WO2023178352A2 - Peptides de lysine et peptides gp38 pour la détection de bactéries - Google Patents

Peptides de lysine et peptides gp38 pour la détection de bactéries Download PDF

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WO2023178352A2
WO2023178352A2 PCT/US2023/064713 US2023064713W WO2023178352A2 WO 2023178352 A2 WO2023178352 A2 WO 2023178352A2 US 2023064713 W US2023064713 W US 2023064713W WO 2023178352 A2 WO2023178352 A2 WO 2023178352A2
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peptide
gram
bacteria
amino acid
cell wall
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PCT/US2023/064713
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WO2023178352A3 (fr
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Ramya Ramadoss
Annette Shoba VINCENT
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Carnegie Mellon University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/245—Escherichia (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/25—Shigella (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/255—Salmonella (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/26—Klebsiella (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/305—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F)
    • G01N2333/31—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/315—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Streptococcus (G), e.g. Enterococci
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/335—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Lactobacillus (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)

Definitions

  • the present disclosure relates generally to the field of bacteria detection and more specifically to peptide sequences derived from conserved regions of bacteriophages specific to a family of bacteria that includes pathogenic strains and the use of these peptides for detection of gram-negative and gram-positive bacteria.
  • Wastewater treatment plants facilitate rapid discovery of bacteriophages predacious towards the native bacteria and could be enlisted for detection and biocontrol of gram-positive and gram-negative bacteria.
  • the present disclosure relates to peptide sequences derived from bacteriophages specific to a particular family of gram-negative or gram-positive bacteria among which pathogenic strains exist.
  • the present disclosure relates to peptide sequences capable of detecting gram-negative and gram-positive bacteria.
  • the peptides of the present disclosure can individually or in combination recognize and bind to ligands on the cell wall surface of gram-negative and gram-positive bacteria.
  • the present disclosure provides a method of detecting gram-negative and grampositive bacteria in aqueous samples.
  • the peptides of the present disclosure may be fused with a reporter tag via a linker amino acid sequence.
  • the tagged peptides of the present disclosure may then be individually or in combination added to any aqueous sample.
  • the sample may then be visually examined to determine the presence of gram-negative and gram-positive bacteria.
  • the present disclosure further provides a kit or tool for detection of gram-negative and gram -positive bacteria.
  • the kit or tool may contain individual or multiple tagged peptides of the present disclosure .
  • the kit or tool may measure an aqueous sample for the presence of gram-negative and grampositive bacteria.
  • the kit or tool may be configured to detect which strains of gram -negative and grampositive bacteria are present in any aqueous sample.
  • FIG. 1 shows the sequences of the GP38 and lysin peptides of the present disclosure and the bacteriophages they were derived from, wherein the critical amino acids important for binding to at least one ligand on the cell wall surface of gram-negative and gram-positive bacteria are shown in bold.
  • FIG. 2A shows the sequences for the N-terminal gp37 attachment domain ( - ) and the C-terminal PGII sandwich domain ( - ) attached by an EFAT linker sequence ( — ) in the GP38 peptide of SEQ ID NO:5.
  • FIG. 2B illustrates a model of the GP38 peptide of SEQ ID NO: 5 fused with the N- terminus of GFP protein via a linker amino acid sequence.
  • FIG. 3 represents a three-dimensional model of the GP38 peptide sequence SEQ ID NO: 5 fused with a linker to GFP protein.
  • the N-terminus is marked as N and the C-terminus is marked as C.
  • FIG. 4 represents a three-dimensional model of the protein-ligand complex formed after binding of the GP38 peptide sequence of SEQ ID NO: 5 with the Tetrasaccharide ligand on a bacteria cell wall.
  • the GP38 peptide of SEQ ID NO: 5 is fused with GFP protein and a linker sequence.
  • FIG. 5 represents a three-dimensional model of the protein-ligand complex formed after binding of the GP38 peptide sequence of SEQ ID NO: 5 with the Wall Technoic Acid ligand on a bacteria cell wall.
  • the GP38 peptide of SEQ ID NO: 5 is fused with GFP protein and a linker sequence.
  • FIG. 6A shows the sequence of a nonspecific peptide, i.e., GFP fused polyalanine peptide sequence via an EFAT linker
  • FIGS. 6B and 6C illustrate ITASSER modelled structures of the nonspecific peptide used to evaluate binding specificity of GFP fused lysin peptides.
  • FIG. 7A shows the sequence of a nonspecific peptide, i.e., GFP fused polyalanine peptide sequence via an EFAT linker, wherein the peptide has extra amino acids (EFQHTGGRY) added
  • FIGS. 7B and 7C illustrate ITASSER modelled structures of the nonspecific peptide used to evaluate binding specificity of GFP fused GP38 peptides.
  • FIG. 8A illustrates the ITASSER modelled structure of CL 1-C600M2 -Lysin with labeled N and C-terminal regions and the catalytic triad Glu(E)-Asp(D)-Thr(T).
  • FIG. 8B shows the Z- score plot generated by ProSA for predicted model quality assessment.
  • FIG. 8C illustrates the superposition of CLl-C600M2-Lysin with AcLys and the catalytic triad. The C-terminal a-helix enriched with positively charged residues is also shown.
  • FIG. 9 shows the phylogenetic tree of phage lysin protein sequences derived from CL 1 - C600M2-Lysin.
  • FIGS. 10A, 10B, and 10C illustrate the 3-Dimensional structure of CL1-C600M2- Lysin complexed with WTA, TS, and PG, respectively. Interacting residues are denoted as sticks and further detailed based on the represented conservation scale with 9 being highly conserved and 1 being highly variable. N and C terminals of each structure is indicated by N and C, respectively.
  • FIGS. 10D, 10E, 10F illustrate Ligplot maps for interacting residues for protein-ligand complexes. The sugar components of PG, P ⁇ (l, 4) linked N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) are labeled.
  • FIG. HA represents a turbidity reduction assay of Escherichia coli strains C, C600, B/R, and HB101.
  • FIG. 11B represents the same strains with 80 ng of purified CL 1-C600M2 -Lysin.
  • FIGS. 12A, 12B, and 12C illustrate the 3 -Dimensional structure of CL1-C600M2- Lysin complexed with ligands WTA, TS, and PG, respectively.
  • FIG. 13 is a summarized list of lysin sequences obtained from a corresponding environment.
  • FIG. 14 shows a Ramachandran Plot generated by PROCHECK for CL1-C600M2- Lysin protein model quality validation.
