95% identity to a sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 16, or a fragment thereof having lysin activity, wherein the lysin polypeptide inhibits the growth, or reduces the population, or kills Gram Negative Bacteria including Escherichia coli.

[00054] In one aspect the present invention provides lysin polypeptides effective against the infections caused by an Escherichia coli selected from the group consisting of but not limited to, Uropathogenic Escherichia coli (UPEC), Shiga toxin-producing Escherichia coli (STEC), Avian Pathogenic Escherichia coli (APEC) and Mammary pathogenic Escherichia coli (MPEC).

[00055] In one embodiment, the present invention provides a process of preparation of lysin polypeptide comprising an amino acid sequence having SEQ ID NO: 1-SEQ ID NO: 16.

[00056] In one embodiment, the present invention provides a process of preparation of lysin polypeptide comprising an amino acid sequence having SEQ ID NO: 1-SEQ ID NO: 16 using a host cell selected from the group consisting of but not limited to prokaryotic cells and eukaryotic cells.

[00057] The host cell can be of mammalian, plant, insect, fungal or bacterial origin.

[00058] The eukaryotic host cells are selected from but not limiting to mammalian cells or other animal cells; avian cells; insect cells; plant cells and fungal cells, [00059] The mammalian cells can be selected from without limitation cells of hamster, rabbit, rat, pig, mouse, or the like.

[00060] The avian cells can be selected from without limitation duck, chicken, quail, or the like.

[00061] The plant cells can be selected from without limitation corn cells, tobacco cells or the like.

[00062] The fungal cells can be selected from without limitation Saccharomyces cerevisiae, Pichia pastoris or the like. [00063] The prokaryotic cells can be selected from without limitation E. coli; and other cells used in the art for the production of monoclonal antibodies and other binding proteins.

[00064] In some embodiments, the present invention provides a composition comprising a lysin polypeptide for prophylaxis and/or treatment of infections caused by Gram Negative Bacteria including Escherichia coli. In certain embodiment the present invention provides a composition comprising a lysin polypeptide comprising an amino acid sequence having at least 80% or at least 85% or at least 90% or at least 95% identity to a sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 16, or a fragment thereof having lysin activity.

[00065] The Escherichia coli is selected from the group consisting of but not limited to Uropathogenic Escherichia coli (UPEC), Shiga toxin-producing Escherichia coli (STEC), Avian Pathogenic Escherichia coli (APEC) and Mammary pathogenic Escherichia coli (MPEC),

[00066] In one embodiment, the present invention provides a composition comprising a lysin polypeptide comprising an amino acid sequence having at least 80% or at least 85% or at least 90% or at least 95% identity to a sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 16, or a fragment thereof having lysin activity for prophylaxis and/or treatment of infections caused by an Escherichia coli selected from the group consisting of, but not limited to, ATCC BAA-2471, BAA-197, 55244, BAA-2452, BAA-2523, 25252, 700609, 700078, BAA-2340, BAA-2469, 11370, 29214, 29181, 25253, 25250, 25922, 700928, ST410, ST131, NA114, PAI IV536, PAI I CFT073, ST405, ST13,CFT073, BL21 DE3, Shiga toxin-producing E. Coli (STEC) strains ATCC 43895, Avian Pathogenic Escherichia coli (APEC) Strain ATCC 11775, CCUG 27532, CCUG 11380, Mammary pathogenic Escherichia (E.) Coli (MPEC) CCUG 36534, BAA- 2440, BAA-2196 and BAA-2215.

In another embodiment, the present invention provides a composition comprising a lysin polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 16, or a fragment thereof and Pharmaceutically-acceptable carrier.

[00067] The pharmaceutically acceptable carrier is selected from but not limiting to, buffer, antioxidant, preservative, isotonic agent and chelating agent.

[00068] In a preferred embodiment of the present invention the composition comprises additional outer membrane permeabilizer (OMP) selected from, but not limited to, EDTA, TRIS, Citric Acid, lactic acid, lactoferrin, polymix and mixture of two or more thereof.

