WO2021010835A1

WO2021010835A1 - Peptides for use in the treatment of cholera

Info

Publication number: WO2021010835A1
Application number: PCT/NL2020/050469
Authority: WO
Inventors: Shuaiqi GUO; Ilja VOETS; Peter Davies; Karl Klose
Original assignee: Technische Universiteit Eindhoven
Priority date: 2019-07-18
Filing date: 2020-07-16
Publication date: 2021-01-21
Also published as: NL2023527B1

Abstract

The present invention relates to means and methods for the treatment of cholera. The current invention also provides for means and methods for the prevention of cholera. In particular, the means and methods of the current invention allows to target the bacteria that cause cholera, Vibrio cholerae, while not targeting other bacteria. Peptides are provided that can interfere with binding of Vibrio cholerae to the intestine thereby disrupting biofilm formation and/or colonization in the intestine can be disrupted, which is useful in the treatment or prevention of cholera. Means and methods to identify such peptides are provided as well.

Description

Title: Peptides for use in the treatment of cholera

Background

Cholera is an acute diarrheal infection caused by ingestion of food or water contaminated with the bacterium Vibrio cholerae. Cholera remains a global threat to public health and an indicator of inequity and lack of social development. Researchers have estimated that every year, there are roughly 1.3 to 4.0 million cases, and 21 000 to 143 000 deaths worldwide due to cholera.

Cholera vaccines are widely used to prevent cholera outbreaks. The first vaccines used against cholera were developed in the late 1800s. They were the first widely used vaccine to be made in a laboratory. Oral cholera vaccines were first introduced in the 1990s. For these oral vaccines, two or three doses are typically recommended. The available types of oral vaccine are generally safe. Mild abdominal pain or diarrhoea may occur. They are safe in pregnancy and in those with poor immune function. They are licensed for use in more than 60 countries. In countries where the disease is common, the vaccine appears to be cost effective. A single dose vaccine is available for those traveling to an area were cholera is common. As of 2010, an injectable cholera vaccine was available in some countries.

Cholera vaccines can help prevent cholera. For the first six months after vaccination they provide about 85 percent protection, which decreases to roughly 50-62 per cent during the first year. After two years the level of protection decreases to less than 50 per cent. When a sufficient part of the population has been immunized, it may provide protection to those who have not been immunized (known as herd immunity). The World Health Organization (WHO) recommends the use of cholera vaccines in combination with other measures among those at high risk.

Once a person has cholera, the patient is typically given antibiotics, if available, and a hydration treatment. Antibiotic regimens for the treatment of cholera include treatments with tetracycline, which has been shown to be effective, and is superior to furazolidone, chloramphenicol and sulfaguanidine in reducing cholera morbidity. Treatment with a single 300 mg dose of doxycycline has shown to be equivalent to tetracycline treatment. Erythromycin is effective for cholera treatment, and appropriate for children and pregnant women. Orfloxacin, trimethoprim-sulfamethoxazole (TMP-SMX), and ciprofloxacin are effective, but doxycycline offers advantages related to ease of administration and comparable or superior effectiveness. Recently, azithromycin has been shown to be more effective than erythromycin and ciprofloxacin, and is an appropriate first line regimen for children and pregnant women.

Antibiotics treatment of the Vibrio cholerae bacterium has the downside that it will also affect the human microbiome, and can induce resistance to the antibiotics. Resistance to tetracycline and other antimicrobial agents in Vibrio cholerae has been demonstrated in both endemic and epidemic cholera settings. Resistance can be acquired through the accumulation of selected mutations over time, or the acquisition of genetic elements such as plasmids, introns, or conjugative elements, which confer rapid spread of resistance. A likely risk factor for antimicrobial resistance is widespread use of antibiotics, including mass distribution for prophylaxis in asymptomatic individuals.

Hence, in spite of the vaccines providing a measure of protection to some extent, and the ability to treat cholera with a combination of antibiotics and rehydration, there remains a need for new and improved means and methods for the treatment and/or prevention of cholera.

Summary of the invention

The current inventors now provide for new and improved means and methods for the treatment and/or prevention of cholera. In particular, the means and methods of the current invention allow specific targeting of the bacteria that cause cholera, Vibrio cholerae. In particular, the current invention relates to means and methods involving peptides that bind to the protein-binding domain of Repeats-ln-Toxin (RTX) adhesin FrhA of Vibrio cholerae. RTX adhesin FrhA of Vibrio cholerae (FrhA adhesin) mediates adherence of the bacterium to epithelial cells to enhance biofilm formation and colonization (Syed et al. , J Bacteriol., 2009). By binding of the peptides of the invention to the FrhA adhesin, biofilm formation and/or colonization in the intestine can be disrupted, which is useful in the treatment or prevention of cholera. Hence, the peptides of the invention that bind to the FrhA adhesin are for use in the treatment of subjects diagnosed or suspected to have cholera, and also for use as a prophylaxis in subjects at risk for cholera. Said peptides are to be delivered to the intestine, where they are to have their action, i.e. interfere with biofilm formation and/or colonization. Any suitable means may be used for delivery of the peptides of the invention to the intestine. When these peptides are delivered to the intestine, peptides need to withstand the environment of the intestine, and also that of the gastric environment through which the peptides may pass upon e.g. oral intake. Hence, the peptides may be chemically modified or comprised in a suitable composition to allow the peptides to have their action in the intestine. Such means also include pharmaceutical formulations suitable for delivery to the intestine. Alternatively, the peptides may be produced on site in the intestine via expression of said peptides by micro-organisms, e.g. a probiotic. The peptide may be secreted by the micro-organism. The peptide may also be comprised in precursor polypeptides (which may be expressed or delivered). The peptide may also be a modified peptide that e.g. is resistant to cleavage or degradation. Furthermore, means and methods are provided for further identification of peptides that can interfere with in binding to the intestine. Accordingly, the current invention provides for means, methods and uses, for peptides that are useful in the treatment or prophylaxis of cholera caused by Vibrio cholerae, wherein said means, methods and uses comprises interfering with the FrhA protein of Vibrio cholerae for binding to the intestine, the formation of a biofilm and/or colonisation.

