Biofilm formation on human immune cells is a multicellular predation strategy of Vibrio cholerae.

Biofilm formation is generally recognized as a bacterial defense mechanism against environmental threats, including antibiotics, bacteriophages, and leukocytes of the human immune system. Here, we show that for the human pathogen Vibrio cholerae, biofilm formation is not only a protective trait but also an aggressive trait to collectively predate different immune cells. We find that V. cholerae forms biofilms on the eukaryotic cell surface using an extracellular matrix comprising primarily mannose-sensitive hemagglutinin pili, toxin-coregulated pili, and the secreted colonization factor TcpF, which differs from the matrix composition of biofilms on other surfaces. These biofilms encase immune cells and establish a high local concentration of a secreted hemolysin to kill the immune cells before the biofilms disperse in a c-di-GMP-dependent manner. Together, these results uncover how bacteria employ biofilm formation as a multicellular strategy to invert the typical relationship between human immune cells as the hunters and bacteria as the hunted.

Interpersonal Gut Microbiome Variation Drives Susceptibility and Resistance to Cholera Infection.

The gut microbiome is the resident microbial community of the gastrointestinal tract. This community is highly diverse, but how microbial diversity confers resistance or susceptibility to intestinal pathogens is poorly understood. Using transplantation of human microbiomes into several animal models of infection, we show that key microbiome species shape the chemical environment of the gut through the activity of the enzyme bile salt hydrolase. The activity of this enzyme reduced colonization by the major human diarrheal pathogen Vibrio cholerae by degrading the bile salt taurocholate that activates the expression of virulence genes. The absence of these functions and species permits increased infection loads on a personal microbiome-specific basis. These findings suggest new targets for individualized preventative strategies of V. cholerae infection through modulating the structure and function of the gut microbiome.

High-Resolution Whole-Genome Analysis of Sister-Chromatid Contacts.

Sister-chromatid cohesion describes the orderly association of newly replicated DNA molecules behind replication forks. It plays an essential role in the maintenance and faithful transmission of genetic information. Cohesion is created by DNA topological links and proteinaceous bridges, whose formation and deposition could be potentially affected by many processes. Current knowledge on cohesion has been mainly gained by fluorescence microscopy observation. However, the resolution limit of microscopy and the restricted number of genomic positions that can be simultaneously visualized considerably hampered progress. Here, we present a high-throughput methodology to monitor sister-chromatid contacts (Hi-SC2). Using the multi-chromosomal Vibrio cholerae bacterium as a model, we show that Hi-SC2 permits to monitor local variations in sister-chromatid cohesion at a high resolution over a whole genome.

Emerging Concepts in Cholera Vaccine Design.

Cholera is a severe diarrheal disease caused by the bacterium Vibrio cholerae and constitutes a significant public health threat in many areas of the world. V. cholerae infection elicits potent and long-lasting immunity, and efforts to develop cholera vaccines have been ongoing for more than a century. Currently available inactivated two-dose oral cholera vaccines are increasingly deployed to both prevent and actively curb cholera outbreaks, and they are key components of the global effort to eradicate cholera. However, these killed whole-cell vaccines have several limitations, and a variety of new oral and nonoral cholera vaccine platforms have recently been developed. Here, we review emerging concepts in cholera vaccine design and implementation that have been driven by insights from human and animal studies. As a prototypical vaccine-preventable disease, cholera continues to be an excellent target for the development and application of cutting-edge technologies and platforms that may transform vaccinology.

A peptide-binding domain shared with an Antarctic bacterium facilitates Vibrio cholerae human cell binding and intestinal colonization.

