Effect of physicochemical and microbiological factors on the development of viable but non-culturable and resuscitation states of Vibrio cholerae.

BACKGROUND: Vibrio cholerae can endure harsh environmental conditions by transitioning into viable but non-culturable (VBNC) form and resuscitate upon return of appropriate conditions. METHOD: In this study, we assessed the impact of physicochemical and microbiological factors, on the development of low temperature-induced VBNC state and subsequent recovery by temperature upshift. RESULTS: In estuarine water, Vibrio cholerae exhibits a slower decline in culturability over a period of 77 days as compared to 10 days in fresh water. When variable cell numbers from different growth phases were used for VBNC induction, it was observed that the higher inoculum size (106-107 cfu ml-1) from the late log phase culture appears to be crucial for entering the VBNC state. Conversely, starved cells could enter the VBNC state with an initial inoculum of 104-105 cfu ml-1, followed by resuscitation as well. The addition of glucose, GlcNAc and mannitol differentially affects progression into VBNC, while the addition of tryptone, yeast extract and casamino acid facilitated early entry into the VBNC state and shortened the length of the recovery period. CONCLUSION: Altogether these findings demonstrated that the ionic strength of water, inoculum size and the availability of nutrients played distinct roles during VBNC induction and resuscitation.

Fuzzy recognition by the prokaryotic transcription factor HigA2 from Vibrio cholerae.

Disordered protein sequences can exhibit different binding modes, ranging from well-ordered folding-upon-binding to highly dynamic fuzzy binding. The primary function of the intrinsically disordered region of the antitoxin HigA2 from Vibrio cholerae is to neutralize HigB2 toxin through ultra-high-affinity folding-upon-binding interaction. Here, we show that the same intrinsically disordered region can also mediate fuzzy interactions with its operator DNA and, through interplay with the folded helix-turn-helix domain, regulates transcription from the higBA2 operon. NMR, SAXS, ITC and in vivo experiments converge towards a consistent picture where a specific set of residues in the intrinsically disordered region mediate electrostatic and hydrophobic interactions while "hovering" over the DNA operator. Sensitivity of the intrinsically disordered region to scrambling the sequence, position-specific contacts and absence of redundant, multivalent interactions, point towards a more specific type of fuzzy binding. Our work demonstrates how a bacterial regulator achieves dual functionality by utilizing two distinct interaction modes within the same disordered sequence.

Genomic characteristics of clinical non-toxigenic Vibrio cholerae isolates in Switzerland: a cross-sectional study.

STUDY AIMS: Although non-toxigenic Vibrio cholerae lack the ctxAB genes encoding cholera toxin, they can cause diarrhoeal disease and outbreaks in humans. In Switzerland, V. cholerae is a notifiable pathogen and all clinical isolates are analysed at the National Reference Laboratory for Enteropathogenic Bacteria and Listeria. Up to 20 infections are reported annually. In this study, we investigated the population structure and genetic characteristics of non-toxigenic V. cholerae isolates collected over five years. METHODS:  V. cholerae isolates were serotyped and non-toxigenic isolates identified using a ctxA-specific PCR. Following Illumina whole-genome sequencing, genome assemblies were screened for virulence and antibiotic resistance genes. Phylogenetic analyses were performed in the context of 965 publicly available V. cholerae genomes. RESULTS: Out of 33 V. cholerae infections reported between January 2017 and January 2022 in Switzerland, 31 were caused by ctxA-negative isolates. These non-toxigenic isolates originated from gastrointestinal (n = 29) or extraintestinal (n = 2) sites. They were phylogenetically diverse and belonged to 29 distinct sequence types. Two isolates were allocated to the lineage L3b, a ctxAB-negative but tcpA-positive clade previously associated with regional outbreaks. The remaining 29 isolates were placed in lineage L4, which is associated with environmental strains. Genes or mutations associated with reduced susceptibility to the first-line antibiotics fluoroquinolones and tetracyclines were identified in 11 and 3 isolates, respectively. One isolate was predicted to be multidrug resistant. CONCLUSIONS:  V. cholerae infections in Switzerland are rare and predominantly caused by lowly virulent ctxAB-negative and tcpA-negative strains. As V. cholerae is not endemic in Switzerland, cases are assumed to be acquired predominantly during travel. This assumption was supported by the phylogenetic diversity of the analysed isolates.