Figures

Figure 1. Domain map of FrhA and amino-acid sequence alignment between the MplBP_PBD (RIII-3) (SEQ ID N0.1) and the FrhA_PBD (RIII-3 like domain) (SEQ ID NO.2). Grey shading indicates residues residues that are conserved between the two sequences, with those that directly participate in binding peptides highlighted (corresponding to positions a (N), b (Y), c (V), d (E), e (T), f (N), g (S), h (D) of FrhA).

Figure 2. Screening of 12 random peptides of different amino-acid sequences by FP (See table 3). (A) the fluorescence polarization of each peptide measured at a protein concentration of 10 mM. (B) FP assay of Peptide 12 from (A) with titration of MplBP_PBD in the presence of CaCI2 (upper line in the graph) and in the presence of excess EDTA (lower line in the graph).

Figure 3: FP assay of four peptides with C-terminal aspartates to MplBP_PBD. With lines from top to bottom representing PepB: FITC-AbGPDSD (SEQ ID NO. 3), PepC: FITC-AbDSTD (SEQ ID NO. 4), PepD: FITC-AbGPDD(SEQ ID NO. 5), and PepA: FITC-AbDSTPD(SEQ ID NO.6), respectively.

Figure 4: Binding of pepB and pepC to the PBD of FrhA. (left) FP assay of the two peptides (PepB (upper line) and PepC (lower line)) with titration of FrhA_PBD. (right) FP competition assays of the unlabelled peptides (DSD and DSTD (SEQ ID NO. 62) against the fluorescent peptides (PepB (upper line) and PepC (lower line)) . The amino-acid sequences of the peptides are indicated.

Figure 5: Fluorescence polarization assays of peptides. On the left, fluorescence polarization assay results of peptides with sequence of AGAXD (SEQ ID NO. 7) are depicted, where X represents each of the 20 different amino acids (left graph). Peptide AGATD (SEQ ID NO.8) showed strongest binding. On the right, fluorescence polarization assay results of peptides with sequence of AGXTD (SEQ ID NO. 9) are depicted, where X represents each of the 20 different amino acids (left graph). Peptide AGWTD (SEQ ID NO.10) showed strongest binding. The EC-50 values of each peptide are listed in Table 4.

Figure 6: X-ray crystal structures of MplBP_PBD in complex with three different peptides: pepA, pepB and pepC. The peptides are coloured in magenta while the protein is coloured in grey. PDB amino acids that interact with the peptides, including the tyrosine that potentially forms tt-p stacking with an aromatic amino acid at position 3, are shown in stick representations.

Detailed description

Bacterial adhesins mediate the adherence of bacteria to other cells, ligands or surfaces to form biofilms that can cause severe infections in humans. The biofilm forming ability of Vibrio cholerae involves the production of Vibrio polysaccharides and carbohydrate-binding matrix proteins Bap1 , RbmA and RbmC. However, relatively little is known about the interactions between Vibrio cholerae and their specific ligands during the bacteria’s initial attachment to various cells or surfaces. Repeats-ln-Toxin (RTX) adhesins are a recently discovered class of large (typically between 0.2 -1.5 MDa) repetitive biofilm- associated proteins produced by many Gram-negative bacteria including several pathogenic species of the Vibrio genus, including Vibrio cholerae (Satchell, Annu Rev Microbiol, 2011 ; Syed et al. , J Bacteriol, 2009). RTX adhesins play an important role in the early stages of biofilm formation by assisting bacteria in binding to surfaces to form microcolonies. However, there is a lack of structural information on FrhA adhesin, i.e. the Vibrio cholerae adhesin or simply FrhA, and its binding partners are unknown.

The current inventors now provide for peptides that can interfere with FrhA binding sites. By interfering with said binding sites, binding of Vibrio cholerae to the intestine can be interfered with. Accordingly, a method is provided for interfering with the binding of Vibrio cholerae to intestinal epithelial cells, comprising interfering with binding sites on an RTX adhesin FrhA protein of Vibrio cholerae with a peptide.

As shown in the examples, short peptides were identified in vitro that have a high affinity to FrhA, i.e. to the binding domain of FrhA (e.g. an EC50 of 100nM or lower). An amino acid sequence of the binding domain of FrhA is shown in Figure 1 and listed below (SEQ ID NO.11).

Hence, the short peptides in accordance with the invention are capable of binding to said FrhA domain, and to FrhA domains having at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID N0.11. Said FrhA domains are from FrhA adhesins from Vibrio cholerae and play a role in adherence to the intestine, i.e. the intestinal wall, which can result in colonization and which can result in the formation of a biofilm. Said FrhA domains preferably are from FrhA adhesins of pathogenic Vibrio cholerae. Preferably said Vibrio cholerae is a Vibrio cholerae from the serogroup 01. Said Vibrio cholerae can also be a Vibrio cholerae 0395 derivative from the serogroup 01.

These peptides are useful for the treatment of cholera, as they have the potential to interfere with the binding of Vibrio cholerae to the intestine. Such peptides may be used as a prophylactic, i.e. in case of an outbreak, or may be used in a human subject being diagnosed or suspected to have cholera. These peptides may be used combined with existing treatments as well.

Hence, in one embodiment, a method is provided for interfering with the binding of Vibrio cholera to intestinal epithelial cells, comprising interfering with binding sites on an RTX adhesin FrhA protein of Vibrio cholerae with a peptide. Peptides that are suitable for interfering with binding sites of an FrhA adhesin are described in the examples. However, the invention is not limited to said peptides, any peptide may suffice, as long as it is capable of interfering with the binding sites such as described in the examples, and which can be identified as described in the examples.

Peptides in accordance with the invention preferably have a length of at least 2 amino acids, more preferably a length of 3 amino acids, even more preferably a length of at least 4 amino acids. More preferably, said peptides may have at least 5 amino acids. Said peptides may have any suitable length. In one embodiment, said peptides may have at most 30 amino acids, preferably at most 20 amino acids, more preferably at most 15 amino acids. In one embodiment, the peptides have a length which is in the range of 2- 15 amino acids. In another embodiment, the peptides have a length which is in the range of 4-15 amino acids.