  • FIG. 15 represents multiple sequence alignment of protein-blast hits along with the residues of CLl-C600M2-Lysin’s interaction with WTA, TS, and PG.
  • FIG. 16 illustrates gel electrophoresis verification of the insertion of CL1-C600M2- Lysin.
  • Lane 4 represents the PCR control.
  • FIG. 17 illustrates confirmation by SDS-PAGE expression and the purity of CL1- C600M2 -Lysin protein.
  • FIG. 18 illustrates a bead-based method of testing peptide binding to planktonic bacteria.
  • FIG. 19 demonstrates binding efficacy of C-terminal GFP tagged lysin peptide of SEQ ID NO: 46 to E. coli C cells as measured by fluorescence microscopy (FIG. 19A).
  • FIG. 19B demonstrates E. coli C and emGFP as a positive control.
  • FIG. 19C demonstrates E. coli C only.
  • FIG. 19D demonstrates E. coli C and GFP -tagged polyaniline.
  • FIG. 20 demonstrates E. coli C with emGFP (FIG. 20A), E. coli C600 with emGFP (FIG. 20B), and E. coli B/R with emGFP (FIG. 20C) under confocal microscopy using transmitted light and a GFP filter.
  • FIG. 21 demonstrates the binding efficacy of a GFP tagged lysin peptide of SEQ ID NO: 46 to E. coli C biofilm bound to either the C-terminal (FIG. 21A) or N-terminal (FIG. 21B) of the GFP tagged lysin peptide as verified under confocal microscopy under transmitted light and a GFP filter.
  • FIG. 22 demonstrates the binding efficacy of a GFP tagged lysin peptide of SEQ ID NO: 46 to E. coli C600 biofilm bound to either the C-terminal (FIG. 22A) or N-terminal (FIG. 22B) of the GFP tagged lysin peptide as verified under confocal microscopy under transmitted light and a GFP filter.
  • FIG. 23 demonstrates the binding efficacy of a GFP tagged lysin peptide of SEQ ID NO: 46 to E. coli B/R biofilm bound to either the C-terminal (FIG. 23A) or N-terminal (FIG. 23B) of the GFP tagged lysin peptide as verified under confocal microscopy under transmitted light and a GFP filter.
  • FIG. 24 represents a gel imaged under UV light of 16R PCR results of SEQ ID NO: 46 after immunomagnetic bead assays.
  • FIG. 25 demonstrates binding efficacy of a C-terminal GFP tagged GP38 peptide of SEQ ID NO: 5 to E. coli C cells as measured by fluorescence microscopy using fluorescent and transmitted imaging (FIG. 25A).
  • FIG. 25B demonstrates E. coli C cells only as a negative control.
  • FIG. 25C demonstrates E. coli C and GFP only.
  • FIG. 26 demonstrates E. coli C with emGFP (FIG. 26A), E. coli C600 with emGFP (FIG. 26B), and E. coli B/R with emGFP (FIG. 26C) under confocal microscopy using transmitted light and a GFP filter.
  • FIG. 27 demonstrates the binding efficacy of a GFP tagged GP38 peptide of SEQ ID NO: 5 to E. coli C biofilm bound to either the C-terminal (FIG. 27A) or N-terminal (FIG. 27B) of the GFP tagged GP38 peptide as verified under confocal microscopy using transmitted light and a GFP filter.
  • FIG. 28 demonstrates the binding efficacy of a GFP tagged GP38 peptide of SEQ ID NO: 5 to E. coli C600 biofilm bound to either the C-terminal (FIG. 28A) or N-terminal (FIG. 28B) of the GFP tagged GP38 peptide as verified under confocal microscopy using transmitted light and a GFP filter.
  • FIG. 29 demonstrates the binding efficacy of a GFP tagged GP38 peptide of SEQ ID NO: 1
  • FIG. 30 represents a gel imaged under UV light of 16R PCR results of purified C- terminal lysin peptide of SEQ ID NO: 46 (FIG. 30A) and N-terminal GP38 peptide of SEQ ID NO: 5 (FIG. 30B) of the present disclosure after immunomagnetic bead assays.
  • SEQ ID NOS:l-9 are GP38 peptide sequences according to the present disclosure.
  • SEQ ID NOS: 10-46 are lysin peptide sequences according to the present disclosure.
  • the present disclosure provides peptides capable of binding to at least one cell wall carbohydrate ligand on a cell wall surface of a bacteria, including, but not limited to, Wall Teichoic Acid and Tetrasaccharides.
  • polypeptide “peptide”, and “protein” are typically used interchangeably herein to refer to a polymer of amino acid residues.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC- IUB Biochemical Nomenclature Commission.
  • the present disclosure includes peptides and functional fragments thereof, as well as mutants and variants having substantial similarity and/or having the same biological function or activity.
  • “Peptide” and “polypeptide” may refer to a peptide or polypeptide that contains 5 or more amino acids, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acids, including but not limited to, 5 - 10 amino acids, 5-20, 5-30, 5-40, 5-60, 5-70, 5-80, 5-90, 5-100, 5-200, 5-300, or 5-400 amino acids. The terms also refer to higher molecular weight polypeptides, such as proteins.
  • the present disclosure relates to peptide sequences derived from bacteriophages specific to a particular family of gram-negative or gram-positive bacteria among which pathogenic strains exist.
  • FIG. 1 details the bacteriophages from which the novel peptide sequences of the present disclosure were derived.
  • the present disclosure relates to a GP38 peptide sequence, a label bound to the GP38 peptide, and a GP38 peptide having a label attached via a linker.
  • the GP38 peptide may comprise at least the amino acids bolded in each of SEQ ID NOS: 1-9 as shown in FIG. 1.
  • the GP38 peptide may comprise an amino acid sequence as set forth in any one of SEQ ID NOS: 1-9.
  • the present disclosure may also comprise an unlabeled GP38 peptide sequence comprising at least the amino acids bolded in each of SEQ ID NOS: 1-9 as shown in FIG. 1.
  • the unlabeled GP38 peptide may comprise an amino acid sequence as set forth in any one of SEQ ID NOS: 1-9.
  • the C-terminal domain of the peptide of the present disclosure may comprise ten conserved glycine-rich motifs interspersed by hypervariable segments. As demonstrated in FIGS. 2A- 2B, the glycine-rich motifs may fold into polyglycine type II helices and assemble into a buried threelayered polyglycine type II sandwich domain.