CRISPR antiphage defence mediated by the cyclic nucleotide-binding membrane protein Csx23.

CRISPR-Cas provides adaptive immunity in prokaryotes. Type III CRISPR systems detect invading RNA and activate the catalytic Cas10 subunit, which generates a range of nucleotide second messengers to signal infection. These molecules bind and activate a diverse range of effector proteins that provide immunity by degrading viral components and/or by disturbing key aspects of cellular metabolism to slow down viral replication. Here, we focus on the uncharacterised effector Csx23, which is widespread in Vibrio cholerae. Csx23 provides immunity against plasmids and phage when expressed in Escherichia coli along with its cognate type III CRISPR system. The Csx23 protein localises in the membrane using an N-terminal transmembrane α-helical domain and has a cytoplasmic C-terminal domain that binds cyclic tetra-adenylate (cA4), activating its defence function. Structural studies reveal a tetrameric structure with a novel fold that binds cA4 specifically. Using pulse EPR, we demonstrate that cA4 binding to the cytoplasmic domain of Csx23 results in a major perturbation of the transmembrane domain, consistent with the opening of a pore and/or disruption of membrane integrity. This work reveals a new class of cyclic nucleotide binding protein and provides key mechanistic detail on a membrane-associated CRISPR effector.

Many anti-viral defence systems generate a cyclic nucleotide signal that activates cellular defences in response to infection. Type III CRISPR systems use a specialised polymerase to make cyclic oligoadenylate (cOA) molecules from ATP. These can bind and activate a range of effector proteins that slow down viral replication. In this study, we focussed on the Csx23 effector from the human pathogen Vibrio cholerae ­ a trans-membrane protein that binds a cOA molecule, leading to anti-viral immunity. Structural studies revealed a new class of nucleotide recognition domain, where cOA binding is transmitted to changes in the trans-membrane domain, most likely resulting in membrane depolarisation. This study highlights the diversity of mechanisms for anti-viral defence via nucleotide signalling.

Sporadic regional re-emergent cholera: a 19th century problem in the 21st century.

Cholera, caused by Vibrio cholerae, is a severe diarrheal disease that necessitates prompt diagnosis and effective treatment. This review comprehensively examines various diagnostic methods, from traditional microscopy and culture to advanced nucleic acid testing like polymerase spiral reaction and rapid diagnostic tests, highlighting their advantages and limitations. Additionally, we explore evolving treatment strategies, with a focus on the challenges posed by antibiotic resistance due to the activation of the SOS response pathway in V. cholerae. We discuss promising alternative treatments, including low-pressure plasma sterilization, bacteriophages, and selenium nanoparticles. The paper emphasizes the importance of multidisciplinary approaches combining novel diagnostics and treatments in managing and preventing cholera, a persistent global health challenge. The current re-emergent 7th pandemic of cholera commenced in 1961 and shows no signs of abeyance. This is probably due to the changing genetic profile of V. cholerae concerning bacterial pathogenic toxins. Given this factor, we argue that the disease is effectively re-emergent, particularly in Eastern Mediterranean countries such as Lebanon, Syria, etc. This review considers the history of the current pandemic, the genetics of the causal agent, and current treatment regimes. In conclusion, cholera remains a significant global health challenge that requires prompt diagnosis and effective treatment. Understanding the history, genetics, and current treatments is crucial in effectively addressing this persistent and re-emergent disease.

Cell-lysis sensing drives biofilm formation in Vibrio cholerae.