In another embodiment, as shown in the example section, peptides that may be in particular suitable for interfering with the binding sites to an FrhA protein have a C-terminal amino acid selected from the group consisting of Isoleucine, Serine, Glutamate, Alanine and Aspartate with a free carboxyl group. A C-terminal Aspartate is defined to be the last amino acid of the peptide at the C-terminus. Preferably the C- terminal amino acid is Aspartate.

In another embodiment, in the peptides in accordance with the invention, the amino acid adjacent to the C-terminal amino acid as defined above is selected from the group consisting of Serine, Threonine, Tryptophan, Tyrosine, Phenylalanine and Histidine. In one embodiment, the C-terminal amino acid is Isoleucine, and the amino acid adjacent to Isoleucine is selected from the group consisting of Serine, Threonine, Tryptophan, Tyrosine, Phenylalanine and Histidine. In one embodiment, the C-terminal amino acid is Serine, and the amino acid adjacent to Isoleucine is selected from the group consisting of Serine, Threonine, Tryptophan, Tyrosine, Phenylalanine and Histidine. In one embodiment, the C-terminal amino acid is Glutamate, and the amino acid adjacent to Isoleucine is selected from the group consisting of Serine, Threonine, Tryptophan, Tyrosine, Phenylalanine and Histidine. In one embodiment, the C-terminal amino acid is Alanine, and the amino acid adjacent to Isoleucine is selected from the group consisting of Serine, Threonine, Tryptophan, Tyrosine, Phenylalanine and Histidine. In one embodiment, the C-terminal amino acid is Aspartate, and the amino acid adjacent to Isoleucine is selected from the group consisting of Serine, Threonine, Tryptophan, Tyrosine, Phenylalanine and Histidine. Preferably, the amino acid adjacent to the C- terminal amino acid as defined above is selected from Serine and Threonine.

In a preferred embodiment, the C-terminus of the peptide in accordance with the invention has Threonine adjacent to a C-terminal Aspartate or Serine adjacent to a C-terminal Aspartate. Hence, the C-terminus of the peptide comprises a Threonine- Aspartate or Serine-Aspartate.

In one embodiment, the amino acid adjacent to the two C-terminal amino acids can be selected to be any amino acid. Preferably, the amino acid adjacent to the two C-terminal amino acids is selected from the group consisting Histidine, Isoleucine, Methionine, Threonine, Valine, Glutamine, Arginine, Tryptophan, Tyrosine, and Phenylalanine. More preferably, the amino acid adjacent to the two C-terminal amino acids is selected from the group consisting of Tryptophan, Tyrosine, and Phenylalanine. Hence, in one preferred embodiment, the C-terminus of the peptide in accordance with the invention comprises Tryptophan-Serine-Aspartate, Tyrosine-Serine-Aspartate, Phenylalanine-Serine-Aspartate, Tryptophan-Threonine-Aspartate, Tyrosine-Threonine- Aspartate or Phenylalanine-Threonine-Aspartate.

Said three C-terminal amino acids are preferably preceded by two amino acids selected from Alanine and Glycine to provide for five C-terminal consecutive amino acids. These five amino acids preferably have a composition as defined above. The tables 1 and 2 below provide a scheme listing preferred compositions of five consecutive amino acids comprised in the C-terminus of the peptide interfering with binding sites of the FrhA adhesin in accordance with the invention. The numbering in the table indicates that the amino acids are in consecutive order and are selected from the amino acids listed in each column. It is understood that the numbering is to indicate the relative position and consecutive order of amino acids comprised in the peptide in accordance with the invention. The peptide in accordance with the invention thus may consist of more than five consecutive amino acids as listed in the tables 1 and 2. As long as the C-terminus of the peptide is accessible thereby allowing binding of the peptide to the adhesin, such a peptide is contemplated herein. The amino acid of position five of said five C-terminal amino acids as listed in the tables preferably is a C-terminal amino acid. The amino acid at position one can be at the N-terminal position, or the peptide may comprise additional amino acids flanking adjacent thereto (numbered e.g. as position 0, and continuing as position -1 , -2, -3 etc.).

Table 1. Peptide compositions suitable for the treatment or prevention of Cholera.

Table 2. Preferred peptide compositions for the treatment or prevention of Cholera.

In one embodiment, the peptide in accordance with the invention for use in a method for interfering with the binding of Vibrio cholerae to intestinal epithelial cells comprises an amino acid sequence selected from the group consisting of AGWTD (SEQ ID NO.10), AGYTD (SEQ ID N0.12) and AGFTD (SEQ ID NO.13). Preferably said peptide comprises an amino acid sequence at the C-terminus selected from the group consisting of AGWTD, AGYTD and AGFTD.

The peptides in accordance with the invention as described above are useful for the treatment of cholera, as they have the potential to interfere with the binding of to the intestine. Preferably, such peptide binds to the protein binding domain of RTX adhesin FrhA of Vibrio cholerae with an EC50 of less than 400 nM, preferably with an EC50 of less than 300 nM, more preferably with an EC50 of less than 200 nM. Even more preferably, a peptide in accordance with the invention binds to the protein binding domain of RTX adhesin FrhA of Vibrio cholerae with an EC50 of less than 100 nM. Most preferably, a peptide in accordance with the invention has an affinity for the protein binding domain of RTX adhesin FrhA of Vibrio cholerae with an EC50 of less than 10 nM. The EC50 of a peptide can be determined using methods known in the art, e.g. as shown in the examples. In one embodiment, the EC50 of a peptide in accordance with the invention for the protein binding domain of RTX adhesin FrhA of Vibrio cholerae is determined with a peptide, preferably a labelled peptide, dissolved in FP buffer (10 mM Hepes, pH 7.4, 150 mM NaCI, 2 mM CaCI2 0.1 % Tween-20, 1 mg/ml_ BSA) to a final concentration of 10 nM in a direct binding assay with the protein binding domain of RTX adhesin FrhA of Vibrio cholerae. In a further embodiment, said protein binding domain of the adhesin has a sequence corresponding with SEQ ID NO. 11.

As said, a peptide in accordance with the invention is to have its action in the intestine. Hence, any means that allows for the peptide in accordance with the invention to have its action in the intestine is contemplated. In one embodiment, the peptide in accordance with the invention is a non-digestible peptide. A non-digestible peptide in accordance with the invention means that the peptide is resistant to enzymatic activity (as present e.g. in the intestine). Said enzymatic activity may degrade the peptides in accordance with the invention thereby preventing the peptides to have their action. Hence, non-digestible in this context is understood to include a strong reduction in peptide degradation as compared to an un-modified peptide consisting of naturally occurring L- amino acids.