Vibrio cholerae, the causative agent of the disease cholera, is responsible for multiple pandemics. V. cholerae binds to and colonizes the gastrointestinal tract within the human host, as well as various surfaces in the marine environment (e.g., zooplankton) during interepidemic periods. A large adhesin, the Flagellar Regulated Hemagglutinin A (FrhA), enhances binding to erythrocytes and epithelial cells and enhances intestinal colonization. We identified a peptide-binding domain (PBD) within FrhA that mediates hemagglutination, binding to epithelial cells, intestinal colonization, and facilitates biofilm formation. Intriguingly, this domain is also found in the ice-binding protein of the Antarctic bacterium Marinomonas primoryensis, where it mediates binding to diatoms. Peptide inhibitors of the M. primoryensis PBD inhibit V. cholerae binding to human cells as well as to diatoms and inhibit biofilm formation. Moreover, the M. primoryensis PBD inserted into FrhA allows V. cholerae to bind human cells and colonize the intestine and also enhances biofilm formation, demonstrating the interchangeability of the PBD from these bacteria. Importantly, peptide inhibitors of PBD reduce V. cholerae intestinal colonization in infant mice. These studies demonstrate how V. cholerae uses a PBD shared with a diatom-binding Antarctic bacterium to facilitate intestinal colonization in humans and biofilm formation in the environment.

ToxR activates the Vibrio cholerae virulence genes by tethering DNA to the membrane through versatile binding to multiple sites.

ToxR, a Vibrio cholerae transmembrane one-component signal transduction factor, lies within a regulatory cascade that results in the expression of ToxT, toxin coregulated pilus, and cholera toxin. While ToxR has been extensively studied for its ability to activate or repress various genes in V. cholerae, here we present the crystal structures of the ToxR cytoplasmic domain bound to DNA at the toxT and ompU promoters. The structures confirm some predicted interactions, yet reveal other unexpected promoter interactions with implications for other potential regulatory roles for ToxR. We show that ToxR is a versatile virulence regulator that recognizes diverse and extensive, eukaryotic-like regulatory DNA sequences, that relies more on DNA structural elements than specific sequences for binding. Using this topological DNA recognition mechanism, ToxR can bind both in tandem and in a twofold inverted-repeat-driven manner. Its regulatory action is based on coordinated multiple binding to promoter regions near the transcription start site, which can remove the repressing H-NS proteins and prepares the DNA for optimal interaction with the RNA polymerase.

Retinal Input to Macaque Superior Colliculus Derives from Branching Axons Projecting to the Lateral Geniculate Nucleus.

The superior colliculus receives a direct projection from retinal ganglion cells. In primates, it remains unknown if the same ganglion cells also supply the lateral geniculate nucleus. To address this issue, a double-label experiment was performed in two male macaques. The animals fixated a target while injection sites were scouted in the superior colliculus by recording and stimulating with a tetrode. Once suitable sites were identified, cholera toxin subunit B-Alexa Fluor 488 was injected via an adjacent micropipette. In a subsequent acute experiment, cholera toxin subunit B-Alexa Fluor 555 was injected into the lateral geniculate nucleus at matching retinotopic locations. After a brief survival period, ganglion cells were examined in retinal flatmounts. The percentage of double-labeled cells varied locally, depending on the relative efficiency of retrograde transport by each tracer and the precision of retinotopic overlap of injection sites in each target nucleus. In counting boxes with extensive overlap, 76-98% of ganglion cells projecting to the superior colliculus were double labeled. Cells projecting to the superior colliculus constituted 4.0-6.7% of the labeled ganglion cell population. In one particularly large zone, there were 5,746 cells labeled only by CTB-AF555, 561cells double labeled by CTB-AF555 and CTB-AF488, but no cell labeled only by CTB-AF488. These data indicate that retinal input to the macaque superior colliculus arises from a collateral axonal branch supplied by ∼5% of the ganglion cells that project to the lateral geniculate nucleus. Surprisingly, there exist no ganglion cells that project exclusively to the SC.

A polyvalent multiepitope protein cross-protects against Vibrio cholerae infection in rabbit colonization and passive protection models.