Matrix-encapsulated communities of bacteria, called biofilms, are ubiquitous in the environment and are notoriously difficult to eliminate in clinical and industrial settings. Biofilm formation likely evolved as a mechanism to protect resident cells from environmental challenges, yet how bacteria undergo threat assessment to inform biofilm development remains unclear. Here we find that population-level cell lysis events induce the formation of biofilms by surviving Vibrio cholerae cells. Survivors detect threats by sensing a cellular component released through cell lysis, which we identify as norspermidine. Lysis sensing occurs via the MbaA receptor with genus-level specificity, and responsive biofilm cells are shielded from phage infection and attacks from other bacteria. Thus, our work uncovers a connection between bacterial lysis and biofilm formation that may be broadly conserved among microorganisms.

Emerging resistome diversity in clinical Vibrio cholerae strains revealing their role as potential reservoirs of antimicrobial resistance.

BACKGROUND: This is a unique and novel study delineating the genotyping and subsequent prediction of AMR determinants of Vibrio cholerae revealing the potential of contemporary strains to serve as precursors of severe AMR crisis in cholera. METHODS AND RESULTS: Genotyping of representative strains, VC1 and VC2 was undertaken to characterize antimicrobial resistance genes (ARGs) against chloramphenicol, SXT, nalidixic acid and streptomycin against which they were found to be resistant by antibiogram analysis in our previous investigation. strAB, sxt, sul2, qace∆1-sul1 were detected by PCR. Genome annotation and identification of ARGs with WGS helped to detect the presence of almG, varG, strA (APH(3'')-Ib), strB (APH(6)-Id), sul2, catB9, floR, CRP, dfrA1 genes. Signatures of resistance determinants and protein domains involved in antimicrobial resistance, primarily, efflux of antibiotics were identified on the basis of 30-100% homology to reference proteins. These domains were predicted to be involved in other metabolic functions on the basis of 100% identity with 100% coverage with reference protein and nucleotide sequences and were predicted to be of a diverse taxonomic origin accentuating the influence of the microbiota on AMR acquisition. Sequence analysis of QRDR (quinolone resistance-determining region) revealed SNPs. Cytoscape v3.8.2 was employed to analyse protein-protein interaction of MDR proteins, MdtA and EmrD-2, with nodes of vital AMR pathways. Vital nodes involved in efflux of different classes of antibiotics were found to be absent in VC1 and VC2 justifying the sensitivity of these strains to most antibiotics. CONCLUSIONS: The study helped to examine the resistome of VC isolated from recent outbreaks to understand the underlying reason of sensitivity to most antibiotics and also to characterize the ARGs in their genome. It revealed that VC is a reservoir of signatures of resistance determinants and serving as precursors for severe AMR crisis in cholera. This is the first study, to our knowledge, which has scrutinized and presented systematically, information on prospective domains which bear the potential of serving as AMR determinants in VC with the help of bioinformatic tools. This pioneering approach may help in the prediction of AMR landfalls and benefit epidemiological surveillance and early warning systems.

Smelly shark, smelly ray: what is infecting you?

AIMS: Although elasmobranchs are consumed worldwide, bacteriological assessments for this group are still sorely lacking. In this context, this study assessed bacteria of sharks and rays from one of the most important landing ports along the Rio de Janeiro coast. METHODS AND RESULTS: Bacteria were isolated from the cloacal swabs of the sampled elasmobranchs. They were cultured, and Vibrio, Aeromonas, and Enterobacterales were isolated and identified. The isolated bacteria were then biochemically identified and antimicrobial susceptibility assays were performed. Antigenic characterizations were performed for Salmonella spp. and Polymerase Chain Reaction (PCR) assays were performed to identify Escherichia coli pathotypes. Several bacteria of interest in the One Health context were detected. The most prevalent Enterobacterales were Morganella morganii and Citrobacter freundii, while Vibrio harveyi and Vibrio fluvialis were the most prevalent among Vibrio spp. and Aeromonas allosacharophila and Aeromonas veronii bv. veronii were the most frequent among Aeromonas spp. Several bacteria also displayed antimicrobial resistance, indicative of Public Health concerns. A total of 10% of Vibrio strains were resistant to trimethoprim-sulfamethoxazole and 40% displayed intermediate resistance to cefoxitin. Salmonella enterica strains displayed intermediate resistance to ciprofloxacin, nalidixic acid and streptomycin. All V. cholerae strains were identified as non-O1/non-O139. The detected E. coli strains did not exhibit pathogenicity genes. This is the first study to perform serology assessments for S. enterica subsp. enterica isolated from elasmobranchs, identifying the zoonotic Typhimurium serovar. Salmonella serology evaluations are, therefore, paramount to identify the importance of elasmobranchs in the epidemiological salmonellosis chain. CONCLUSIONS: The detection of several pathogenic and antibiotic-resistant bacteria may pose significant Public Health risks in Brazil, due to high elasmobranch consumption rates, indicating the urgent need for further bacteriological assessments in this group.