  • the hypervariable segments may determine host receptor specificity by forming 180-degree P-tum loops interspersing the ends of polyglycine type II helices, which are highly accessible for potential receptor interaction at the ends of the polyglycine sandwich.
  • the C-terminal domain of the peptide may allow for the detection of gram-negative and gram-positive bacteria.
  • the present disclosure also relates to a lysin peptide sequence, a label bound to the lysin peptide, and a lysin peptide having a label attached via a linker.
  • the lysin peptide may comprise at least those amino acids bolded in each of SEQ ID NOS : 10-46 as shown in FIG. 1.
  • the lysin peptide may comprise an amino acid sequence as set forth in any one of SEQ ID NOS: 10-46.
  • the present disclosure may also comprise an unlabeled lysin peptide sequence comprising at least the amino acids bolded in each of SEQ ID NOS: 10-46 as shown in FIG. 1.
  • the unlabeled lysin peptide may comprise an amino acid sequence as set forth in any one of SEQ ID NOS: 10-46.
  • any of the peptides of the present disclosure may include a linker sequence that provides attachment of a label such as a reporter tag.
  • the linker sequence may be any known in the art for attaching a label to a peptide.
  • Exemplary linker sequences comprise short peptides, such as peptides having 3 or more amino acids, such as at least 3 amino acids and up to 100 amino acids.
  • the linker sequences may include short stretches of repeating amino acids, such as amino acids that form a-helices or other three-dimensional structures (e.g., beta-pleats, hairpin turns, and the like).
  • the linker sequence may comprise at least 3 or 4 amino acids, such as the linker sequence EFAT (see FIGS. 2A and 3A).
  • the label may include any label capable of fusion with the linker sequence or directly to the peptides.
  • the label of the present disclosure may be capable of qualitative or quantitative detection.
  • the fusion of the linker and/or label has substantially no effect of the ability of the peptide to bind to Wall Teichoic Acid and/or Tetrasaccharide on a cell wall surface.
  • a preferred label is a fluorescent reporter tag, such as GFP protein.
  • the peptide of the present disclosure as set forth in any one of SEQ ID NOS: 1-46 may be fused with the N-terminus of GFP protein via a linker amino acid sequence described hereinabove.
  • the peptides of the present disclosure as set forth in any one of SEQ ID NOS: 1-46 may be capable of binding to Wall Technoic Acid on a cell wall surface.
  • the C-terminal cell wall binding region of each peptide may bind to Wall Technoic Acid to form a protein-ligand complex (see FIG. 5)
  • the peptides of the present disclosure as set forth in any one of SEQ ID NOS: 1-46 may be capable of binding to Tetrasaccharide on a cell wall surface.
  • the C-terminal cell wall binding region of each peptide may bind to Tetrasaccharide to form a protein-ligand complex (see FIG. 4), while the N-terminal region of each peptide provides catalytic activity.
  • the peptides of the present disclosure as set forth in any one of SEQ ID NOS: 1-46 may be capable of binding to Wall Technoic Acid and/or Tetrasaccharide on a cell wall surface of at least one bacteria.
  • the present inventors have defined regions of the peptides that are key to recognition and/or binding to the cell wall of bacteria. Those key residues, or cell wall binding region of the peptides of the present disclosure comprise of at least those amino acid sequences bolded in SEQ ID NOS: 1-46 (see FIG. 1). Accordingly, the present disclosure also relates to peptides comprising at least the amino acids that are bolded in SEQ ID NOS: 1-46 of FIG. 1. The present disclosure also relates to a fragment thereof or a polypeptide that is a functionally equivalent variant or a functionally active variant of the amino acid sequence of SEQ ID NOS: 1-46 or the fragment thereof.
  • the present disclosure also relates to a fragment thereof or a polypeptide that is a functionally equivalent variant or a functionally active variant of the amino acid sequence of SEQ ID NOS: 1-46 or the fragment thereof, wherein the amino acids that are bolded in SEQ ID NOS: 1-46 of FIG. 1 are conserved or have at least 95, 96, 97, 98, or 99% amino acid sequence identity.
  • the present disclosure also relates to a fragment thereof or a polypeptide that is substantially similar to the amino acid sequence of SEQ ID NO: 1-46 or the fragment thereof.
  • the present disclosure also relates to a fragment thereof or a polypeptide that is substantially similar to the amino acid sequence of SEQ ID NOS: 1-46 or the fragment thereof, wherein the amino acids that are bolded in SEQ ID NOS: 1-46 of FIG. 1 are conserved.
  • peptides as forth in any one of SEQ ID NOS: 1-46 may comprise amino acid substitutions that have little to no effect on the recognition and/or binding of the peptides to the bacterial cell wall (e.g., conservative substitutions). For example, conservative substitutions outside of the bolded regions in the peptides shown in FIG. 1 are possible.
  • a polypeptide sequence is considered to be “homologous” to one another if the percent amino acid sequence identity is at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%.
  • the present disclosure also provides methods of detecting gram-negative and grampositive bacteria in aqueous samples.
  • a peptide as set forth in any one of SEQ ID NOS: 1-46 may be fused with a label and added individually or in combination with another peptide of the present disclosure to an aqueous sample.
  • the labeled peptide(s) may be added to one or more aqueous samples and measured depending on the type of label used.
  • the preferred label is a fluorescent reporter tag such as GFP protein, a detectable level of fluorescence indicates the presence of bacteria.
  • Binding of the labeled peptide(s) to bacteria may result in aggregation of the labeled peptide(s) on the cell wall surface, resulting in fluorescent spots indicative of bacteria.
  • the aqueous sample may be measured using microscopic analysis or any other type of suitable qualitative or quantitative detection.
  • the presence of multiple peptides may enable detection of multiple strains of gram-negative and/or gram-positive bacteria in one or more aqueous samples.
  • the methods of the present disclosure may also be configured to provide determination of only gram-negative or gram-positive bacteria, levels of the gram-negative and/or gram-positive bacteria, and even specific strains of gram-negative and/or grampositive bacteria present in any aqueous sample.
  • the present disclosure also provides a kit or tool for detection of gram-negative and gram-positive bacteria.
  • the kit or tool may include one or more peptides as set forth in any one of SEQ ID NOS: 1-46.
  • the peptides may be labeled, such as with a reporter tag as described herein, e.g., the label may be attached directly to the peptide or linked via a linker.