Using epitope- and structure-based multiepitope fusion antigen vaccinology platform, we constructed a polyvalent protein immunogen that presents antigenic domains (epitopes) of Vibrio cholerae toxin-coregulated pilus A, cholera toxin (CT), sialidase, hemolysin A, flagellins (B, C, and D), and peptides mimicking lipopolysaccharide O-antigen on a flagellin B backbone. Mice and rabbits immunized intramuscularly with this polyvalent protein immunogen developed antibodies to all of the virulence factors targeted by the immunogen except lipopolysaccharide. Mouse and rabbit antibodies exhibited functional activities against CT enterotoxicity, CT binding to GM1 ganglioside, bacterial motility, and in vitro adherence of V. cholerae O1, O139, and non-O1/non-O139 serogroup strains. When challenged orogastrically with V. cholerae O1 El Tor N16961 or a non-O1/non-O139 strain, rabbits IM immunized with the immunogen showed a 2-log (99%) reduction in V. cholerae colonization of small intestines. Moreover, infant rabbits born to the mother immunized with the protein immunogen acquired antibodies passively and were protected from bacterial intestinal colonization (>2-log reduction), severe diarrhea (100%), and mild diarrhea (88%) after infection with V. cholerae O1 El Tor (N16961), O1 classical (O395), O139 (Bengal), or a non-O1/non-O139 strain. This study demonstrated that this polyvalent cholera protein is broadly immunogenic and cross-protective, and an adult rabbit colonization model and an infant rabbit passive protection model fill a gap in preclinical efficacy assessment in cholera vaccine development.

ER-transiting bacterial toxins amplify STING innate immune responses and elicit ER stress.

The cGAS/STING sensor system drives innate immune responses to intracellular microbial double-stranded DNA (dsDNA) and bacterial cyclic nucleotide second messengers (e.g., c-di-AMP). STING-dependent cell-intrinsic responses can increase resistance to microbial infection and speed pathogen clearance. Correspondingly, STING activation and signaling are known to be targeted for suppression by effectors from several bacterial pathogens. Whether STING responses are also positively regulated through sensing of specific bacterial effectors is less clear. We find that STING activation through dsDNA, by its canonical ligand 2'-3' cGAMP, or the small molecule DMXAA is potentiated following intracellular delivery of the AB5 toxin family member pertussis toxin from Bordetella pertussis or the B subunit of cholera toxin from Vibrio cholerae. Entry of pertussis toxin or cholera toxin B into mouse macrophages triggers markers of endoplasmic reticulum (ER) stress and enhances ligand-dependent STING responses at the level of STING receptor activation in a manner that is independent of toxin enzymatic activity. Our results provide an example in which STING responses integrate information about the presence of relevant ER-transiting bacterial toxins into the innate inflammatory response and may help to explain the in vivo adjuvant effects of catalytically inactive toxins.

Ethiopia National Cholera Elimination Plan 2022-2028: Experiences, Challenges, and the Way Forward.