[00069] The pharmaceutical compositions containing the lysin polypeptide comprising amino acid sequence having SEQNID NO: 1-16 of the invention, maybe in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs; sterile injectable aqueous or oleaginous suspension; suppositories; topical use, for example creams, ointments, jellies, solutions or suspension or the like including mouth washes and gargles. These dosage forms can be prepared by any method known in the art using the polypeptide of the present invention combined with non-toxic pharmaceutically acceptable excipients.

[00070] In one embodiment, the present invention provides composition comprising lysin polypeptide comprising amino acid sequence having SEQ ID NO: 1-16 further comprising an antibiotic suitable for the treatment of Gram Negative Bacterial infection.

The antibiotic can be selected from, but not limited to, cephalosporins, fluoroquinolones, aminoglycosides, imipenem, broad- spectrum penicillins with or without P-lactamase inhibitors, trimethoprim- sulfamethoxazole or the mixtures thereof.

[00071] The cephalosporins can be selected from without limitations ceftriaxone - cefotaxime and ceftazidime; the fluoroquinolones can be selected from without limitations ciprofloxacin and levofloxacin; the aminoglycosides can be selected from without limitations gentamicin and amikacin; the broad- spectrum penicillins with or without P-lactamase inhibitors can be selected from without limitations amoxicillin-clavulanic acid and piperacillin-tazobactam. [00072] In one embodiment, the present invention provides use of composition comprising a lysin polypeptide comprising an amino acid sequence having at least 80% or at least 85% or at least 90% or at least 95% identity to a sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 16 for prophylaxis and/or treatment of infections caused by Gram Negative Bacteria including Escherichia coli.

[00073] In one embodiment, the present invention provides use of a composition comprising a lysin polypeptide comprising an amino acid sequence having at least 80% or at least 85% or at least 90% or at least 95% identity to a sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 16 for prophylaxis and treatment of infection caused by the Escherichia coli selected from, but not limited to, Uropathogenic Escherichia coli (UPEC), Shiga toxin-producing Escherichia coli (STEC), Avian Pathogenic Escherichia coli (APEC) and Mammary pathogenic Escherichia coli (MPEC).

[00074] In another embodiment, the present invention provides a method of inhibiting the growth, or reducing the population, or killing of at least one species of Gram-negative bacteria, the method comprising contacting the bacteria with a composition containing an effective amount of a lysin polypeptide comprising an amino acid sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 16, or active fragments thereof, wherein the lysin polypeptide has the property of inhibiting the growth, or reducing the population, or killing Gram Negative Bacteria including Escherichia coli.

[00075] In one more related embodiment, the present invention provides a method of treating a bacterial infection caused by an Escherichia coli selected from the group consisting of but not limited to, Uropathogenic Escherichia coli (UPEC), Shiga toxin-producing Escherichia coli (STEC), Avian Pathogenic Escherichia coli (APEC) and Mammary pathogenic Escherichia coli (MPEC), comprising administering to a subject diagnosed with, at risk for, or exhibiting symptoms of a bacterial infection, a composition containing an effective amount of a lysin polypeptide comprising an amino acid sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 16, or an active fragment thereof, wherein the lysin polypeptide has the property of inhibiting the growth, or reducing the population, or killing Uropathogenic Escherichia coli (UPEC).

[00076] In one embodiment, the present invention provides a method of treating topical or systemic bacterial infection caused by Gram Negative Bacteria Escherichia coli by administering effective amount of a composition comprising lysin polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16.

[00077] In one embodiment, the present invention provides a method of treating bacterial infection caused by Gram Negative Bacteria Escherichia coli by administering effective amount of a composition comprising lysin polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16 through an oral route or an intravenous route.

[00078] In one embodiment, the composition can be administered orally, nasally, topically, subcutaneously, intramuscularly, intravenously, or by other modes of administration.