Clinical surveillance systems obscure the true cholera infection burden in an endemic region.

Our understanding of cholera transmission and burden largely relies on clinic-based surveillance, which can obscure trends, bias burden estimates and limit the impact of targeted cholera-prevention measures. Serological surveillance provides a complementary approach to monitoring infections, although the link between serologically derived infections and medically attended disease incidence-shaped by immunological, behavioral and clinical factors-remains poorly understood. We unravel this cascade in a cholera-endemic Bangladeshi community by integrating clinic-based surveillance, healthcare-seeking and longitudinal serological data through statistical modeling. Combining the serological trajectories with a reconstructed incidence timeline of symptomatic cholera, we estimated an annual Vibrio cholerae O1 infection incidence rate of 535 per 1,000 population (95% credible interval 514-556), with incidence increasing by age group. Clinic-based surveillance alone underestimated the number of infections and reported cases were not consistently correlated with infection timing. Of the infections, 4 in 3,280 resulted in symptoms, only 1 of which was reported through the surveillance system. These results impart insights into cholera transmission dynamics and burden in the epicenter of the seventh cholera pandemic, where >50% of our study population had an annual V. cholerae O1 infection, and emphasize the potential for a biased view of disease burden and infection risk when depending solely on clinical surveillance data.

The Biofilm Lifestyle Shapes the Evolution of ß-Lactamases.

The evolutionary relationship between the biofilm lifestyle and antibiotic resistance enzymes remains a subject of limited understanding. Here, we investigate how ß-lactamases affect biofilm formation in Vibrio cholerae and how selection for a biofilm lifestyle impacts the evolution of these enzymes. Genetically diverse ß-lactamases expressed in V. cholerae displayed a strong inhibitory effect on biofilm production. To understand how natural evolution affects this antagonistic pleiotropy, we randomly mutagenized a ß-lactamase and selected for elevated biofilm formation. Our results revealed that biofilm evolution selects for ß-lactamase variants able to hydrolyze ß-lactams without inhibiting biofilms. Mutational analysis of evolved variants demonstrated that restoration of biofilm development was achieved either independently of enzymatic function or by actively leveraging enzymatic activity. Taken together, the biofilm lifestyle can impose a profound selective pressure on antimicrobial resistance enzymes. Shedding light on such evolutionary interplays is of importance to understand the factors driving antimicrobial resistance.

Vibriosis in South Asia: A systematic review and meta-analysis.