A chemical modification that can make peptides resistant to digestion includes the incorporation of one or more D-amino acids in the peptides in accordance with the invention.

In another embodiment, a peptide in accordance with the invention is produced from a precursor polypeptide. In this embodiment, a peptide, or a plurality of peptides, is comprised in a larger peptide, i.e. a precursor peptide. Said larger peptide being subjected to cleavage to thereby release the peptide in accordance with the invention from the precursor peptide. Such cleavage may be via autocatalytic cleavage, or via proteases. Preferably, said cleavage occurs extracellular, i.e. in the environment wherein the peptide is to interfere with the FrhA adhesin. Preferably, the peptide or the plurality of peptides is cleaved from the precursor polypeptide peptide by extracellular proteases. It is understood that when a plurality of peptides is to be released from the precursor polypeptide, the plurality of peptides may comprises a plurality of different peptides in accordance with the invention, i.e. peptides having different amino acid sequences and having high affinity to FrhA adhesin.

In another aspect of the invention, a gene construct for expression of a peptide in accordance with the invention, or of a precursor polypeptide in accordance with the invention is provided. A gene construct in accordance with the invention is a nucleotide sequence that encodes for said peptide or said precursor polypeptide and further comprises elements, which are known in the art, for expression of the peptide or precursor polypeptide in a cell (such as promoter sequences, introns, polyA, transcription initiation site, transcription termination sequence, ribosomal binding site, translation start and stop sites, etc.). The gene construct can be used to express the peptide in accordance with the invention in the intestine. The gene construct and/or peptide or precursor polypeptide preferably provides for expression of the peptide such that the peptide is secreted from the cell in which it is produced. The peptide may be secreted itself, or the peptide may be comprised in a precursor polypeptide from which, once the precursor polypeptide is secreted, the peptide is released. This way, the peptide can be produced and delivered on site in the intestine. The gene construct can also be used in manufacturing of the peptides in accordance with the invention.

In one embodiment, the gene construct for expression of a peptide in accordance the invention, or of a precursor polypeptide in accordance with the invention is provided to a cell. The gene construct may be provided to a cell comprised in a vector. The vector may be any suitable vector for delivery of gene constructs to a cell. The vector may be a plasmid. The vector may be viral vector. In another or further embodiment, the gene construct for expression of a peptide in accordance the invention, or of a precursor polypeptide in accordance with the invention is comprised in a cell, preferably in the genome of the cell.

It is understood that the genetic information of a cell may be modified such that it expresses the peptide or precursor polypeptide as described herein, e.g. by insertion of a sequence encoding the peptide or precursor polypeptide in the genome of the cell. Hence, it may not be necessary to introduce a complete gene construct in a cell, but one can rely on endogenous sequences of the cell such that after the introduction of the sequence encoding the peptide or precursor polypeptide in the genome, the cell is capable of producing said peptide or precursor polypeptide. Hence, it is understood that the gene construct may comprise endogenous sequences of the cell.

In another embodiment, the cell from which the peptide or precursor polypeptide as described herein is produced is a micro-organism. Preferably, said micro- organism comprises a gene construct for expression of a peptide or of a precursor polypeptide in accordance with the invention. Said micro-organism may be used for manufacturing of the peptide of precursor polypeptide. Preferably, said micro-organism is to produce the peptide or precursor polypeptide in the intestine. The peptide or precursor polypeptide preferably is provided to the lumen and epithelium of the gut. Most preferably, said peptide or precursor polypeptide is secreted from the micro-organism. Preferred suitable micro-organisms are probiotic micro-organisms, such as lactobacillus. Preferably, the probiotic micro-organism is a human probiotic micro-organism. Suitable means and methods to have peptides or precursor polypeptides produced that may be contemplated are known in the art (e.g. Shaw et al. , Immunology 2000, 100 pp.510-518; Maassen et al. , Vaccine, 2003, 21 pp. 4685-4693)

In a further embodiment, a composition is provided comprising a peptide, a precursor polypeptide, or a micro-organism, as described herein. Such a composition preferably is suitable for oral intake. Preferably, the composition comprises an enteric coating. Said composition may be a pharmaceutical composition. Said composition preferably is suitable for use in the treatment of cholera or for the prophylactic treatment for cholera as described herein.

As said, the peptide in accordance with the invention is for use in the prevention or treatment of cholera. Accordingly, the peptides, the precursor polypeptides, or micro-organisms as described herein are for use in a medical treatment. Said medical treatment may comprise a prophylactic treatment. Said medical treatment comprises a treatment of cholera.

In one embodiment, the peptides, the precursor polypeptides, or micro organisms as described herein are for use in a medical treatment, which use comprises interfering with binding of Vibrio cholerae to the intestine. It is understood that in said use, the peptides in accordance with the invention are to interfere with binding of Vibrio cholerae to the intestine, said peptides produced e.g. from the precursor polypeptides or micro-organisms. In a further embodiment, the peptides, the precursor polypeptides, or micro-organisms as described herein are for use in a medical treatment, which use comprises disrupting the formation of a biofilm of Vibrio cholerae. It is understood that in said use, the peptides in accordance with the invention are to disrupt the formation of a biofilm of Vibrio cholerae, said peptides produced e.g. from the precursor polypeptides or micro-organisms. In another further embodiment, the peptides, the precursor polypeptides, or micro-organisms as described herein are for use in a medical treatment, which use comprises preventing colonisation with

Vibrio cholerae. It is understood that in said use, the peptides in accordance with the invention are to prevent colonisation with Vibrio cholerae, said peptides produced e.g. from the precursor polypeptides or micro-organisms.

In one embodiment, a method is provided for identifying peptides that interfere with the binding of Vibrio cholerae to intestinal epithelial cells, comprising

- providing one or more peptides;

- providing the protein binding domain of RTX adhesin FrhA of Vibrio cholerae ;

- contacting the one or more peptides with the protein binding domain of RTX adhesin FrhA of Vibrio cholerae ;

- identifying a peptide that binds to the protein binding domain of RTX adhesin FrhA of Vibrio cholerae.