[00079] While the disclosure has been described and illustrated with reference to certain preferred embodiments thereof, those skilled in the art will appreciate that various changes, modifications and substitutions can be made therein without departing from the spirit and scope of the present disclosure. For example, the specific pharmacological responses observed may vary according to and depending on the particular active compound selected or whether there are present pharmaceutical carriers, as well as the type of formulation and mode of administration employed, and such expected variations or differences in the results are contemplated in accordance with the objects and practices of the present disclosure.

Examples

[00080] The disclosure is further illustrated by the following examples which in no way should be construed as being further limiting. One skilled in the art will readily appreciate that the specific methods and results described are merely illustrative.

Materials

[00081] pET28b (Novagen, US), DH5a strain (ThermoFisher, US), E. coli BL21 (DE3) strain (ThermoFisher, US), T7E strain (ProteoGenix, France), Rosetta DE3 strain (Novagen, US) Arctic strain (Agilent, US), LB medium (Sigma, US) isopropyl B-D-l -thiogalactopyranoside (IPTG, Sigma, US), Ni-NTA chromatography (Qiagen, US), ATCC 25922 (US) and ATCC 700928 (US).

Example 1

Identification of prophage-encoded lysin (Endolysin) sequences

[00082] Figure 1 describes the strategy to identify the 16 prophage -encoded lysin sequences targeting peptidoglycan in E. coli. The In-silico analysis showed the Lysozyme like domain’ is the predominant (56%) domain in the 16 lysin sequences. The predicted lysins are either modular or globular. Lysins with cationic and hydrophobic residues at the C-terminal end and cationic residues in the catalytic domains indicate bactericidal activity. Consequently, based on the characteristics, 7 Endolysin polypeptide sequences are shortlisted and taken forward for cloning, expression and purification as well as for further evaluation of antibacterial activity using in vitro assays.

Example 2

Cloning, expression and purification of Endolysins

[00083] As described Ma et al, 2017 [Qiang Ma . Zhimin Guo . Chencheng Gao . Rining Zhu . Shuang Wang . Ling Yu . Wanhai Qin . Xiaojing Xia . Jingmin Gu . Guangmou Yan . Liancheng Lei. Enhancement of the direct antimicrobial activity of Lysep3 against Escherichia coli by inserting cationic peptides into its C terminus. Antonie Van Leeuwenhoek. 2017 Mar;110(3):347-355. doi: 10.1007/sl0482-016-0806-2], the lysin sequences were cloned, expressed and purified with minor modifications from the reported method. The sequences were chemically synthesized with optimization for E. coli system. The cDNA was cloned into expression vector pET28b (Novagen). After the transformation of DH5a strain (ThermoFisher), the positive clone strain was extracted to obtain enough expression plasmids. The expression plasmid was transformed into BL21 (DE3) strain (ThermoFisher), T7E strain (ProteoGenix), Rosetta DE3 strain (Novagen) and Arctic strain (Agilent) respectively, in the LB medium (Sigma) and the culture was continued until the logarithmic phase. Induction was performed with ImM Isopropyl B-D-l -thiogalactopyranoside (IPTG, Sigma) at 37°C for 3-4 hours. Uninduced samples were also kept with the same conditions. After centrifugation of both the induced and uninduced cultures, pellets were collected for SDS PAGE analysis and the purification of the proteins respectively.

[00084] All the expressed His-Tag seven lysin proteins were purified using Ni- NTA chromatography (Qiagen). SDS PAGE analysis (Figure 2) was utilized to confirm the molecular weight of the proteins whereas Western blotting (Figure 3) was done to confirm the protein identity and specificity using anti His-Tag antibody [Qiang Ma . Zhimin Guo . Chencheng Gao . Rining Zhu . Shuang Wang . Ling Yu . Wanhai Qin . Xiaojing Xia . Jingmin Gu . Guangmou Yan . Liancheng Lei. Enhancement of the direct antimicrobial activity of Lysep3 against Escherichia coli by inserting cationic peptides into its C terminus. Antonie Van Leeuwenhoek. 2017 Mar;110(3):347-355. doi: 10.1007/sl0482-016-0806-2], The maximum bactericidal activity observed was of lysin polypeptide having amino acid sequence of SEQ ID NO: 10 therefore it was selected for further experiments. Figures 4, 5 and 6 show the SDS PAGE results of the induced fraction, NiNTA purified protein and the western blot results of lysin polypeptide having amino acid sequence of SEQ ID NO: 10 respectively.