OBJECTIVES: South Asia remains home to foodborne diseases caused by the Vibrio species. We aimed to compile and update information on the epidemiology of vibriosis in South Asia. METHODS: For this systematic review and meta-analysis, we searched PubMed, Web of Science, EMBASE, and Google Scholar for studies related to vibriosis in South Asia published up to May 2023. A random-effects meta-analysis was used to estimate the pooled isolation rate of non-cholera-causing Vibrio species. RESULTS: In total, 38 studies were included. Seven of these were case reports and 22 were included in the meta-analysis. The reported vibriosis cases were caused by non-O1/non-O139 V. cholerae, V. parahaemolyticus, V. fluvialis, and V. vulnificus. The overall pooled isolation rate was 4.0% (95% confidence interval [CI] 3.0-5.0%) in patients with diarrhea. Heterogeneity was high (I2 = 98.0%). The isolation rate of non-O1/non-O139 V. cholerae, V. parahaemolyticus, and V. fluvialis were 9.0 (95% CI 7.0-10.0%), 1.0 (95% CI 1.0-2.0%), and 2.0 (95% CI: 1.0-3.0%), respectively. Regarding V. parahaemolyticus, O3:K6 was the most frequently isolated serotype. Cases peaked during summer. Several studies reported antibiotic-resistant strains and those harboring extended-spectrum beta-lactamases genes. CONCLUSIONS: This study demonstrates a high burden of infections caused by non-cholera-causing Vibrio species in South Asia.

Vibrio cholerae arrests intestinal epithelial proliferation through T6SS-dependent activation of the bone morphogenetic protein pathway.

To maintain an effective barrier, intestinal progenitor cells must divide at a rate that matches the loss of dead and dying cells. Otherwise, epithelial breaches expose the host to systemic infection by gut-resident microbes. Unlike most pathogens, Vibrio cholerae blocks tissue repair by arresting progenitor proliferation in the Drosophila model. At present, we do not understand how V. cholerae circumvents such a critical antibacterial defense. We find that V. cholerae blocks epithelial repair by activating the growth inhibitor bone morphogenetic protein (BMP) pathway in progenitors. Specifically, we show that interactions between V. cholerae and gut commensals initiate BMP signaling via host innate immune defenses. Notably, we find that V. cholerae also activates BMP and arrests proliferation in zebrafish intestines, indicating an evolutionarily conserved link between infection and failure in tissue repair. Our study highlights how enteric pathogens engage host immune and growth regulatory pathways to disrupt intestinal epithelial repair.

Mathematical modeling of cholera dynamics with intrinsic growth considering constant interventions.

A mathematical model that describes the dynamics of bacterium vibrio cholera within a fixed population considering intrinsic bacteria growth, therapeutic treatment, sanitation and vaccination rates is developed. The developed mathematical model is validated against real cholera data. A sensitivity analysis of some of the model parameters is also conducted. The intervention rates are found to be very important parameters in reducing the values of the basic reproduction number. The existence and stability of equilibrium solutions to the mathematical model are also carried out using analytical methods. The effect of some model parameters on the stability of equilibrium solutions, number of infected individuals, number of susceptible individuals and bacteria density is rigorously analyzed. One very important finding of this research work is that keeping the vaccination rate fixed and varying the treatment and sanitation rates provide a rapid decline of infection. The fourth order Runge-Kutta numerical scheme is implemented in MATLAB to generate the numerical solutions.

Whole genome sequence of Vibrio cholerae NB-183 isolated from freshwater in Ontario, Canada harbors a unique gene repertoire.

OBJECTIVE: Vibrio cholerae is an enteric pathogen that poses a significant threat to global health. It causes severe dehydrating diarrheal disease cholera in humans. V. cholerae could be acquired either from consuming contaminated seafood or direct contact with polluted waters. As part of a larger program that assesses the microbial community profile in aquatic systems, V. cholerae strain NB-183 was isolated and characterized using a combination of culture- and whole-genome sequencing-based approaches. DATA DESCRIPTION: Here we report the assembled and annotated whole-genome sequence of a V. cholerae strain NB-183 isolated from a recreational freshwater lake in Ontario, Canada. The genome was sequenced using short-read Illumina systems. The whole-genome sequencing yielded 4,112,549 bp genome size with 99 contigs with an average genome coverage of 96× and 47.42% G + C content. The whole genome-based comparison, phylogenomic and gene repertoire indicates that this strain harbors multiple virulence genes and biosynthetic gene clusters. This genome sequence and its associated datasets provided in this study will be an indispensable resource to enhance the understanding of the functional, ecological, and evolutionary dynamics of V. cholerae.