  • the kit or tool may include one or more peptides and one or more labels that may be combined to provide a labeled peptide, and optionally a linker as described herein.
  • An aqueous sample may be tested using the kit or tool of the present disclosure and measured for the presence of gram-negative and/or gram- positive bacteria.
  • kits and tools of the disclosure may also include instructions and additional reagents useful for executing the methods for detection.
  • the kit or tool of the present disclosure may be configured to detect only gram-positive or gram-negative bacteria, or even specific strains of bacteria present in an aqueous sample.
  • the kits and tools may be configured to detect a level of bacterial contamination in an aqueous sample.
  • the kits and tools may further comprise standards that allow quantification of the bacterial content in the sample.
  • the present disclosure provides peptides capable of binding indiscriminately to bacteria in the planktonic and biofilm state.
  • the present disclosure also provides peptides capable of binding to the cell wall of a specific gram-negative or gram-positive bacteria in the planktonic and biofilm state.
  • the term “and/or” includes any and all combinations of one or more of the associated listed items.
  • a term “or” is intended to mean an inclusive “or” rather than an exclusive “or.” That is, unless specified otherwise, or clear from context, “X employs A or B” is intended to mean any of the natural inclusive permutations. That is, if X employs A; X employs B; or X employs both A and B, then “X employs A or B” is satisfied under any of the foregoing instances.
  • bacteria refers to a domain of prokaryotic organisms. Bacteria include at least 11 distinct groups as follows: (1) Gram -positive (gram+) bacteria, of which there are two major subdivisions: (i) high G+C group (Actinomycetes, Mycobacteria, Micrococcus, others) (ii) low G+C group (Bacillus, Clostridia, Lactobacillus, Staphylococci, Streptococci, Mycoplasmas); (2) Proteobacteria, e.g., Purple photosynthetic+non-photosynthetic Gram -negative bacteria (includes most “common” Gram-negative bacteria); (3) Cyanobacteria, e.g., oxygenic phototrophs; (4) Spirochetes and related species; (5) Planctomyces,- (6) Bacteroides, Flavobacteria; (7) Chlamydia,' (8) Green sulfur bacteria; (
  • Gram-negative bacteria includes, but is not limited to cocci, nonenteric rods, and enteric rods.
  • the genera of Gram-negative bacteria include, for example, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, and Fusobacterium.
  • Gram-positive bacteria includes cocci, nonsporulating rods, and sporulating rods.
  • the genera of Gram-positive bacteria include, but is not limited to, Actinomyces, Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus, and Streptomyces.
  • label refers to any atom or molecule which may be used to provide a detectable or quantifiable signal.
  • a “label” may include a reporter tag, such as Green Fluorescent Protein (GFP). Labels may provide signals by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, mass spectrometry, binding affinity, hybridization radiofrequency, nanocrystals, and the like. “Qualitative or quantitative” detection refers to visual or automated assessments based upon the magnitude (strength) or number of signals generated by the label.
  • the term “substantial similarity” when referring to a peptide or fragment thereof, indicates that, when optimally aligned with appropriate amino acid insertions or deletions, there is amino acid sequence identity in at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or at least about 90%, or at least about 95%, 96%, 97%, 98%, or 99% of the amino acid residues, as measured by any well-known algorithm of sequence identity, such as BLAST.
  • the term “functionally equivalent variant” or “functionally active variant” of a peptide of the present disclosure refers to a sequence resulting from modification of the sequence of a peptide of the present disclosure by insertion, deletion, or substitution of one or more amino acids or nucleotides within the sequence or at either or both distal ends of the sequence, and which modification does not affect (in particular impair) the binding of the sequence.
  • the functionally active peptide variant as used according to this disclosure would still have the predetermined binding specificity, though this could be changed e.g., to change the fine specificity to a specific binding site in a bacterium.
  • the functionally active variant a) comprises the critical amino acids necessary for binding to the cell wall of a bacteria; b) is derived from the peptide and comprises at least one amino acid substitute, addition, and/or deletion, wherein the functionally active variant has a sequence identity to the critical amino acids of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and at least 95%, 96%, 97%, 98%, and 99%.
  • the functionally active variant may be obtained by sequence alterations in the peptide sequence, wherein the sequence alterations retain a function of the unaltered peptide sequence. Such sequence alterations may include, but are not limited to, (conservative) substitutions, additions, deletions, mutations, and insertions.
  • the variant of the peptide is functionally active in the context of this disclosure if the activity of the peptide amounts to at least 10%, preferably at least 25%, more preferably at least more preferably 50%, even more preferably at least 70%, still more preferably at least 80%, especially at least 90%, particularly at least 95%, and most preferably at least 99% of the biological activity of the peptide as used according to the present disclosure, including the peptide sequence without sequence alteration (i.e. the original peptide).
  • Conservative substitutions are those that take place within a family of amino acids that are related in their side chains and chemical properties. Examples of such families are amino acids with basic side chains, with acidic side chains, with non-polar aliphatic side chains, with non-polar aromatic side chains, with unchanged polar side chains, with small side chains, with large side chains, etc.
  • the peptide sequence as defined above may be modified by a variety of chemical techniques to produce derivatives having essentially the same activity (as defined above for fragments and variants) as the peptide, and optionally having other desirable properties.
  • percent (%) amino acid sequence identity with respect to the peptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide sequence, after aligning the sequence and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • Those skilled in the art may determine appropriate parameters for measuring alignment, including any algorithms such as BLAST needed to achieve maximal alignment over the full length of the sequences being compared.
  • compositions, materials, components, elements, features, integers, operations, and/or process steps described herein also specifically includes embodiments consisting of, or consisting essentially of, such recited compositions, materials, components, elements, features, integers, operations, and/or process steps.
  • the alternative embodiment excludes any additional compositions, materials, components, elements, features, integers, operations, and/or process steps
  • any additional compositions, materials, components, elements, features, integers, operations, and/or process steps that materially affect the basic and novel characteristics may be excluded from such an embodiment, but any compositions, materials, components, elements, features, integers, operations, and/or process steps that do not materially affect the basic and novel characteristics may be included in the embodiment.
  • the term “about” refers to values within an order of magnitude, potentially within 5 -fold or 2-fold of a given value. Notwithstanding the approximations of numerical quantities stated herein, the numerical quantities described in specific examples of actual measured values may be reported as precisely as possible. Any numerical values, however, inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
  • Aspect 1 A peptide capable of binding at least one cell wall carbohydrate ligand on a cell wall surface of at least one bacteria, wherein the peptide comprises the amino acid sequence as set forth in any one of SEQ IDS NO: 1-46.