Example 3

In-vitro assays determining the antibacterial activity of lysins using E. coli BL21 DE3 cells

[00085] Spot on Lawn Assay, Turbidity Reduction Assay and Log Killing Assay were performed respectively and as described previously with slight modifications [Qiang Ma . Zhimin Guo . Chencheng Gao . Rining Zhu . Shuang Wang . Ling Yu . Wanhai Qin . Xiaojing Xia . Jingmin Gu . Guangmou Yan . Liancheng Lei. Enhancement of the direct antimicrobial activity of Lysep3 against Escherichia coli by inserting cationic peptides into its C terminus. Antonie Van Leeuwenhoek. 2017 Mar;110(3):347-355. doi: 10.1007/sl0482-016-0806-2 and Lucia Blasco, Anton Ambroa, Rocio Trastoy, Ines Bleriot, Miriam Moscoso, Laura Fernandez- Garcia, Elena Perez-Nadales, Felipe Fernandez-Cuenca, Julian Torre-Cisneros, Jesus Oteo-Iglesias, Antonio Oliver, Rafael Canton, Tim Kidd, Ferran Navarro, Elisenda Miro, Alvaro Pascual, German Bou, Luis Martinez-Martinez & Maria Tomas. In vitro and in vivo efficacy of combinations of colistin and different endolysins against clinical strains of multi-drug resistant pathogens. Sci Rep. 2020;10(l):7163. doi: 10.1038/s41598-020-64145-7; Yu Larpin, Frank Oechslin, Philippe Moreillon, Gregory Resch, Jose Manuel Entenza , Stefano Mancini. In vitro characterization of PlyE146, a novel phage lysin that targets Gram-negative bacteria. PLoS One. 2018 Feb 6;13(2):e0192507. doi: 10.1371/journal.pone.0192507].

[00086] Using spot on lawn assay, 3 out of 7 lysins (9-12 pg) showed lysis of E. coli cells (BL21 DE3). The maximum bactericidal activity was observed with lysin polypeptide having amino acid sequence of SEQ ID NO: 10. Turbidimetric reduction assay showed a 74.94% drop in ODeoonm in E. coli cells treated with lysin seq 5 (15 pM) after 3 hrs. of incubation at 37°C (Figure 7). Log killing assay showed 4 log 10 reduction in the lysin polypeptide having amino acid sequence of SEQ ID NO: 10 treated cells in figure 8 and 9 respectively. Lysozyme kit based assay demonstrates higher than/comparable lysozyme activity of lysin polypeptide having amino acid sequence of SEQ ID NO: 10 with the positive control (Figure 10).

Example 4

Spot on lawn assay determining the antibacterial activity of lysin using E. coli CLSI ATCC 25922 and ATCC UPEC 700928 strain