Total Synthesis of Vibrio Cholerae O43 Tetrasaccharide Repeating Unit.

Vibrio cholerae is a pathogen responsible for the deadly pandemic - cholera. The glycans present on the surface of various strains of V. cholerae are considered as potential vaccine candidates. The tetrasaccharide repeating unit (RU) of V. cholerae O43 is decorated with less-explored rare deoxy amino sugars like d-quinosamine and d-viosamine, along with a rare amino acid, N-acetyl-l-allothreonine. Herein, we report a detailed account of the total synthesis of V. cholerae O43 tetrasaccharide RU. In our earlier attempt, while a one-pot assembly of trisaccharide was successful, the final coupling with a fully functionalized d-viosamine donor was low yielding. The successful route involved employing the Fmoc-protected d-viosamine building block as a donor and a late-stage amide bond formation of the tetrasaccharide.

A Vibrio cholerae viral satellite maximizes its spread and inhibits phage by remodeling hijacked phage coat proteins into small capsids.

Phage satellites commonly remodel capsids they hijack from the phages they parasitize, but only a few mechanisms regulating the change in capsid size have been reported. Here, we investigated how a satellite from Vibrio cholerae, phage-inducible chromosomal island-like element (PLE), remodels the capsid it has been predicted to steal from the phage ICP1 (Netter et al., 2021). We identified that a PLE-encoded protein, TcaP, is both necessary and sufficient to form small capsids during ICP1 infection. Interestingly, we found that PLE is dependent on small capsids for efficient transduction of its genome, making it the first satellite to have this requirement. ICP1 isolates that escaped TcaP-mediated remodeling acquired substitutions in the coat protein, suggesting an interaction between these two proteins. With a procapsid-like particle (PLP) assembly platform in Escherichia coli, we demonstrated that TcaP is a bona fide scaffold that regulates the assembly of small capsids. Further, we studied the structure of PLE PLPs using cryogenic electron microscopy and found that TcaP is an external scaffold that is functionally and somewhat structurally similar to the external scaffold, Sid, encoded by the unrelated satellite P4 (Kizziah et al., 2020). Finally, we showed that TcaP is largely conserved across PLEs. Together, these data support a model in which TcaP directs the assembly of small capsids comprised of ICP1 coat proteins, which inhibits the complete packaging of the ICP1 genome and permits more efficient packaging of replicated PLE genomes.

Spatial constraints and stochastic seeding subvert microbial arms race.

Surface attached communities of microbes grow in a wide variety of environments. Often, the size of these microbial community is constrained by their physical surroundings. However, little is known about how size constraints of a colony impact the outcome of microbial competitions. Here, we use individual-based models to simulate contact killing between two bacterial strains with different killing rates in a wide range of community sizes. We found that community size has a substantial impact on outcomes; in fact, in some competitions the identity of the most fit strain differs in large and small environments. Specifically, when at a numerical disadvantage, the strain with the slow killing rate is more successful in smaller environments than in large environments. The improved performance in small spaces comes from finite size effects; stochastic fluctuations in the initial relative abundance of each strain in small environments lead to dramatically different outcomes. However, when the slow killing strain has a numerical advantage, it performs better in large spaces than in small spaces, where stochastic fluctuations now aid the fast killing strain in small communities. Finally, we experimentally validate these results by confining contact killing strains of Vibrio cholerae in transmission electron microscopy grids. The outcomes of these experiments are consistent with our simulations. When rare, the slow killing strain does better in small environments; when common, the slow killing strain does better in large environments. Together, this work demonstrates that finite size effects can substantially modify antagonistic competitions, suggesting that colony size may, at least in part, subvert the microbial arms race.

Identification of promoter activity in gene-less cassettes from Vibrionaceae superintegrons.