Cholera remains a significant public health concern in Ethiopia. More than 15.9 million Ethiopians, constituting 15% of the total population, live in areas with a history of recurrent cholera outbreaks. The last 9 years of national cholera surveillance data show the country has been experiencing cholera outbreaks every year. The current cholera outbreak, starting in August 2022, has affected the entire country, with 841 reported cases and a 3.13% case fatality rate (CFR) in 2022, and >30 000 cases with nearly a 1.4% CFR in 2023. In line with "Ending Cholera-A Global Roadmap to 2030," the government of Ethiopia is committed to eliminate cholera in the country and has prepared its "National Cholera Elimination Plan (NCP): 2022-2028" with aims to achieve zero local transmission in cholera hotspot areas by 2028 and 90% fatality reduction from the recent (2020-2022) average of 1.8% CFR. The plan is multisectoral, has a clear coordination platform, contains all interventions with in-depth situational analysis, is concordant with existing plans and strategies, and is cascaded at the regional level and implemented with existing government and public structures. Nationwide, total 118 cholera hotspot woredas (districts) were identified, and a comprehensive situation analysis of the existing cholera outbreak response capacity was assessed. This multisectoral and multiyear NCP has forecasted around US$404 million budget estimates with >90% allocated to improving the country's water, sanitation, and hygiene (US$222 million; 55% of total NCP budget) and case management (US$149 million; 37%). The cholera vaccination strategy included in the NCP exhibited a 5-year oral cholera vaccine (OCV) introduction plan with 2 doses (30 604 889 doses) and single dose (3 031 266 doses) in selected cholera hotspot areas. However, its implementation is challenged due to a lack of financial support, inability to get the requested vaccine for targeted hotspot woredas (due to the current shortage of doses in the OCV global stockpile), recurrent cholera outbreaks, and high humanitarian needs in the country. It is recommended to have a sustainable financial mechanism to support implementation, follow the requested vaccine doses, and reorganize the planned coordination platform to foster the implementation.

Coverage of Two-Dose Preemptive Cholera Mass Vaccination Campaign in High-Priority Hotspots in Shashemene, Oromia Region, Ethiopia.

BACKGROUND: Cholera is a public health priority in Ethiopia. The Ethiopian National Cholera Plan elaborates a multi-year scheme of oral cholera vaccine (OCV) use. Aligned with this, a preemptive OCV campaign was conducted under our Ethiopia Cholera Control and Prevention project. Here, we present the OCV vaccination outcomes. METHOD: Cholera high-priority hotspots in the Oromia Region, Shashemene Town (ST) and Shashemene Woreda (SW), were selected. Four kebelles (Abosto, Alelu, Arada, and Awasho) in ST and 4 clusters (Faji Gole, Harabate, Toga, and Chabi) in SW were study sites with OCV areas nested within. A total of 40 000 and 60 000 people in ST and SW, respectively, were targeted for a 2-dose OCV (Euvichol-Plus) campaign in 11-15 May (first round [R1]) and 27-31 May (second round [R2]) 2022. Daily administrative OCV coverage and a coverage survey in 277 randomly selected households were conducted. RESULTS: The administrative OCV coverage was high: 102.0% for R1 and 100.5% for R2 in ST and 99.1% (R1) and 100.0% (R1) in SW. The coverage survey showed 78.0% (95% confidence interval [CI]: 73.1-82.9) of household members with 2-dose OCV and 16.8% (95% CI: 12.4-21.3) with no OCV in ST; and 83.1% (95% CI: 79.6-86.5) with 2-dose OCV and 11.8% (95% CI: 8.8-14.8) with no OCV in SW. The 2-dose coverages in 1-4-, 5-14-, and ≥15-year age groups were 88.3% (95% CI: 70.6-96.1), 88.9% (95% CI: 82.1-95.7), and 71.3% (95% CI: 64.2-78.3), respectively, in ST and 78.2% (95% CI: 68.8-87.7), 91.0% (95% CI: 86.6-95.3), and 78.7% (95% CI: 73.2-84.1) in SW. CONCLUSIONS: High 2-dose OCV coverage was achieved. Cholera surveillance is needed to assess the vaccine impact and effectiveness.

Dissecting Water, Sanitation, and Hygiene (WaSH) to Assess Risk Factors for Cholera in Shashemene, Oromia Region, Ethiopia.