[00087] Using spot on lawn assay, lysin of polypeptide having amino acid sequence of SEQ ID NO: 10 showed bactericidal activity of E. coli cells against ATCC 25922 and ATCC UPEC 700928 strains, however pre-treatment using EDTA as an OMP on both the strains/culture was performed. The procedure of pre-treatment was performed as described previously with slight modifications [Lucia Blasco, Anton Ambroa, Rocio Trastoy, Ines Bleriot, Miriam Moscoso, Laura Fernandez-Garcia, Elena Perez-Nadales, Felipe Fernandez-Cuenca, Julian Torre-Cisneros, Jesus Oteo -Iglesias, Antonio Oliver, Rafael Canton, Tim Kidd, Ferran Navarro, Elisenda Miro, Alvaro Pascual, German Bou, Luis Martmez- Martfnez & Maria Tomas. In vitro and in vivo efficacy of combinations of colistin and different endolysins against clinical strains of multi-drug resistant pathogens. Sci Rep. 2020;10(l):7163. doi: 10.1038/s41598-020-64145-7], The pre-treatment was performed on the mid log phase cells of both ATCC 25922 strain and ATCC UPEC 700928 strain. The growing culture of both the strains was centrifuged at 3000 rpm for 10 mins and the respective pellets were dissolved in 20mM Tris, and 0.3mM EDTA, pH 8.5 (approx.) and kept at 37°C, for 20 mins. After pretreatment, two washes with 5ml LB media were given, to both the pretreated cells (of ATCC 25922 and ATCC 700928), and while giving washes, centrifugation was performed at 12000 rpm for 10 mins. Subsequently, the respective pellets (of both ATCC 25922 and ATCC 700928) were dissolved 2ml LB media. Finally, respective suspensions were mixed and poured with soft agar and left to solidify. 18.6pg of purified lysin of sequence ID 10 and as duplicate was taken for spotting for both the strains (ATCC 25922 and ATCC 700928) respectively. The plates were kept overnight at 37°C and zone of inhibition was observed next day. The bactericidal activity of lysin polypeptide having amino acid sequence of SEQ ID NO: 10 (denoted by zone of inhibition) against both ATCC 700928 strain and ATCC 25922 UPEC strain are shown in Figure 11 and 12 respectively.

Sequence Listings:

>SEQ ID NO: 1

MNSAIAEIQRMLINGGFSVGKSGADGIYGPATKSALEKCIQKANSTNGGN TLLTQTQLDKIFPIGAKANRNSKFLGPLNNLFLKTDINTPNRIAGFLSQIGV ESAEFKYVRELGNDAYFDKYDTGSLAKKLGNTPEKDGDGAKYKGRGLIQ ITGYANYRDCGKALGIDLINHPELLEQPEYAVLSAGWYWQSRDINAACD ADDIVKITKLVNGGTNHLSERTAYYNKAKSVLNS