Integrons are genetic platforms that acquire new genes encoded in integron cassettes (ICs), building arrays of adaptive functions. ICs generally encode promoterless genes, whose expression relies on the platform-associated Pc promoter, with the cassette array functioning as an operon-like structure regulated by the distance to the Pc. This is relevant in large sedentary chromosomal integrons (SCIs) carrying hundreds of ICs, like those in Vibrio species. We selected 29 gene-less cassettes in four Vibrio SCIs, and explored whether their function could be related to the transcription regulation of adjacent ICs. We show that most gene-less cassettes have promoter activity on the sense strand, enhancing the expression of downstream cassettes. Additionally, we identified the transcription start sites of gene-less ICs through 5'-RACE. Accordingly, we found that most of the superintegron in Vibrio cholerae is not silent. These promoter cassettes can trigger the expression of a silent dfrB9 cassette downstream, increasing trimethoprim resistance >512-fold in V. cholerae and Escherichia coli. Furthermore, one cassette with an antisense promoter can reduce trimethoprim resistance when cloned downstream. Our findings highlight the regulatory role of gene-less cassettes in the expression of adjacent cassettes, emphasizing their significance in SCIs and their clinical importance if captured by mobile integrons.

Total Synthesis of a Conjugation-Ready Tetrasaccharide Repeating Unit of Vibrio cholerae O:3 O-antigen Polysaccharide.

Herein, we report the first total synthesis of the tetrasaccharide repeating unit of Vibrio cholerae O:3 O-antigen polysaccharide. The highly complex tetrasaccharide contains rare amino sugars such as d-bacillosamine and l-fucosamine, highly labile sugar ascarylose, and higher carbon sugar d-d-heptose. Stereoselective glycosylation of the notoriously reactive ascarylose with d-d-heptose, poor nucleophilicity of the axial C4-OH of l-fucosamine, and amide coupling are the key challenges encountered in the total synthesis, which was completed via a longest linear sequence of 23 steps in 4.2% overall yield.

Sugar-binding and split domain combinations in repeats-in-toxin adhesins from Vibrio cholerae and Aeromonas veronii mediate cell-surface recognition and hemolytic activities.

Many pathogenic Gram-negative bacteria use repeats-in-toxin adhesins for colonization and biofilm formation. In the cholera agent Vibrio cholerae, flagellar-regulated hemagglutinin A (FrhA) enables these functions. Using bioinformatic analysis, a sugar-binding domain was identified in FrhA adjacent to a domain of unknown function. AlphaFold2 indicated the boundaries of both domains to be slightly shorter than previously predicted and assisted in the recognition of the unknown domain as a split immunoglobulin-like fold that can assist in projecting the sugar-binding domain toward its target. The AlphaFold2-predicted structure is in excellent agreement with the molecular envelope obtained from small-angle X-ray scattering analysis of a recombinant construct spanning the sugar-binding and unknown domains. This two-domain construct was probed by glycan micro-array screening and showed binding to mammalian fucosylated glycans, some of which are characteristic erythrocyte markers and intestinal cell epitopes. Isothermal titration calorimetry further showed the construct-bound l-fucose with a Kd of 21 µM. Strikingly, this recombinant protein construct bound and lysed erythrocytes in a concentration-dependent manner, and its hemolytic activity was blocked by the addition of l-fucose. A protein ortholog construct from Aeromonas veronii was also produced and showed a similar glycan-binding pattern, binding affinity, erythrocyte-binding, and hemolytic activities. As demonstrated here with Hep-2 cells, fucose-based inhibitors of this sugar-binding domain can potentially be developed to block colonization by V. cholerae and other pathogenic bacteria that share this adhesin domain.IMPORTANCEThe bacterium, Vibrio cholerae, which causes cholera, uses an adhesion protein to stick to human cells and begin the infection process. One part of this adhesin protein binds to a particular sugar, fucose, on the surface of the target cells. This binding can lead to colonization and killing of the cells by the bacteria. Adding l-fucose to the bacteria before they bind to the human cells can prevent attachment and has promise as a preventative drug to protect against cholera.