BACKGROUND: Cholera outbreaks have afflicted Ethiopia, with nearly 100 000 cases and 1030 deaths reported from 2015 to 2023, emphasizing the critical need to understand water, sanitation, and hygiene (WaSH) risk factors. METHODS: We conducted a cross-sectional household (HH) survey among 870 HHs in Shashemene Town and Shashemene Woreda, alongside extracting retrospective cholera case data from the Ethiopian Public Health Institute database. Relationships between WaSH and sociodemographic/economic-levels of HHs were examined. WaSH status and cholera attack rates (ARs) were described at kebele-level using geospatial mapping, and their association was statistically analyzed. RESULTS: Access to basic drinking water, sanitation, and hygiene facilities was limited, with 67.5% (95% confidence interval, 64.4-70.6), 73.4% (70.3-76.3), and 30.3% (27.3-33.3) of HHs having access, respectively. Better WaSH practices were associated with urban residence (adjusted odds ratio, 1.7, [95% confidence interval, 1.1-2.7]), higher educational levels (2.7 [1.2-5.8]), and wealth (2.5 [1.6-4.0]). The association between cholera ARs and at least basic WaSH status was not statistically significant (multiple R2 = 0.13; P = .36), although localized effects were suggested for sanitation (Moran I = 0.22; P = .024). CONCLUSIONS: Addressing gaps in WaSH access and hygiene practices is crucial for reducing cholera risk. Further analyses with meaningful covariates and increased sample sizes are necessary to understand the association between cholera AR and specific WaSH components.

Retrospective Analysis of Cholera/Acute Watery Diarrhea Outbreaks in Ethiopia From 2001 To 2023: Incidence, Case Fatality Rate, and Seasonal and Multiyear Epidemic Patterns.

BACKGROUND: The Ethiopian government has developed the multisectoral cholera elimination plan (NCP) with an aim of reducing cholera incidence and case fatality rate (CFR). To better understand and monitor the progress of this plan, a comprehensive review of national cholera epidemiology is needed. METHODS: Reported data on cholera/acute watery diarrhea (AWD) cases in the past 20 years were extracted from the Ethiopian Public Health Institute and World Health Organization databases. Descriptive statistics, Pearson χ2, and logistic regression analyses were conducted. RESULTS: From January 2001 to November 2023, a total of 215 205 cholera/AWD cases, 2355 deaths with a cumulative CFR of 1.10% (95% confidence interval [CI], 1.092-1.095), and a mean annual incidence rate of 8.9/100 000 (95% CI, 6.5-11.3) were reported. Two major upsurges of cholera epidemics were found in the last two decades with mean attack rate (AR) of 20.57/100 000 in 2006-2010 and 14.83/100 000 in 2016-2020. Another resurgence of outbreaks occured in 2021-2023 (mean AR, 8.63/100 000). In 2015-2023, 54.0% (53 990/99 945) of cases were aged 15-44 years. National cholera CFR (3.13% [95% CI: 2.1-4.5]) was the highest in 2022. The 2015-2023 cumulative cholera CFR was different across regions: Benishangul Gumuz (6.07%), Gambela (1.89%), Sidama (1.42%), Southern Nation, Nationalities, and Peoples' (1.34%), Oromia (1.10%), and Amhara (1.09%). Cholera/AWD patients in older adults (≥45 years), severe dehydration, peak rainy season (June-August), and outpatients were associated with higher risk of death. CONCLUSIONS: Cholera has been a public health problem in Ethiopia with case fatalities still above the global target. Case management needs to be improved particularly in outpatients and older populations. Outbreak preparedness should be rolled out well in advance of the typical rainy seasons. Significant investments are essential to advance the cholera surveillance system at healthcare setting and community level. Underlying factors of cholera deaths per areas should be further investigated to guide appropriate interventions to meet the NCP target by 2028.

Comprehensive Review on the Use of Oral Cholera Vaccine (OCV) in Ethiopia: 2019 to 2023.