  • Aspect 2 The peptide according to aspect 1, wherein the at least one cell wall carbohydrate ligand is at least one Tetrasaccharide.
  • Aspect 3 The peptide according to any of the foregoing aspects, wherein the at least one cell wall carbohydrate ligand is at least one Wall Teichoic Acid.
  • Aspect 4 The peptide according to any of the foregoing aspects, wherein the at least one cell wall carbohydrate ligand is a combination of at least one Tetrasaccharide and at least one Wall Teichoic Acid.
  • Aspect 5 The peptide according to any of the foregoing aspects, wherein the at least one bacteria is a gram-positive bacteria.
  • Aspect 6 The peptide according to any of the foregoing aspects, wherein the at least one bacteria is a gram-negative bacteria.
  • Aspect 7 The peptide according to any of the foregoing aspects, wherein the at least one bacteria is a combination of at least one gram-positive bacteria and at least one gram-negative bacteria.
  • Aspect 8 The peptide according to any of the foregoing aspects, wherein the grampositive bacteria are Staphlyococcus, Streptococcus, Enterococcus, Lactococcus, Lactobacillus, and Mycobacterium.
  • Aspect 9 The peptide according to any of the foregoing aspects, wherein the gramnegative bacteria are Pseudomonas, Enterobacter, Salmonella, Escherichia coli, Klebsiella, Shigellae, and Citrobacter.
  • Aspect 10 The peptide according to any of the foregoing aspects, wherein the peptide comprises a label forming a labeled peptide.
  • Aspect 11 The peptide according to any of the foregoing aspects, wherein the label is attached to at least one peptide via a linker amino acid sequence.
  • Aspect 12 The peptide according to any of the foregoing aspects, wherein the linker amino acid sequence comprises at least 3 or more amino acids.
  • Aspect 13 The peptide according to any of the foregoing aspects, wherein the label is GFP protein.
  • Aspect 14 The peptide according to any of the foregoing aspects, wherein the peptide is fused with the N-terminus of GFP protein via a linker amino acid sequence.
  • Aspect 15 The peptide according to any of the foregoing aspects, wherein the peptide comprises at least the amino acids bolded in FIG. 1.
  • Aspect 16 The peptide according to any of the foregoing aspects, wherein the peptide is capable of binding to Tetrasaccharides on a cell wall surface, wherein the peptide comprises at least the amino acids bolded in FIG. 1.
  • Aspect 17 The peptide according to any of the foregoing aspects, wherein the peptide is capable of binding to Wall Teichoic Acid and Tetrasaccharides on a cell wall surface, wherein the peptide comprises at least those amino acids bolded in SEQ ID NOS: 1-46 as shown in FIG. 1.
  • Aspect 18 The peptide according to any of the foregoing aspects, wherein the label is selected from any label capable of fusion with the peptide directly or via a linker sequence, wherein the label is selected from a group consisting of GFP protein, superfolder green fluorescent protein, alkaline phosphatase, DsRed-Express2, citrine, red fluorescent protein (RFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), mCherry , or any of the site specific labeling systems disclosed in Lotze et al. (Lotze et al., 2016, Peptide-tags for site specific labelling in vitro and in vivo, Mol. BioSyst. 12, 1731).
  • a method of detecting bacteria comprising: providing at least one peptide comprising the amino acid sequence as set forth in any one of SEQ ID NOS: 1-46; attaching a label to the at least one peptide to form a labeled peptide; adding the labeled peptide to an aqueous sample; and detecting a qualitative or quantitative signal in the aqueous sample, wherein the signal indicates a presence of at least one gram-positive or at least one gram-negative bacteria.
  • Aspect 20 The method of aspect 19, wherein the labeled peptide binds to at least one cell wall carbohydrate ligand of the at least one gram-positive or at least one gram-negative bacteria.
  • Aspect 21 The method according to any of the foregoing aspects, wherein the at least one cell wall carbohydrate ligand is at least one Tetrasaccharide.
  • Aspect 22 The method according to any of the foregoing aspects, wherein the at least one cell wall carbohydrate ligand is at least one Wall Teichoic Acid.
  • Aspect 23 The method according to any of the foregoing aspects, wherein the at least one cell wall carbohydrate ligand is a combination of at least one Tetrasaccharide and at least one Wall Teichoic Acid.
  • Aspect 24 The method according to any of the foregoing aspects, wherein the label is attached to the at least one peptide via a linker amino acid sequence.
  • Aspect 25 The method according to any of the foregoing aspects, wherein the linker amino acid sequence comprises at least 3 or more amino acids.
  • Aspect 26 The method according to any of the foregoing aspects, wherein the label is GFP protein.
  • Aspect 27 The method according to any of the foregoing aspects, wherein the peptide is fused with the N-terminus of GFP protein via a linker amino acid sequence.
  • Aspect 28 The method according to any of the foregoing aspects, wherein the quantitative signal is by fluorescence, radioactivity, colorimetry, binding affinity, or mass spectrometry.
  • Aspect 29 The method according to any of the foregoing aspects, wherein the qualitative signal is a visual change in color.
  • Aspect 30 The method according to any of the foregoing aspects, wherein the grampositive bacteria are Staphlyococcus, Streptococcus, Enterococcus, Lactococcus, Lactobacillus, and Mycobacterium.
  • Aspect 31 The method according to any of the foregoing aspects, wherein the gramnegative bacteria are Pseudomonas, Enterobacter, Salmonella, Escherichia coli, Klebsiella, Shigellae, and Citrobacter.
  • Aspect 32 A kit comprising: at least one labeled peptide comprising an amino acid sequence as set forth in any one of SEQ ID NOS: 1-46, wherein the at least one labeled peptide is added to an aqueous sample, wherein the aqueous sample is measured for a qualitative signal or a quantitative signal; wherein the kit detects the presence of gram-negative and gram-positive bacteria.
  • Aspect 33 The kit of aspect 32, wherein the qualitative signal is a visual change in color.
  • Aspect 34 The kit according to any of the foregoing aspects, wherein the quantitative signal is a signal resulting from fluorescence, radioactivity, colorimetry, binding affinity, or mass spectrometry.
  • Aspect 35 The kit according to any of the foregoing aspects, comprising at least two labeled peptides comprising an amino acid sequence as set forth in any one of SEQ ID NOS: 1-46.