As shown in the examples, this method allows identification of suitable peptide candidates that have a high affinity to the protein binding domain of RTX adhesin FrhA of Vibrio cholerae, and that thereby can interfere with the binding of Vibrio cholerae to the intestine, also interfering with biofilm formation and/or preventing colonisation of the intestine. Hence, the current invention is not restricted to the peptides as identified herein, but also provides the means and methods to provide for further peptides that are useful in the treatment or prevention of cholera. Said further peptides may be made in accordance with the general design rules as described herein, but may not necessarily be limited thereto and thus may include randomly generated peptides. It is also understood that once a suitable candidate is identified, based on this candidate, further variations may be made taking the identified candidate as a starting point, similarly to as described in the example section.

The protein binding domain of the RTX adhesin FrhA of Vibrio cholerae may be a protein binding domain having at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID N0.11. Said protein binding domain preferably is from an FrhA adhesin of pathogenic Vibrio cholerae. Preferably said protein-binding domain is from an FrhA adhesin of a Vibrio cholerae from the serogroup 01 or a Vibrio cholerae 0395 derivative from the serogroup 01.

The peptide candidates preferably are labelled. Any labelling that allows identification of interaction with the protein binding domain can be contemplated. For example, a fluorescent label may be preferred. The peptides and the protein binding domain interaction step can be a step in which one peptide interacts with a provided protein binding domain, such as described in the examples. Preferably, the contacting step is carried out in the presence of Ca2+. In addition to having an interaction with one candidate peptide and one protein binding domain e.g. in one well (as described in the example section), one can also carry out the interaction step with a plurality of candidate peptides and/or protein binding domains. Hence, one can also have an interaction step in which a plurality of peptides interact with a protein binding domain, i.e. peptides of different amino acid sequences. The plurality of peptides can than have labels that allow to directly identify a peptide that interacts with the protein binding domain (e.g. by using different fluorescent labels, each label identifying a peptide sequence). Alternatively, the plurality of peptides may carry the same label, and in a subsequent step the peptide candidate(s) that have high affinity to the peptide binding domain may be identified, e.g. because the sequences and/or composition of the plurality of peptides is known. Hence, preferably, the one or more peptides are labelled, most preferably with a FITC label.

Preferably, a peptide (or peptides) is identified that has an EC50 of less than 100 nM, more preferably of less than 10 nM. Once such a peptide is identified, the amino acid sequence of said peptide is provided and said sequence information is subsequently used for the manufacturing of a peptide, or a precursor polypeptide or micro-organism as described herein which is suitable for the prevention or treatment of cholera.

As used in the description of the invention, clauses and clauses appended claims, the singular forms“a”,“an” and“the” are used interchangeably and intended to include the plural forms as well and fall within each meaning, unless the context clearly indicates otherwise. Also, as used herein,“and/or” refers to and encompasses any and all possible combinations of one or more of the listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

As used herein, the term“about” will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used,“about” will mean up to plus or minus 10% of the particular term. Examples

Introduction

Recently, the structure of a 1.5-MDa RTX adhesin Mp\BP was provided (Shuaiqi Guo et al. , Science Advances, 2017). This Mp\BP adhesin binds to ice (i.e. Ice- Binding Protein). The adhesins Mp\BP and FrhA share a similar domain architecture: an N-terminal region that anchors the adhesin to the outer cell membrane, and a long extender region that projects different C-terminal ligand-binding domains away the cell membrane. Furthermore, the peptide-binding domain (PBD) of Mp\BP has 65% sequence identity to that of FrhA, and the amino-acid residues that participate in binding peptides are conserved between the two proteins. In a structure based approach, utilizing information from both Mp\BP and FrhA, peptides that can bind to FrhA were identified. Subsequent sequential optimisation of these molecules resulted in peptides with low nano-molar affinities for FrhA.

Materials and Methods

Protein expression, purification and crystallization

PBDs of Mp\BP and FrhA were expressed in Escherichia coli BL21 (DE3) cells using a pET28a vector, purified using Ni-NTA affinity and size-exclusion chromatography, and then concentrated to 5 mg/ ml. The Mp\BP_PBD crystallized using the‘microbatch-under-oil’ method by mixing equal volumes of the concentrated protein with a precipitant solution in the presence of 2 mM various peptides. Crystals of Mp\BP_PBD grew in the presence of 0.1 M calcium chloride, 0.1 M sodium acetate (pH 4.6), 30% (w/v) PEG 400. High-resolution datasets were collected from the 08ID-1 beamline of the Canadian Light Source synchrotron facilities, while the dataset of RIC was collected at the Petra III DESY beamline (Hamburg, Germany).

Solid phase peptide synthesis

Peptides were synthesized using solid phase peptide synthesis. Per peptide, 100 pmole was synthesized. The peptides were synthesized from the C-termini to the N-termini. The C-terminal amino acid was coupled to a Wang-resin and protected with a Fmoc-group. Each amino acid to be coupled, was protected with a Fmoc-group. Before each coupling the N-terminus was deprotected by removing the Fmoc-group using 20% v/v piperidine in DMF. For each coupling 4 equivalents of HBTU, OxymaPure and Fmoc- protected amino acid and 8 equivalents of DIPEA dissolved in 5 mL DMF was used. Some peptides were coupled to a fluorescent dye. The dye was coupled to the N-terminus of the peptide. Before the dye-coupling the peptide was coupled to a linker-molecule. The linker-molecule was b-alanine. The fluorescent dye, fluorescein isothiocyanate (FITC), was coupled using 7 equivalents of FITC, 14 equivalents of DIPEA and 2 ml_ of DMF. After the peptides were finished they were cleaved off the resin. This was done using a solution of 95/2.5/2.5 (v/v) TFA/mQ(H20)/TIS. These reagents also cause deprotection of tBu- protected amino acid side chains. After cleavage the peptide was precipitated in ice-cold diethyl ether, stored in the freezer for 10 minutes and then centrifuged at 2000 rpm for 10 min. Then the supernatant was decanted and the remaining pellet was air-dried.