BACKGROUND: Cholera outbreaks in Ethiopia necessitate frequent mass oral cholera vaccine (OCV) campaigns. Despite this, there is a notable absence of a comprehensive summary of these campaigns. Understanding national OCV vaccination history is essential to design appropriate and effective cholera control strategies. Here, we aimed to retrospectively review all OCV vaccination campaigns conducted across Ethiopia between 2019 and 2023. METHODS: The OCV request records from 2019 to October 2023 and vaccination campaign reports for the period from 2019 to December 2023 were retrospectively accessed from the Ethiopia Public Health Institute (EPHI) database. Descriptive analysis was conducted using the retrospective data collected. RESULTS: From 2019 to October 2023, Ethiopian government requested 32 044 576 OCV doses (31 899 576 doses to global stockpile; 145 000 doses to outside of stockpile). Around 66.3% of requested doses were approved; of which 90.4% were received. Fifteen OCV campaigns (12 reactive and 3 pre-emptive) were conducted, including five two-dose campaigns with varying dose intervals and single-dose campaigns partially in 2019 and entirely in 2021, 2022 and 2023. Overall vaccine administrative coverage was high; except for Tigray region (41.8% in the 1st round; 2nd round didn't occur). The vaccine administrative coverage records were documented, but no OCV coverage survey data was available. CONCLUSIONS: This study represents the first comprehensive review of OCV campaigns in Ethiopia spanning the last five years. Its findings offer valuable insights into informing future cholera control strategies, underscoring the importance of monitoring and evaluation despite resource constraints. Addressing the limitations in coverage survey data availability is crucial for enhancing the efficacy of future campaigns.

Healthcare Seeking Behavior and Disease Perception Toward Cholera and Acute Diarrhea Among Populations Living in Cholera High-Priority Hotspots in Shashemene, Ethiopia.

BACKGROUND: Healthcare seeking behavior (HSB) and community perception on cholera can influence its management. We conducted a cross-sectional survey to generate evidence on cholera associated HSB and disease perception in populations living in cholera hotspots in Ethiopia. METHODS: A total of 870 randomly selected households (HHs) in Shashemene Town (ST) and Shashemene Woreda (SW) participated in our survey in January 2022. RESULTS: Predominant HHs (91.0%; 792/870) responded "primary health center" as the nearest healthcare facility (HCF). Around 57.4% (247/430) of ST HHs traveled <30 minutes to the nearest HCF. In SW, 60.2% (265/440) of HHs travelled over 30 minutes and 25.9% (114/440) over 4 km. Two-thirds of all HHs paid

Vibrio mimicus Lineage Carrying Cholera Toxin and Vibrio Pathogenicity Island, United States and China.

Vibrio mimicus bacteria have caused sporadic cases and outbreaks of cholera-like diarrhea throughout the world, but the association of lineages with such events is unexplored. Genomic analyses revealed V. mimicus lineages carrying the virulence factors cholera toxin and toxin coregulated pilus, one of which has persisted for decades in China and the United States.

Spatiotemporal Modeling of Cholera, Uvira, Democratic Republic of the Congo, 2016-2020.

We evaluated the spatiotemporal clustering of rapid diagnostic test-positive cholera cases in Uvira, eastern Democratic Republic of the Congo. We detected spatiotemporal clusters that consistently overlapped with major rivers, and we outlined the extent of zones of increased risk that are compatible with the radii currently used for targeted interventions.

Peri-weaning cholera toxin consumption suppresses chemically-induced carcinogenesis in mice.

Gastrointestinal bacteria are known to have an impact on local and systemic immunity, and consequently either promote or suppress cancer development. Following the notion that perinatal bacterial exposure might confer immune system competency for life, we investigated whether early-life administration of cholera-toxin (CT), a protein exotoxin of the small intestine pathogenic bacterium Vibrio cholerae, may shape local and systemic immunity to impart a protective effect against tumor development in epithelia distantly located from the gut. For that, newborn mice were orally treated with low non-pathogenic doses of CT and later challenged with the carcinogen 7,12-dimethylbenzanthracene (DMBA), known to cause mainly mammary, but also skin, lung and stomach cancer. Our results revealed that CT suppressed the overall incidence and multiplicity of tumors, with varying efficiencies among cancer types, and promoted survival. Harvesting mouse tissues at an earlier time-point (105 instead of 294 days), showed that CT does not prevent preneoplastic lesions per se but it rather hinders their evolution into tumors. CT pretreatment universally increased apoptosis in the cancer-prone mammary, lung and nonglandular stomach, and altered the expression of several cancer-related molecules. Moreover, CT had a long-term effect on immune system cells and factors, the most prominent being the systemic neutrophil decrease. Finally, CT treatment significantly affected gut bacterial flora composition, leading among others to a major shift from Clostridia to Bacilli class abundance. Overall, these results support the notion that early-life CT consumption is able to affect host's immune, microbiome and gene expression profiles toward the prevention of cancer.