>SEQ ID NO: 2

MNSAIAEIQRMLINGGFSVGKSGADGIYGPATKSALEKCIQKANSTNGGN

TLLTQAQLDKIFPIGAKANRNSKFLEPLNNLFLKTDINTTNRIAGFLSQIGV

ESAEFKYVREEGNDAYFDKYDTGNEAKKEGNTPEKDGDGAKYKGRGEI

QITGYANYRDCGKALGIDLINHPELLEQPEYAVLSAGWYWQSRNINAAC DADDIVKITKLVNGGTNHLSERTAYYNKAKSVLNS

>SEQ ID NO: 3

MQTSPDGIALIKKFEGCRLTAYPDPGTGDAPWTIGYGWTHPVDGKPVKRGMTID

QQTADRLLKTGLVGYENDVLKVVRVKLTQGQFDALVSFAYNVGSRALSTSTLL KKLNAGDIKGAADEFLRWNKSGGKVMPGLTNRRKAERALFLS

>SEQ ID NO: 4

MQTSPDGIALIKKFEGCRLTAYPDPGTGDAPWTIGYGLTHPVDGKPVKRGMTID

QQTADRLLKTGLVGYENDVLKVVRVKLTQGQFDALVSFAYNVGSRALSTSTLL KKLNAGDIKGAADEFLRWNKSGGKVMPGLTNRRKAERALFLS

>SEQ ID NO: 5

MQISDKGIALIKQFEGCKLTAYQDSVGVWTIGYGWTQPVDGKPIRAGMTIKQET

AERLLKTGLVSYESDVSRLVKVGLTQGQFDALVSFTYNLGARSLSTSTLLRKLNA

GDYAGAADEFLRWNKAGGKVLNGLTRRREAERALFLS

>SEQ ID NO: 6

MNPTLRNKLVGAIVGGSGAITIAAVMLGNADGLEGRRYYAYQDVVGVWTVCD

GHTGTDIRRGHRYTDKECDNLLKADLRKVASAIDPLIKVRIPEPTRAALYSFTYN

VGSGAFASSTLLKKLNSGDVPGACKELQRWTYAGGKQWKGLITRREIEREVCE WGQK

>SEQ ID NO: 7

MGSRAKLSAAVLGLVLAGAPASVILDQFLNEKEGNSLTAYKDGSGIWTICRGAT

TVDGKPVTPGMRLSPEKCNQVNASELNKALAWVDRNIQVPLTEPQKAGIASFCP

YNIGPGKCFPSTFYKRINAGDRKGACEAIRWWIKDGGRDCRLTKGQKNGCYGQ VERRDQESALVCWGIEQ

>SEQ ID NO: 8

MQTSPDGIALIKKFEGCRLTAYPDPGTGDAPWAIGYGWTHPVDGKPVKRGMTID

QQTADRLLKTGLVGYENDVLKVVRVKLTQGQFDALVSFAYNNGSRALSTSTLL KKLNAGDIKGAADEFLRWNKSGGKVMPGLTNRRKAERALFLS

>SEQ ID NO: 9

MNKDQFIKATGITPALADKWYQCIVDTMKEFGIDTPKRQAHFLAQIGTESNGFRS

VQESLNYSVSGLQIFGSRLTEPQRQHLGRKPGEMALSPARQEAIANIVYGGRYGN

TQNGDGWKYRGRGLKQITFKDNYSACGKALNLDLVANPDLLLQDLNAARSAG WFWKANNCNQFADTGDVKGLTRRINGGFNGLQDRIDRTNKAEAVLK

>SEQ ID NO: 10 MVEINNQRKAFLDMLAWSEGTDNGRQKTRNHGYDVIVGGELFTDYSDHPRKLV TLNPKLKSTAAGRYQLLSRWWDAYRKQLGLKDFSPKSQDAVALQQIKERGALP MIDRGDIRQAIDRCSNIWASLPGAGYGQFEHKADSLIAKFKEAGGTVREIEV

>SEQ ID NO: 11

MAAILRMFSRGRDVIKLQQLLNKNVIISRLVEDGIFGSDTFNAVKIFQRHNGLND

YGIVGPETWQKLNRDPFVRRSSVGSSVGSSSLIERGKLTYDSEGNNIPGSIYYSRVI

HWPGTIFSGVTLGRGYDMGDRTESSILQDMLTAGIEADTARKISLARSYKGSAAG

AFVANNKRDIGEITEEQQINLFNHIYTDYISRTIWNYNRWTSDTTAAKHWDELDQ

PIQEVLIDFVYQGFTKGPRPMLAGSNNDKQELINYIRNTPGISRYEQGRHRADFLE RN

>SEQ ID NO: 12

MTRTLTHDQQQAAALYLGIPLAALQAVQEVEARSHGFLPDGRPALLFERHIMYR

QLKSHGLDADRLAQNYPELVNKSAGGYQGGSREHYRLNLAKQIHSTAAIESASW

GLFQIMGFHWKALGYASAADFEKQMNDSEQMQLDAFVRFVEANSKIHDAMKV QNWPEFARRYNGPQYKRNQYDTKLAIAFEKFSQAAA

>SEQ ID NO: 13

MSTFKPLKTLTSRRQVLKAGLAALTLSGMSQAIAKDEPLKTSNGHSKPKAKKSG

GKRVVVLDPGHGGIDTGAIGRNGSKEKHVVLAIAKNVRSILRNHGIDARLTRSGD

TFIPLYDRVEIAHKHGADLFMSIHADGFTNPKAAGASVFALSNRGASSAMAKYLS

ERENRADEVAGKKATDKDHLLQQVLFDLVQTDTIKNSLTLGSHILKKIKPVHKL HSRNTEQAAFVVLKSPSVPSVLVETSFITNPEEERLLGTAAFRQKIATAIAEGVISY FHWFDNQKAHSRKR