Fluorescence polarization

The affinities of the different peptides for the PBDs were determined using fluorescence polarization (FP). The FITC-labeled peptides were dissolved in FP buffer (10 mM Hepes, pH 7.4, 150 mM NaCI, 2 mM CaCI2 0.1 % Tween 20, 1 mg/ml_ BSA). Dilution series of the PBDs were made on Corning black round-bottom 384-well plates, and their polarization was measured on a Tecan Infinite F500 plate reader (excitation 485 nm, emission 535 nm). The FP assay was performed as a direct binding assay and a competitive binding assay. The FP assay was performed in a 384-well plate measuring with the TECAN plate-reader. For the direct binding assay the polarization was measured by titrating the protein while keeping the FITC-labeled peptides at a concentration of 10 nM. For the competitive binding assay, both labeled and unlabeled peptides were used. The polarization was measured by titrating unlabeled peptide against the labelled peptides. The protein concentration remained constant, which was the EC80 value determined for each FITC-labeled peptide from the direct binding FP assay. The concentration of labeled peptide was kept at 10 nM. The concentration of unlabeled peptide typically started at 2 mM.

Results

Structure-based approach for designing peptide inhibitors for FrhA

The crystal structure of the MplBP_PBD indicated that the three C-terminal residues (Thr-Pro-Asp) from a symmetry-related molecule are stably coordinated via Ca2+ in the protein’s ligand-binding groove (Fig. 1). This crystal structure was used as a starting point to first identify ligands of MplBP_PBD. A six amino-acid peptide with a sequence of FITC-Alab-Asp-Ser-Thr-Pro-Asp (SEQ ID NO.6) was synthesized, and designated as “pepA”. The fluorescent probe was linked to the N-terminal Alab as it is solvent exposed and does not influence the binding. In addition, a screening of 12 different peptides indicated that one peptide with significant binding to the protein has a C-terminal aspartate residue (Fig.2A; Table 3). The interaction is in a Ca2+-dependent manner.

Table 3. Sequences of peptides listed on the x-axis of Fig. 2A (peptides 1-12 corresponding with SEQ ID NOs. 14-25, respectively).

FP showed that phosphorylated peptide with a sequence of FITC- RHKKLMFKpTEGPDSD (peptide 12, SEQ ID N0.25) has an EC50 of 760 nM in the presence of Ca2+, whereas the excess of EDTA abolished the peptide-protein interaction (Fig.2B). With the hypothesis that the binding module is located at the C terminus of the protein, a short peptide encompassing only the last five amino acids were synthesized with a fluorescent probe attached at the N terminus. The resulting peptide has a sequence of FITC- Alab-Gly-Pro-Asp-Ser-Asp (SEQ ID NO.3), which is designated as “pepB”. During the synthesis of pepA (SEQ ID NO.6) and pepB, two variants were unintentionally made, which have sequences of FITC- Alab-Asp-Ser-Thr-Asp (pepC) (SEQ ID NO. 4), and FITC- Alab-Gly-Pro-Asp-Asp (pepD) (SEQ ID NO.5), respectively. All four peptides contain a C-terminal aspartate, and their affinity to the adhesins was tested in FP assays.

Intriguingly, pepA, which is the sequence originally identified in the crystal structure of MplBP_PBD showed the weakest binding of 27 mM, whereas its variant, pepC, binds at least 200-fold stronger with an EC50 of 110 nM (Fig.3). PepB binds with an EC50 of 159 nM, which is roughly five-fold stronger than its longer and phosphorylated counterpart (760 nM), and 20-fold stronger than pepD. Close inspection of the amino-acid sequence of the peptides suggested that besides the aspartate at the 1st position, residues at position 2 also played a role in binding MplBP_PBD. The strongest binders pepC and pepB contain small polar residues of threonine and serine at their position 2, respectively. The intermediate binder pepD has an aspartate while the weakest binder pepA has a proline at the same position. It appears that the negative charge of the aspartate in conjunction with the rigid conformation of proline at the 2nd position can be disruptive for the peptide-protein interaction.

Next, it was tested if the peptides identified in binding the PBP of Mp\BP could also bind FrhA. When testing pepB and pepC with the FrhA_PBD with FP, it was determined they bind the protein with EC50 of 370 nM and 333 nM, respectively (Fig.4). Competition assays with FP showed that FrhA complex with fluorescently-labelled pepB and pepC could be displaced by their unlabelled counterparts at low micro-molar concentrations. This further confirms that binding potency arises from the amino-acid sequence rather than that of hydrophobic fluorescent probe FITC, which could non- specifically interact with a protein. /nip

Sequential optimisation to provide low nano-molar affinities

Subsequently, the“hit” molecules were sequentially optimized. It appeared that C-terminal Aspartate (position 1) is of importance in the interaction as its carboxyl group is directly coordinated by the Ca2+ ion, while residues at positions 2 and 3 can be optimized to improve the potency. To obtain an optimal residue at the 2nd position, we fixed the 1st position with a C-terminal aspartate while varying its neighbouring residue with the 20 different amino acids. Residues from positions 3 to 5 were fixed with a sequence of Alanine-Glycine-Alanine, resulting in 20 peptides with a consensus sequence of AGAXD (X can be any residue) (SEQ ID NO.7). Most of these 20 peptides bind to FrhA with low micro-molar EC50 (Fig. 5 and Table 2). In line with results that threonine at position 2 improves the binding while proline is disruptive, AGATD (SEQ ID NO.8) was the optimal binder with an EC50 of 40 nM that is at least 3-fold stronger than any of the other peptides. In contrast, AGAPD (SEQ ID NO.38) and was the weakest binder with an EC50 of 10 mM.

With this information, we now fixed the residues at positions 1 and 2 with Asp and Thr, and proceeded to screen for the optimal amino acid for position 3 (AGXTD). While all peptides of AGXTD bind more strongly with affinity in nanomolar ranges, the three with aromatic side-chains stood out. In particular, AGYTD and AGWTD have an EC50 of 7 nM and 5 nM, respectively. These peptides are expected to have potent inhibitory activity to the adhesin.

