Vibrio cholerae motility is associated with inter-animal transmission

Campbell, Ian W.; Dehinwal, Ruchika; Morano, Alexander A.; Dailey, Katherine G.; Zingl, Franz G.; Waldor, Matthew K.

doi:10.1038/s41467-025-62984-4

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Published: 27 August 2025

Vibrio cholerae motility is associated with inter-animal transmission

Nature Communications volume 16, Article number: 7989 (2025) Cite this article

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Abstract

Outbreaks of cholera are caused by the highly transmissive pathogen Vibrio cholerae. Infant mouse studies have elucidated many aspects of V. cholerae pathogenesis; however, the components of pathogenesis that feed-forward to promote transmission have remained enigmatic because animal models routinely bypass the mechanisms of inter-animal transmission by directly inoculating cultured bacteria into the stomach. Here, a transposon screen reveals that inactivation of the V. cholerae motility-linked gene motV increases infant mouse intestinal colonization. Compared to wild-type V. cholerae, a ΔmotV mutant, which exhibits heightened motility in the form of constitutive straight swimming, localizes to the crypts earlier in infection and over a larger area of the small intestine. Aberrant localization of the mutant is associated with an increased number of V. cholerae initiating infection, and elevated pathogen burden, diarrhea, and lethality. Moreover, the deletion of motV causes V. cholerae to transmit from infected suckling mice to naïve littermates more efficiently. Even in the absence of cholera toxin, the ΔmotV mutant continues to transmit between animals, although less than in the presence of toxin, indicating that phenotypes other than cholera toxin-driven diarrhea contribute to transmission. Collectively, this work provides experimental evidence linking intra-animal bottlenecks, colonization, and disease to inter-animal transmission.

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Introduction

Cholera is a severe diarrheal disease caused by Vibrio cholerae, a highly motile Gram-negative rod¹. Individuals become exposed to the pathogen by ingesting contaminated water or food, or through close person-to-person spread^2,3,4. After ingestion, the pathogen colonizes and rapidly proliferates in the small intestine (SI). During its growth in the SI, V. cholerae secretes cholera toxin, an AB₅-type protein toxin whose activities largely account for the secretory diarrhea characteristic of cholera⁵.

Orogastric inoculation of 3–5-day-old infant mice with toxigenic V. cholerae has been used to model cholera for over five decades^6,7,8. The disease observed in infant mice mirrors many aspects of human infection, including toxin-dependent diarrheal disease⁹. Studies in this model have yielded many critical insights into the genes and processes that contribute to V. cholerae pathogenicity^10,11. For example, toxin co-regulated pilus (TCP), the pathogen’s signature colonization factor, was initially identified in infant mice¹² and was subsequently demonstrated to be critical for human infection⁵.

Infant mouse studies also revealed that V. cholerae motility and chemotaxis play important and often opposite roles in intestinal colonization. The pathogen has a single polar sheathed flagellum whose counterclockwise rotation causes straight swimming. Chemotactic input causes random reorientation by briefly reversing the spin of the flagellum to clockwise rotation¹³. Nonmotile mutants lacking a flagellum or with an inactive flagellum (e.g., ΔmotAB) exhibit marked defects in colonizing infant mice^14,15. Conversely, chemotaxis mutants with an active flagellum often exhibit heightened colonization¹⁵. For example, V. cholerae chemotaxis mutants that cannot change the direction of flagellar rotation from counterclockwise to clockwise (e.g., ΔcheY-3) exhibit longer stretches of straight swimming and have increased intestinal colonization in infant mice, particularly in the proximal SI¹⁶. These V. cholerae motility and chemotaxis phenotypes have not been tested in infant rabbits, another model of V. cholerae pathogenesis¹⁷, or in humans. However, it has been observed that V. cholerae shed in human feces are motile and transcriptionally repress several chemotaxis genes¹⁸.

Cholera epidemics are often characterized as ‘explosive’ because of the rapid spread of the disease to exposed individuals¹. Animal colonization models have elucidated many aspects of pathogenesis that could contribute to the characteristic transmissibility of V. cholerae, including pathogen colonization factors that facilitate replication within the intestine, a host-associated increase in pathogen infectivity (aka hyperinfectivity), and the role of virulence genes in diarrhea¹⁰. However, the mechanisms resulting in pathogen transmission are usually not captured in animal models because infections are initiated by directly inoculating cultured bacteria into the stomach, limiting our knowledge of the factors that govern V. cholerae transmissibility.

Here, we determine which V. cholerae genes modify colonization of infant mice using a transposon loss-of-function screen. We find that inactivation of motV, a mutation that increases V. cholerae motility in liquid¹⁹, increases pathogen burden in the SI. Compared to wild-type V. cholerae, a ΔmotV mutant evades the host bottleneck, resulting in more cells initiating infection by localizing to a broader range of the intestinal crypts, and causing more diarrhea. We leverage the phenotypes of the motV mutant to investigate how events within the intestine feed-forward to govern transmission by adapting the infant mouse model to directly quantify transmission between pups. We find that deletion of motV causes V. cholerae to transmit more efficiently, even in the absence of cholera toxin-dependent diarrhea. Thus, V. cholerae motility modifies its transmissibility.

Results

Genome-scale screen to identify V. cholerae genes modifying infant mouse colonization

Transposon insertion site sequencing (Tn-seq) is a powerful approach for genome-scale identification of bacterial genes that modify growth in diverse conditions^20,21,22. In animal-based studies, Tn-seq screens are generally carried out to identify mutants that fail to colonize, suggesting that the transposon-disrupted gene promotes bacterial growth in the host^23,24,25,26. However, Tn-seq has not yet been used to define the genes required for V. cholerae colonization of infant mice.