Genomic and functional insights into antibiotic resistance genes floR and strA linked with the SXT element of Vibrio cholerae non-O1/non-O139.

The emergence and spread of antibiotic-resistant bacterial pathogens are a critical public health concern across the globe. Mobile genetic elements (MGEs) play an important role in the horizontal acquisition of antimicrobial resistance genes (ARGs) in bacteria. In this study, we have decoded the whole genome sequences of multidrug-resistant Vibrio cholerae clinical isolates carrying the ARG-linked SXT, an integrative and conjugative element, in their large chromosomes. As in others, the SXT element has been found integrated into the 5'-end of the prfC gene (which encodes peptide chain release factor 3 involved in translational regulation) on the large chromosome of V. cholerae non-O1/non-O139 strains. Further, we demonstrate the functionality of SXT-linked floR and strAB genes, which confer resistance to chloramphenicol and streptomycin, respectively. The floR gene-encoded protein FloR belongs to the major facilitator superfamily efflux transporter containing 12 transmembrane domains (TMDs). Deletion analysis confirmed that even a single TMD of FloR is critical for the export function of chloramphenicol. The floR gene has two putative promoters, P1 and P2. Sequential deletions reveal that P2 is responsible for the expression of the floR. Deletion analysis of the N- and/or C-terminal coding regions of strA established their importance for conferring resistance against streptomycin. Interestingly, qPCR analysis of the floR and strA genes indicated that both of the genes are constitutively expressed in V. cholerae cells. Further, whole genome-based global phylogeography confirmed the presence of the integrative and conjugative element SXT in non-O1/non-O139 strains despite being non-multidrug resistant by lacking antimicrobial resistance (AMR) gene cassettes, which needs monitoring.

Tailored multivalent peptide targeting the B-subunit pentamer of cholera toxin inhibits its intestinal toxicity by inducing aberrant transport of the toxin in cells.

Cholera toxin (Ctx) is a major virulence factor produced by Vibrio cholerae that can cause gastrointestinal diseases, including severe watery diarrhea and dehydration, in humans. Ctx binds to target cells through multivalent interactions between its B-subunit pentamer and the receptor ganglioside GM1 present on the cell surface. Here, we identified a series of tetravalent peptides that specifically bind to the receptor-binding region of the B-subunit pentamer using affinity-based screening of multivalent random-peptide libraries. These tetravalent peptides efficiently inhibited not only the cell-elongation phenotype but also the elevated cAMP levels, both of which are induced by Ctx treatment in CHO cells or a human colon carcinoma cell line (Caco-2 cells), respectively. Importantly, one of these peptides, NRR-tet, which was highly efficient in these two activities, markedly inhibited fluid accumulation in the mouse ileum caused by the direct injection of Ctx. In consistent, NRR-tet reduced the extensive Ctx-induced damage of the intestinal villi. After NRR-tet bound to Ctx, the complex was incorporated into the cultured epithelial cells and accumulated in the recycling endosome, affecting the retrograde transport of Ctx from the endosome to the Golgi, which is an essential process for Ctx to exert its toxicity in cells. Thus, NRR-tet may be a novel type of therapeutic agent against cholera, which induces the aberrant transport of Ctx in the intestinal epithelial cells, detoxifying the toxin.