DOI: 10.5061/dryad.f1vhhmh8p
title: Peptide-based ligand antagonists block a <em>Vibrio cholerae</em> adhesin

description: Vibrio cholerae, the causative agent of cholera, uses surface proteins
 such as the repeats-in-toxin (RTX) adhesin FrhA to colonize hosts and
 initiate infection. Blocking bacterial adhesion represents a promising
 therapeutic strategy to treat infections without promoting drug
 resistance. FrhA contains a peptide-binding domain (PBD) that is key for
 hemagglutination, human epithelial cell binding, and V. cholerae biofilm
 formation. Previous studies identified a lead pentapeptide ligand with the
 sequence Ala-Gly-Tyr-Thr-Asp (AGYTD) that blocks V. cholerae colonization
 of the mouse small intestine at high micromolar concentrations. A
 structure-guided approach has now identified a minimal D-amino
 acid-containing tripeptide motif with higher affinity for the FrhA-PBD and
 predicted metabolic stability. Our results contribute to the development
 of anti-adhesion strategies to combat infections.
description: # Peptide-based ligand antagonists block a *Vibrio cholerae* adhesin
 Dataset DOI:
 [10.5061/dryad.f1vhhmh8p](https://doi.org/10.5061/dryad.f1vhhmh8p) ##
 Description of the data and file structure ### Microscale thermophoresis
 Microscale thermophoresis (MST) was performed in the presence of the
 fluorescently labeled FrhA~Split-PBD~ using unlabeled ligands such as
 AGYTD. FrhA~Split-PBD~ was labeled using the primary amine-based labeling
 kit of Red-NHS 2nd Generation (NanoTemper Technologies, San Francisco, CA,
 USA). The labeling reaction was performed in MST buffer (50 mM HEPES pH 8,
 150 mM NaCl, 5 mM CaCl~2~) with a protein concentration of 20 µM (molar
 ratio dye:protein ≈ 3:1). The reaction mixture was incubated at room
 temperature for 1 h in a dark environment for protection from
 photobleaching. Subsequently, the unreacted dye was removed from the
 labeled protein using the supplied desalting column, which was
 equilibrated with MST buffer. The degree of labeling (DOL) was determined
 using UV/Vis spectrophotometry at 650 and 280 nm—a DOL of 0.5 was
 typically achieved. After dialysis of the labeled protein against a buffer
 containing 50 mM Tris-HCl pH 9, 150 mM NaCl, 5 mM CaCl~2~, and 0.05% v/v
 Tween-20, the post-dialysis buffer was filtered and used to solubilize the
 ligands. A series of sixteen 1:1 dilutions of the ligands was prepared,
 resulting in ligand concentrations ranging from 440 μM to 13.4 nM. After 1
 h incubation, the samples were loaded into standard monolith NT.115
 capillaries from NanoTemper Technologies. The MST measurements were
 conducted using a Monolith NT.115 instrument at an ambient temperature of
 ~ 22 °C, with instrument parameters set to 20% excitation power and 40%
 MST power. Three independently prepared replicates were performed for each
 ligand. The NanoTemper MO.Affinity Analysis software (version 2.3,
 NanoTemper Technologies) was used to analyze the fluorescence data, fit
 sigmoidal binding models, and provide the dissociation constants. In the
 software, the K~d~ values were determined by plotting the change in
 normalized fluorescence [ΔF~norm~(‰) = F~1~/F~0~] against the logarithm of
 the concentrations of peptide ligands, where F~1~ and F~0~ respectively
 correspond to the heated and baseline regions of the thermophoresis traces
 [43]. The time between F~1~ and F~0~ was set to 20 seconds for analysis.
 ### Multiple Sequence Alignment Protein sequence alignment of the PBD and
 split domains of FrhA with their homologs in *Mp*IBP. The sequences
 forming the domains were determined by X-ray crystal structures
 (FrhA_Split_PBD PDB ID: 9YBQ; *Mp*IBP \_Split_PBD PDB ID: 6X5W). An
 asterisk (*) indicates positions which have a fully conserved residue. A
 colon (:) indicates conservation between groups of strongly similar
 properties, and a period (.) indicates conservation between groups of
 weakly similar properties. A blank space indicates no similarity at the
 position. Residues lining the ligand-binding pocket are indicated by red
 boxes. The alignment was generated by Clustal Omega on the EMBL-EBI Job
 Dispatcher. The sequences share 60.1% identity, and the residues lining
 the ligand-binding pocket are conserved. ### Files and variables ####
 File: Data_FEBSL-25-0854.zip **Description:**  This ZIP file contains two
 directories. The MST directory contains the following files: * FrhA_PBD
 20s_CompareResultsRawData.xlsx * Export-Table 1.csv * Fit-Values-Table
 1.csv The XLSX file contains two sheets. CSV files are respectively
 exported from sheet 1 and sheet 2 from the ELSX file.  The CSV files
 contain no additional information, but the XLSX contains graphs of plotted
 data.  The XLSX file contains data of MST experiments of FrhA_Split-PBD
 with all ligands shown in the paper. Both analysis results and raw
 measurements are included. The following variables are relevant for data
 re-processing: * Peptide Names (AGYTD, GYTD, YTD ...): The ligands tested
 and described in the main publication. * Target (FrhA 38 kDa): The
 macromolecule/protein construct described in the main publication. *
 TargetConcentration: The concentration of FrhA 38 kDa in M. * Bound: The
 fluorescence level in the Kd model is considered as the ligand fully bound
 in arbitrary units. * Unbound: The fluorescence level in the Kd model is
 considered as the ligand fully unbound in arbitrary units. * Kd: The
 dissociation constant determined by the Kd model in M. * TargetConc: Same
 as TargetConcentration. The value for all runs should be 0.00000008 M. *
 Standard Deviation: The standard deviation of Kd. * Dose: Concentration of
 the ligand/peptide in M. * [Peptide name]: The raw fluorescence level in
 arbitrary units. * DeltaFNorm: normalized change in fluorescence in
 arbitrary units. Note that Dose, [peptide name], and DeltaFNorm
 in Export-Table 1.csv show the fitted data, while these variables show the
 raw data in Fit-Values-Table 1.csv. The Multiple Sequence Alignment
 directory contains the following: Clustal Omega _ Job Dispatcher _
 EMBL-EBI.html  The HTML file can be opened by most browsers and requires
 the files in the directory to display the multiple sequence alignment
 results. The HTML file, when opened, displays the full sequence
 of FrhA_Split_PBD and MpIBP_Split_PBD. This serves as a permanent record
 of the multiple sequence alignment. The main data are the protein sequence
 and their alignment. Under the amino acid sequences, a row of symbols
 shows the amino acid matching. The symbol "*" indicates an exact
 amino acid match. The symbol " ": indicates strong amino acid
 similarity. The symbol ". indicates weak amino acid similarity. The
 space between them shows no amino acid similarity.