  • Aspect 36 The kit according to any of the foregoing aspects, wherein the gram -positive bacteria are Staphlyococcus, Streptococcus, Enterococcus, Lactococcus, Lactobacillus, and Mycobacterium .
  • Aspect 37 The kit according to any of the foregoing aspects, wherein the gram- negative bacteria are Pseudomonas, Enterobacter, Salmonella, Escherichia coli, Klebsiella, Shigellae, and Citrobacter.
  • Aspect 38 A peptide or fragment thereof capable of binding at least one cell wall carbohydrate ligand on a cell wall surface of at least one bacteria, wherein the peptide is a functionally equivalent variant of the amino acid sequence as set forth in any one of SEQ IDS NO: 1-46.
  • Aspect 39 A peptide or fragment thereof capable of binding at least one cell wall carbohydrate ligand on a cell wall surface of at least one bacteria, wherein the peptide is a functionally equivalent variant of the amino acid sequence as set forth in any one of SEQ IDS NO: 1-46, and wherein the bolded amino acids in FIG. 1 are conserved or have at least 95, 96, 97, 98, or 99 percent amino acid sequence identity.
  • Aspect 40 A peptide or fragment thereof capable of binding at least one cell wall carbohydrate ligand on a cell wall surface of at least one bacteria, wherein the peptide is a functionally active variant of the amino acid sequence as set forth in any one of SEQ IDS NO: 1-46.
  • Aspect 41 A peptide or fragment thereof capable of binding at least one cell wall carbohydrate ligand on a cell wall surface of at least one bacteria, wherein the peptide is a functionally active variant of the amino acid sequence as set forth in any one of SEQ IDS NO: 1-46, and wherein the bolded amino acids in FIG. 1 are conserved or have at least 95, 96, 97, 98, or 99 percent amino acid sequence identity.
  • Aspect 42 A peptide or fragment thereof capable of binding at least one cell wall carbohydrate ligand on a cell wall surface of at least one bacteria, wherein the peptide is substantially similar to the amino acid sequence of SEQ ID NOS: 1-46.
  • Aspect 42 A peptide or fragment thereof capable of binding at least one cell wall carbohydrate ligand on a cell wall surface of at least one bacteria, wherein the peptide is substantially similar to the amino acid sequence of SEQ ID NOS: 1-46, and wherein the amino acids that are bolded in SEQ ID NOS: 1-46 in FIG. 1 are conserved.
  • Aspect 43 A peptide according to any of the foregoing aspects, comprising at least one conservative substitution outside of the bolded regions in SEQ ID NOS: 1-46 as shown in FIG. 1.
  • Aspect 44 A peptide according to any of the foregoing aspects, comprising at least one conservative substitution outside of the bolded regions in SEQ ID NOS: 1-46 as shown in FIG. 1, wherein the peptide is homologous to the peptide of SEQ ID NOS: 1-46.
  • Aspect 45 A peptide according to any of the foregoing aspects, comprising at least one conservative substitution inside of the bolded regions in SEQ ID NOS: 1-46 in FIG. 1, wherein the peptide is a functionally equivalent variant or a functionally active variant of SEQ ID NOS: 1-46.
  • Aspect 46 A peptide according to any of the foregoing aspects, wherein the peptide is modified by a chemical technique, wherein the peptide is a derivative of SEQ ID NOS: 1-46, and wherein the peptide is a functionally equivalent variant or a functionally active variant of SEQ ID NOS: 1-46.
  • Aspect 47 A peptide according to any of the foregoing aspects, comprising at least one substitution, addition, deletion, mutation, or insertion, wherein the peptide is a functionally equivalent variant or a functionally active variant of SEQ ID NOS: 1-46.
  • Aspect 48 A peptide according to any of the foregoing aspects, comprising at least one substitution, addition, deletion, mutation, or insertion, wherein the peptide is a functionally equivalent variant or a functionally active variant of SEQ ID NOS: 1-46, and wherein the functionally active variant has a percent amino acid sequence identity to the critical amino acids bolded in FIG. 1 of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and at least 95%, 96%, 97%, 98%, and 99% amino acid sequence identity.
  • GFP protein was fused with a poly alanine peptide sequence via an SGLRS linker sequence (see FIG. 7A). Extra amino acids were added at the end of the poly alanine sequence. As shown in FIGS. 7B and 7C, no significant interaction of the tagged peptide with Tetrasaccharide or Wall Teichoic Acid was found.
  • Lysin protein sequences derived from the genomes of Escherichia Phage C600M2 (Protein ID: UCJ01465) and Escherichia Phage CL1 (Protein ID: UCJ01321) were identical (FIG. 13). Both lysin protein sequences were termed as CL 1-C600M2 -Lysin hereafter. Domain Analysis of CL1- C600M2-Lysin was done using conserveed Domain Database (CDD) (Lu et al., 2020). Further, the protein sequence of CL 1-C600M2 -Lysin was subjected to protein-blast (accessed on 13 October 2021) against Refseq Protein database to identify homologous proteins.
  • CDD conserveed Domain Database
  • SH-alRT Shimodaira-Hasegawa like approximate likelihood ratio test
  • SBS Standard bootstrap support
  • the Standard bootstrap support (SBS) values for the ML analysis was estimated by concatenation of the generated bootstrap trees after 100 iterations with same alignment and substitution model mentioned above.
  • a consensus tree using the original MUSCLE alignment input file was created.
  • the support values UF/SH- aLRT/SBS were mapped to the ML tree and further annotated using Interactive Tree Of Life (iTOL) online tool (Letunic and Bork, 2021).
  • iTOL Interactive Tree Of Life
  • the interacting residues were identified using LigPlot V .2.2 (Laskowski and Swindells, 2011) and PDBsum (Laskowski et al., 2018) and visualized using Chimera (Pettersen et al., 2004) and PyMOL (Schrodinger, LLC) visualization software.
  • the docked proteinligand complexes were also visualized by embedding the ConSurf (Glaser et al., 2003; Landau et al., 2005) output derived by using the protein model of CLl-C600M2-Lysin and the multiple sequence MUSCLE alignment (Section 2.2) as input.
  • Table 3 List of candidate chemical entities chosen as ligands for molecular docking studies with CL 1-C600M2 -Lysin.