Table 4: EC50 values determined by fluorescence polarization for peptides with amino-acid sequences of AGAXD (SEQ ID NO.7 ) on the left (SEQ ID NOs. 8 and 26- 44); and AGXTD (SEQ ID NO.9 ) on the right (SEQ ID NOs. 10, 12-13 and 45-61). The peptides that resulted in the highest EC50 bolded and highlighted (AGA-T-D (SEQ ID NO.8); AG-F-TD (SEQ ID NO.13); AG-W-TD (SEQ ID NO.10); and AG-Y-TD (SEQ ID

Peptide-protein interactions

In addition to the structure that shows the interaction of pepA with MplBP_PBD, we solved the X-ray crystal structures of the adhesin in complex with pepB and pepC at 2-A resolution. The PBD has an oblong b-sandwich fold, with the peptide binding cavity formed at one tip of the structure. Ca²⁺1 and Ca²⁺2 in the interior of the cavity are stably coordinated by the six and seven protein ligands, respectively. The octa- coordination of the two Ca²⁺ ions in the cavity were accomplished by binding to the peptides, where the carboxyl group of the aspartate contributes to Ca²⁺1 , whereas one of these oxygens simultaneously interacts with Ca²⁺2 (Fig.6).

At the 2nd position of pepB, serine has its side-chain hydroxyl group form a salt bridge with the side-chain amine of an asparagine residue of the PBD, while the main- chains also interact between these two residues (Fig.6B). As proline at the same position did not form these interactions, pepA binds over 100-fold weaker than that of pepB (Fig.6A). As for pepC, it has an identical binding mode as pepB, with the threonine side- chain hydroxyl anchored to the protein by interacting with an asparagine at the edge of the peptide-binding cavity. In contrast to pepB, the methyl group restrains the free rotation of the hydroxyl group of the threonine, which can explain why peptides with threonine at position 2 binds the PBD stronger than that of serine.

The peptide-protein complex structures have well-defined electron density extended to the 2^nd position. The density at the 3^rd position is too ambiguous to determine the conformation of its side chain, with little or no density for the following amino acids. This suggests the peptide is flexible beyond the 3rd position, thereby contributing little to the peptide-protein interaction. After fixing the 1^st and 2^nd positions with aspartate and threonine, aromatic residues showed the highest binding at the 3^rd position (Fig.5, right panel). Intriguingly, a tyrosine residue is located at the gate of the peptide-binding cavity of both the PBDs of Mp\BP and FrhA (Fig.6). Given its close proximity to the 3rd position of the complexed peptide, it is possible that the tyrosine of the protein interacts with an aromatic residue from the peptide via tt-p stacking, which can significantly enhance the interaction.

Interference of peptides with binding of Vibrio cholerae to cells The experiments below are to test disruption by the peptides of the binding of Vibrio cholerae to various cells. The mouse intestinal colonisation assay can indicate peptides that substantially reduce colonisation of Vibrio cholerae to the mouse intestine, indicating efficacy of the peptides for treating or preventing cholera.

Hemagglutination and hemolysis assays.

The hemagglutination and hemolysis assays are performed as described by (Gardel and Mekalanos, 1996, Infect. Immun. 64:2246-2255) with the following alterations. Vibrio cholerae strains are grown to an OD600 of 0.6 to 0.8 at 37°C, and then bacterial cells are pelleted, washed twice, and resuspended in KRT buffer at a concentration of 10¹⁰ CFU/ml. Bacterial cells are serially diluted in a round-bottomed 96- well microtiter plate. Human type“O” red blood cells are harvested by centrifugation, washed twice, and resuspended in KRT buffer at a 2% concentration. Red blood cells (0.2 ml) are added to 0.1 ml bacteria, the plate is incubated at room temperature for 30 to 60 min, and the hemagglutination titer is recorded. The titer is the reciprocal of the greatest dilution at which hemagglutination occurred. The hemolysis assay is performed in exactly the same manner as the hemagglutination assay; the plates are incubated for 20 h, at which time the OD540 in the supernatants is measured. The same procedure is repeated in the presence of 2 mM of a peptide (e.g. AGYTD) to assess peptide inhibition of Vibrio cholerae binding to red-blood cells.

HEp-2 cell binding assay

The HEp-2 cell binding assay is performed as described by (Gardel and Mekalanos, 1996, Infect. Immun. 64:2246-2255), with minor changes. Briefly, HEp-2 cells are grown in a 24-well plate on glass coverslips in Dulbecco’s modified Eagle’s medium (Gibco) with 10% FBS. Vibrio cholerae strains are grown to an OD600 of 0.2 to 0.4 at 37°C. Bacterial cells are then pelleted, washed twice, resuspended in a buffer that contains 20 mM Tris (pH7.5), 150 mM NaCI and 5 mM CaCI2 (Buffer A), and added to a monolayer of HEp-2 cells at a multiplicity of infection of 50:1. After incubation for 45 min at 30°C, the medium is aspirated, and wells are washed four times with Buffer A. The cells are then fixed by methanol for 5 min and stained with Giemsa (1 :12.5) for 25 min. Cells are mounted on a glass slide in FluorSave (Calbiochem) and imaged with a Zeiss Axiovert 200 fluorescence microscope. Twenty HEp-2 cells are counted for each bacterial strain, and the experiment is performed three separate times, with similar results. The same procedure is repeated in the presence of 2 mM of a peptide (e.g. AGYTD) to assess peptide inhibition of Vibrio cholerae binding to HEp-2 cells. Mouse intestinal colonization assays.

All animals are handled in strict accordance with good animal practice as defined by the relevant national and/or local animal welfare bodies. The competition assay for intestinal colonization in 5-day-old CD-1 suckling mice is as described by Gardel and Mekalanos (Gardel and Mekalanos, 1996, Infect. Immun. 64:2246-2255). The inocula consists of ~ 10⁵ CFU for both wild-type and mutant strains. The competitive indices are calculated by dividing the ratio of mutant to wild-type bacteria in the output by the ratio of mutant to wild-type bacteria in the input. For adult mouse colonization studies, 5- to 6- week-old female C57BL/6 mice (Harlan, Indianapolis, IN) are anesthetized, and then 50 mL of 8.5% (wt/vol) NaHC03 is administered intragastri cally, followed immediately by treatment with 50 mL of bacterial suspension in Buffer A, using a 22-gauge feeding needle. At 24 or 48 h post-inoculation, mice are euthanized, and small intestines are removed, weighed, and homogenized in Buffer A. The homogenates are serially diluted in Buffer A and plated on LB streptomycin agar plates. The detection limit is 100 CFU in the small intestine. The same procedure is repeated in the presence of 2 mM of a peptide (e.g. AGYTD) to assess peptide inhibition of Vibrio cholerae colonizing the mouse intestine.