>SEQ ID NO: 14

MMNIRYPVRKADGRDYKNYDELLTDIRKNTHGWWLLGVNRYWHGGIHVGASS

SPASVLNQETPEKSVPLQFMMDGEVVAWRVNRDYAAIECYQERPLRQSGTFVLV

KSVYKPDEQDESSWLTLYQLYMHIAPLSEFPKRPLYRVTQKGHGVRMRKYSHH

DDSREIAPDVQANKHGHPKTLTQGDTLAVLQQKSFLLEQRPEPFALVQRLQDGK

PAGELFWVSMRPEYLEPDGQCYVCLPDWMHSALNHGVFDDVVVPPVPLKVMV

KAGDAVGFLGAQDLADEDNYPQIITTDYKAHIELLSLDEHVPDVVANMKGIKTG

KQFIKLKLKRPLYLRNGEGEESTFEQMSAITRADAGKIIPRDVTYPFTDKTGVTYF

QIRPHTWMHQNDVEQLSQHDLAGLNFHCIEAEHTTDFTRTLDECWLIDALKSIRS

HFDGEKGPQFAQAKMFYDSLIHNAENRRPPTPYPDKSLDQYLFGALHTNQMNIP

EYARRLIVKHDSSWHSTRDDARWSSVCKSRDESPVVKMANGGFLDATHWMDK

VPPFASQRSVWHFHPLEFPEILRSAEFPRTPINGVLTPIEFIHFYNGDKIDDTDYEE A

AKGLECEVAAIKAVAKTETGSYGSYFKFEDNDDYVPAILFERHHFHKYTNGKYD

QFEDISNPVAGGYGATSIQYAKLVKAYTLDKKAALKSASWGKFQILASNYATAG YASPEDFVFALSKSEKNQLKAFVSFIKADRVLLHSIRTKDWLSFAQRYNGPRQKG YDLKMERNYNALL

>SEQ ID NO: 15

MSLKFIDISNWQAGFPVSTAGIDAVIVKATEGVGFTDRCCDGFVQQAIARGLPWG

FYHFARDNGATAEADYFISQTENYFTAGIPVLDWEGVYDEHGNLIFNQPVEWVN

TFVRRVHEKTGVWPWIYANPWRFNQGGVEPNCGRWVAGYPAGGITDINYGMA NNLPASYDVGSVCAWQFSSSVRIPGYNGNVDGDVFYGDAAAWGKYANPNGAT APLPAPQPSTPQGSTLELACRVMRGEYGNGDARKSALGARYNEVQGFIDHIASAS ASALAEEVKAGKYGNGDVRKQALGSRYNEVQAIVNGGNASAGRTYTVKAGDT

LSGIAAKYGTSYQVLAQINGIANPNLIYAGQTIKLP

>SEQ ID NO: 16

MEINTEIAIAWMSARQGKVSYSMDYRDGPNSYDCSSSVYYALRSAGASSAGWA

VNTEYMHDWLIKNGYELIAENVDWNAVRGDIAIWGMRGHSSGAGGHVVMFIDP

ENIIHCNWANNGITVNNYNQTAAASGWMYCYVYRLKSGASTQGKSLDTLVKET

LAGNYGNGEARKAVLGNQYEAVMSVINGKTTTNQKTVDQLVQEVIAGKHGNG

EARKKSLGSQYDAVQKRVTELLKKQPSEPFKAQEVNKPTETKTSQTELTGQATA

TKEEGDLSFNGTILKKAVLDKILGNCKKHDILPSYALTILHYEGLWGTSAVGKAD

NNWGGMTWTGQGNRPSGVTVTQGSARPSNEGGHYMHYASVDDFLTDWFYLLR

AGGSYKVSGAKTFSEAIKGMFKVGGAVYDYAASGFDSYIVGASSRLKAIEAENG

SLDKFDKATDIGDGSKDKIDITIEGIEVTINGITYELTKKPV