  • CLl-C600M2-Lysin coding sequence was chemically synthesized (Integrated DNA Technologies) and cloned into the pRSET-emGFP expression vector (ThermoFisher Scientific) using BamHl and EcoRl restriction sites. This incorporated a N-terminal polyhistidine tag (His6). A stop codon was incorporated prior to the emGFP tag at the C-terminal resulting in CLl-C600M2-Lysin with His Tag of molecular weight 21.269 KDa. Successful cloning was verified using PCR (FIG. 16).
  • the bacteria were pelleted at 5000 x g for 5 minutes and resuspended in 4ml B-PER reagent (ThermoFisher Scientific) containing lx protease inhibitor cocktail per gram cell pellet. Following incubation at room temperature for 10 min, the suspension was homogenized by sonicating 20 seconds, followed a 1 -minute rest on ice which is repeated for a total 4 bursts. The lysate was centrifuged at 15,000 x g for 5 min at 4°C to separate the soluble proteins from the insoluble proteins. Ni-NTA chromatography was used to purify the his-tagged CL 1-C600M2 -Lysin from the soluble proteins fraction.
  • lysin sequences shared identity ranging from 98 % to 96 %. Since, lysins sharing high similarity with CL1- C600M2-Lysin were involved in host-pathogen interactions of multiple species in the Enterobacteriaceae family, this could indicate the potency of CL 1-C600M2 -Lysin against multiple hosts. [0160] Lysin from Staphylococcus phage SAI shared 96% identity with CLl-C600M2-Lysin. The affinity of CL 1-C600M2 -Lysin to bacterial cell wall receptors was predicted using molecular docking studies.
  • Table 2 summarizes the interacting residues with each ligand and the corresponding binding affinity values. Sequential interacting amino acid residues could probably suggest an interacting motif in CLl-C600M2-Lysin that facilitates binding to the corresponding ligand.
  • the motif Argl41-Alal44-Aspl45-Leul48-Tyrl52 could be the motif involved in recognition of gram- positive bacterial cell wall with Tyrl52 forming a hydrogen bond of length 3.14 A (FIG. 10D).
  • CL 1-C600M2 -Lysin identical lysin gene products identified from two novel Myoviridae bacteriophages, Escherichia Phage C600M2 and Escherichia Phage CL1, which were isolated from wastewater treatment plant in the State of Qatar.
  • CL 1-C600M2 -Lysin was analyzed in-silico as prospective “enzybiotics” for water-treatment, followed by experimental assessment of the lytic activity of CL 1-C600M2 -Lysin using turbidimetric reduction assay.
  • DNA sequences for GP38 peptide SEQ ID NO: 5 and lysin peptide SEQ ID NO: 46 were ordered as gBlocks Gene Fragments from Integrated DNA Technologies.
  • the plasmid vector pRSET-emGFP (Life Technologies) was used with the restriction enzymes EcoRI and BamHI.
  • the plasmid vectors pEGFP and pET15-b (Novagen) were used alongside the restriction enzymes Ndel and BamHI. Both cloning resulted in the peptides being tagged with fluorescent protein for visualization and with 6x His- tag to aid purification.
  • the cloned plasmids were transformed to E.coli DH5-a cells for plasmid propagation.
  • a DNA sequence of poly alanine peptide, a sequence of 14 alanine amino acids was also ordered as gBlocks Gene Fragments from Integrated DNA Technologies, such that upon insertion of the gene fragment into the plasmid vector pRSET-emGFP yielded a poly alanine peptide with GFP protein tagged at the C-terminus of the peptide.
  • microscopy and bead-based capture were used to test the peptides and methods of the present disclosure on spiked samples: microscopy and bead-based capture.
  • microscopy bacteria in the planktonic form and on biofilm were analyzed using green fluorescence filter.
  • Planktonic samples were prepared by centrifuging an overnight culture of Escherichia coli C and C600 at 5,000 rpm for 3 minutes to separate the cells from the media. The pellet was resuspended in 1 ml dilution fluid and 10 pL of the purified peptide was added. The mixture was incubated in a 37°C shaker for 30 minutes.
  • the sample was centrifuged at 5,000 rmp for 3 minutes and the pellet containing cells was resuspended in dilution fluid. T successfully bound peptide pelleted with the bacteria. A small amount of the resuspended sample was smeared on a microscope slide and covered with a coverslip.
  • Biofilm samples were prepared by first growing different cultures of E. coli C, C600 and B/R in rich media overnight. The sample was diluted 100-fold into fresh media from which 100 pL was added into a small petri dish containing a coverslip. The petri dish was incubated in a shaker at 37°C for 24 hours. After biofilm formation, the coverslip was removed and placed on 10 pL of peptide samples that were pipetted on a microscope slide. The biofilm with the peptide was incubated for 30 minutes at room temperature (25°C). The prepared planktonic and biofilm samples were imaged using the EVOS fluorescence microscope and a confocal fluorescence microscope. For both preparation methods, negative controls were prepared where one had no peptide added, and the other control had standard emGFP-His tag protein instead of the peptide of the present disclosure.
  • FIG. 18 For the bead-based approach, a schematic of the procedure is shown in FIG. 18. 25 pL of Anti-GFP mAb-Magnetic Beads (JSR Life Sciences) and 25 pL of the purified peptide of the present disclosure were added in an Eppendorf tube. The ratio of GFP-tagged peptides with immunomagnetic beads that have an anti-GFP antibody was 1: 1. The sample was vortexed for 15 minutes. Then 1 mb of overnight culture of E. coli C600 diluted in dilution fluid to mimic bacteria contaminated in water was added to the beads using sterile technique. The sample was incubated at 37°C in a shaker at 200 rpm for 30 minutes. The tube was placed on a magnetic rack and the supernatant was transferred to a clean Eppendorf tube.
  • FIG. 19 The binding efficacy of C-terminal emGFP tagged lysin peptide of SEQ ID NO: 46 to planktonic E.coli C cells was visualized by fluorescence microscopy (FIG. 19) for E. coli C and emGFP-tagged lysin peptide of the present disclosure (FIG. 19A), E. coli C and emGFP (control) (FIG. 19B), E. coli C only (control) (FIG. 19C), and E. coli C and GFP -tagged polyalanine (FIG. 19D).
  • FIG. 20 demonstrates E. coli C biofilm (FIG. 20A), E. coli C600 biofilm (FIG. 20B), and E. coli C600 biofilm (FIG. 20C) with emGFP were measured under confocal microscopy using transmitted light and a GFP filter.