Claims

1. A method for interfering with the binding of Vibrio cholerae to intestinal epithelial cells, comprising interfering with binding sites on an RTX adhesin FrhA protein of Vibrio cholerae with a peptide.

2. A method according to claim 1 , wherein said peptide has a length of 4-15 amino acids.

3. A method according to claim 1 or claim 2, wherein said peptide has a C-terminal Aspartate.

4. A method according to claim 3, wherein said peptide has at the C- terminus Threonine-Aspartate or Serine-Aspartate.

5. A method according to claim 3 or claim 4, wherein said peptide has at the C-terminus, Tyrosine, Tryptophan or Phenylalanine, followed by Threonine- Aspartate or Serine-Aspartate.

6. A method according to any one of claims 1-4, wherein said peptide comprises an amino acid sequence selected from the group consisting of AGWTD (SEQ ID NO. 10), AGYTD (SEQ ID NO. 12) and AGFTD (SEQ ID NO. 13).

7. A method according to any one of claims 1-4, wherein said peptide comprises an amino acid sequence at the C-terminus selected from the group consisting of AGWTD (SEQ ID NO. 10), AGYTD (SEQ ID NO. 12) and AGFTD (SEQ ID NO. 13).

8. A method according to any one of claims 1-7, wherein at least one of said peptides is produced by a microorganism.

9. A method according to claim 8, wherein said peptide is produced from a precursor polypeptide comprising cleavage by extracellular proteases.

10. A peptide that binds to the protein-binding domain of RTX adhesin FrhA of Vibrio cholerae with an EC50 of less than 100 nM.

11. A peptide that has an affinity for the protein-binding domain of RTX adhesin FrhA of Vibrio cholerae with an EC50 of less than 10 nM.

12. A peptide according to any one of claims 1 1 , wherein said peptide has a length of 4-15 amino acids.

13. A peptide according to claim 11 or claim 12 that binds to the protein-binding domain of RTX adhesin FrhA of Vibrio cholerae having a C-terminal Aspartate.

14. A peptide according to claim 13, wherein said peptide comprises at the C-terminus Threonine-Aspartate or Serine-Aspartate.

15. A peptide according to any one of claims 13 and 14, wherein said peptide comprises at the C-terminus, Tyrosine, Tryptophan or Phenylalanine, followed by Threonine-Aspartate or Serine-Aspartate.

16. A peptide comprising an amino acid sequence selected from the group consisting of AGWTD (SEQ ID NO. 10), AGYTD (SEQ ID NO. 12) and AGFTD (SEQ ID NO. 13).

17. A peptide comprising an amino acid sequence at the C-terminus selected from the group consisting of AGWTD (SEQ ID NO. 10), AGYTD (SEQ ID NO. 12) and AGFTD (SEQ ID NO. 13).

18. A peptide according to any one of claims 10-17, wherein said peptide is non-digestible.

19. A precursor polypeptide comprising a peptide according to any one of claims 10-17, wherein the peptide can be released by cleavage.

20. A precursor polypeptide comprising a plurality of peptides according to any one of claims 10-17, wherein the plurality of peptides can be released by cleavage.

21. A gene construct for expression of a peptide in accordance with any one of claims 10-17, or of a precursor polypeptide in accordance with claim 19 or 20.

22. A micro-organism comprising the gene construct according to claim

21.

23. A micro-organism according to claim 22, wherein said peptide or precursor polypeptide is secreted from the micro-organism.

24. A micro-organism according to claim 22 or claim 23, wherein said micro-organism is a probiotic, preferably a lactobacillus.

25. A micro-organism according to claim 22-24, wherein said micro organism is a human probiotic.

26. A peptide according to any one of claims 10-18, a precursor polypeptide according to any one of claims 19 and 20, or a micro-organism according to any one of claims 22-25, for use in a medical treatment.

27. A use according to claim 26, wherein said treatment is a prophylactic treatment.

28. A use according to claim 26 or 27, comprising the treatment of cholera.

29. A peptide according to any one of claims 10-18, a precursor polypeptide according to any one of claims 19 and 20, or a micro-organism according to any one of claims 22-25, for use in a medical treatment, wherein said use comprises interfering with binding of Vibrio cholerae to the intestine.

30. A peptide according to any one of claims 10-18, a precursor polypeptide according to any one of claims 19 and 20, or a micro-organism according to any one of claims 22-25, for use in a medical treatment, wherein said use comprises disrupting the formation of a biofilm of Vibrio cholerae.

31. A peptide according to any one of claims 10-18, a precursor polypeptide according to any one of claims 19 and 20, or a micro-organism according to any one of claims 22-25, for use in a medical treatment, wherein said use comprises preventing colonisation with Vibrio cholerae.

32. A composition comprising a peptide according to any one of claims 10-18, a precursor polypeptide according to any one of claims 19 and 20, or a micro organism according to any one of claims 22-25.

33. A composition according to claim 30, wherein said composition is for oral intake and preferably comprises an enteric coating.

34. A composition according to claim 32 or claim 33, wherein said composition is a pharmaceutical composition.

35. A method for identifying peptides that interfere with the binding of Vibrio cholerae to intestinal epithelial cells, comprising

- providing one or more peptides;

- providing the protein binding domain of RTX adhesin FrhA of Vibrio cholerae;

- contacting the one or more peptides with the protein-binding domain of RTX adhesin FrhA of Vibrio cholerae;

36. A method according to claim 35, wherein the one or more peptides are labelled, preferably with a FITC label.

37. A method according to claim 35 or claim 36, wherein the contacting step is carried out in the presence of Ca2+.

38. A method according to any one of claims 35-37, wherein the one or more peptides are peptides in accordance with any one of claims 10-18.

39. A method according to any one of claims 35-38, wherein a peptide is identified to interfere with said binding by having an EC50 of less than 100 nM.

40. A method according to claim 39, comprising the step of providing the amino acid sequence of said identified peptide, further comprising the step of synthesizing said peptide.