  • E. coli C biofilm was prepared. After formation of the biofilm, the C-terminus (FIG. 21A) and N-terminus (FIG. 21B) of GFP labeled lysin peptide of SEQ ID NO: 46 or control emGFP were left to incubate for 30 minutes before washing with PBS and imaging under transmitted light and a GFP filter.
  • E. coli C600 biofilm was prepared. After formation of the biofilm, the C-terminus (FIG. 22A) and N-terminus (FIG. 22B) of GFP labeled lysin peptide of SEQ ID NO: 46 or control emGFP were left to incubate for 30 minutes before washing with PBS and imaging under transmitted light and a GFP filter.
  • E. coli B/R biofilm was prepared. After formation of the biofilm, the C-terminus (FIG. 23A) and N-terminus (FIG. 23B) of GFP labeled lysin peptide of SEQ ID NO: 46 or control emGFP were left to incubate for 30 minutes before washing with PBS and imaging under transmitted light and a GFP filter.
  • FIG. 24 demonstrates 16S PCT results after immunomagnetic bead assays for the GFP labeled lysin peptide of SEQ ID NO: 46.
  • Lane 1 was the C-terminal peptide beads sample.
  • Lane 2 was the C-terminal peptide supernatant sample (Cs).
  • Lane 5 was the N-terminal peptide beads sample (N).
  • Lane 6 was the N-terminal supernatant sample (Ns).
  • Lane 7 was a no peptide control loaded (NPC) along with its corresponding supernatant (NPCs) in Lane 8.
  • Lane 9 was a PCT control that consisted of a no template control.
  • Lane 10 was a bacteria DNA control.
  • FIG. 25A demonstrates green fluorescence indicating that the peptide has bound to the bacteria cells.
  • FIG. 25B demonstrates a negative control with no green fluorescence, proving the bacterial cells do not have fluorescing abilities on their own.
  • FIG. 25C shows a second negative control with no green fluorescence, indicating that GFP alone does not bind to the bacteria cells. The green fluorescence seen was from background noise, as it was not on live cells.
  • FIG. 26 demonstrates E. coli C biofilm (FIG. 26A), E. coli C600 biofilm (FIG. 26B), and E. coli C600 biofilm (FIG. 26C) with emGFP were measured under confocal microscopy using transmitted light and a GFP filter.
  • E. coli C biofilm was prepared. After formation of the biofilm, the C-terminus (FIG. 27A) and N-terminus (FIG. 27B) of GFP labeled GP38 peptide of SEQ ID NO: 5 or control emGFP were left to incubate for 30 minutes before washing with PBS and imaging under transmitted light and a GFP filter.
  • E. coli C600 biofilm was prepared. After formation of the biofilm, the C-terminus (FIG. 28A) and N-terminus (FIG. 28B) of GFP labeled GP38 peptide of SEQ ID NO: 5 or control emGFP were left to incubate for 30 minutes before washing with PBS and imaging under transmitted light and a GFP filter.
  • E. coli B/R biofilm was prepared. After formation of the biofilm, the C-terminus (FIG. 29A) and N-terminus (FIG. 29B) of GFP labeled GP38 peptide of SEQ ID NO: 5 or control emGFP were left to incubate for 30 minutes before washing with PBS and imaging under transmitted light and a GFP filter.
  • FIG. 30A demonstrates 16S PCT results after immunomagnetic bead assays for the GFP labeled lysin peptide of SEQ ID NO: 46.
  • Lane 1 was a 100 bp DNA ladder.
  • Lane 2 was a polyalanine -GFP peptide coated beads samples.
  • Lane 3 was a polyalanine-GFP peptide coated supernatant sample.
  • Lane 4 was an N-terminal lysin peptide beads sample.
  • Lane 5 was an N-terminal lysin peptide supernatant sample.
  • Lane 5 was a no peptide control loaded along with its corresponding supernatant in Lane 6.
  • Lane 7 was a PCR control.
  • 30B demonstrates 16S PCT results after immunomagnetic bead assays forthe GFP labeled GP38 peptide of SEQ ID NO: 5.
  • the gel was imaged under UV light.
  • Lane 1 was a 100 bp DNA ladder.
  • Lane 2 was a polyalanine-GFP peptide coated beads sample.
  • Lane 3 was a polyalanine-GFP peptide coated supernatant sample.
  • Lane 4 was an N-terminal GP38 peptide of SEQ ID NO:5 beads sample.
  • Lane 5 was a no peptide control loaded along with its corresponding supernatant.
  • Lane 6 was a PCR control.
  • Adibi M., Mobasher, N., Ghasemi, Y ., Mohkam, M., Mobasher, M.A., 2017. Isolation, purification and identification of E. coli 0157 phage for medical purposes. Trends in Pharmaceutical Sciences 3, 43-48.
  • ModelTest-NG A New and Scalable Tool for the Selection of DNA and Protein Evolutionary Models. Molecular Biology and Evolution 37, 291-294. https://doi.org/10.1093/molbev/mszl89
  • LigPlot+ multiple ligand-protein interaction diagrams for drug discovery.
  • Novel antibody-antibiotic conjugate eliminates intracellular S. aureus. Nature, https://doi.org/10.1038/naturel6057 Letunic, I., Bork, P., 2021.
  • Interactive Tree Of Life (iTOL) v5 an online tool for phylogenetic tree display and annotation. Nucleic acids research 49, W293— W296.
  • CDD/SPARCLE the conserved domain database in 2020. Nucleic acids research 48, D265— D268.
  • IQ-TREE 2 New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. Molecular Biology and Evolution 37, 1530-1534. https://d0i.0rg/l 0.1093/molbev/msaa015

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Abstract

L'invention concerne un procédé permettant de détecter des bactéries. Un aspect donné à titre d'exemple est conçu pour : fournir au moins un peptide qui comprend la séquence d'acides aminés présentée dans une séquence quelconque parmi les SEQ ID NOS : 1-46 ; fixer un marqueur audit ou auxdits peptides pour former un peptide marqué ; ajouter le peptide marqué à un échantillon aqueux ; et détecter un signal qualitatif ou quantitatif dans l'échantillon aqueux, le signal indiquant la présence d'au moins une bactérie à Gram positif ou d'au moins une bactérie à Gram négatif. Un peptide capable de se lier à au moins un ligand glucidique de paroi cellulaire sur une surface de paroi cellulaire d'au moins une bactérie, le peptide comportant une séquence d'acides aminés qui est telle que présentée dans une séquence quelconque parmi les SEQ ID NOS : 1-46.
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