Colonization bottlenecks can render Tn-seq studies uninterpretable because the bottleneck causes a stochastic loss of mutants from the population, which can obscure the identity of mutants lost due to selective pressure in the host²⁷. However, recent studies measuring the bottleneck restricting the V. cholerae population during colonization of infant mice suggest that Tn-seq is possible in this model; using genomically-barcoded V. cholerae, Gillman et al. (2021) demonstrated that when given a high dose (10⁸ colony forming units, CFU), ~10⁵ unique bacterial cells survive the bottleneck and expand in the SI of infant CD1 mice²⁸. High-density Tn-seq libraries created with a mariner transposon generally contain ~10⁵ unique mutants, approximately the same number of cells that survive the bottleneck. We used this insight to carry out a Tn-seq screen for V. cholerae genes that modify growth in the infant mouse intestine.

We orally inoculated ~5 × 10⁷ CFU of a dense transposon library containing ~1.1 × 10⁵ unique mutants created in a 2010 V. cholerae clinical isolate from Haiti²⁹ into postnatal day 4 Crl:CD1(ICR) mice (henceforth P4 CD1). 18-hours after inoculation, we recovered between 1.7 to 2.5 × 10⁴ of 1.1 × 10⁵ unique mutants from the SI of 3 pups (Fig S1A), indicating that the bottleneck caused a substantial loss of mutant diversity. To increase the recoverable mutants, we pooled the SIs of 10 P4 mice (one litter) and recovered 3.3 × 10⁴ unique mutants, covering ~35% of the average gene’s potential mutagenesis sites (Fig S1A, B). This degree of mutant loss can be accommodated by our analytical pipeline, which uses simulation-based normalization to model and compensate for the random loss of diversity caused by population bottlenecks^27,30.

Using this approach, we identified the genes that promote V. cholerae survival in the infant mouse SI (Supplementary Data 1). Transposon insertions in genes that facilitate growth in the murine intestine but not when cultured in LB media are found on the left side of the volcano plot in Fig. 1A. The hits on the left side of the plot validate this application of Tn-seq. Transposon insertions in many genes previously demonstrated to be critical for intestinal colonization in this model, such as genes required for the biogenesis of the type IV pilus TCP (shown in orange in Fig. 1A, B), had markedly reduced abundance in vivo. Within the tcp locus, the reduction in the abundance of insertions in genes such as tcpA³¹, the major subunit of the pilus, and tcpF, a secreted and essential colonization factor³², was greater than 1000-fold; in contrast transposon insertions were not depleted in tcpI during colonization, a gene that is not required for colonization and that has been linked to reduced TcpA expression³³, illustrating the specificity of the Tn-seq screen.

**Fig. 1: Tn-seq screen reveals that disruption of *motV* increases *V. cholerae* infant mouse colonization.**

Flagellar-based motility is generally thought to promote V. cholerae intestinal colonization in infant mice¹³, and insertions in many of the genes linked to the biogenesis and spinning of the pathogen’s flagellum had reduced abundance in animal samples relative to growth in LB (Fig. 1A, B). In contrast, there was no reduction in insertions in the flagellin genes flaABCDE, which are the building blocks of the flagellar filament. The lack of a phenotype associated with disruption of flaABCDE may be due to functional redundancy between the flagellins. However, unlike flaBCDE, flaA was previously determined to be necessary and sufficient for motility in another V. cholerae strain³⁴, and it is unclear why flaA is not required for colonization here.

Intriguingly, several genes were also identified on the right side of the volcano plot (Fig. 1A). Insertions in these genes were more abundant in vivo than in vitro, suggesting that these genes antagonize V. cholerae growth in the intestine. Within the initial LB library, no individual mutant was represented by more than 0.02% of sequencing reads. Mutants that became more abundant in the animals were visualizable as peaks in transposon abundance, with individual mutants comprising >0.4% of reads (Figs. 1C, S1C). Four of these genes, cheA−2, cheR-2, cheV-3, and cheY-3 (Fig. 1A), are linked to chemotaxis, which was previously demonstrated to limit colonization of infant mice¹³. motV insertions were among the most enriched in vivo (Fig. 1A, C). motV is encoded by a small subset of flagellated bacteria¹⁹ and the molecular function of MotV is unknown. MotV was previously demonstrated to control motility and associate with HubP¹⁹, the V. cholerae cell pole organizer³⁵. Without motV, V. cholerae cannot reverse directions (tumble). Cells lacking motV have increased motility in liquid, constitutively swimming straight at elevated speeds. Conversely, cells lacking motV cannot swim in soft agar, which requires reversing direction¹⁹.

MotV’s role in V. cholerae infection has not been studied; no information indicates whether this gene has a role in human infection, and defined motV mutants have not been tested in the infant mouse or infant rabbit models. Furthermore, Tn-seq studies in infant rabbits differ on the role of motV during infection^27,36,37,38, indicating a need for further investigation.

Deletion of motV elevates V. cholerae intestinal colonization

To corroborate that motV antagonizes V. cholerae colonization in infant mice, we created a motV deletion mutant (ΔmotV). As expected, the ΔmotV strain had severely impaired motility in soft agar, like that observed in a ΔcheY-3 mutant (Fig S2A, B), and the provision of a plasmid-borne copy of motV (pmotV) restored soft agar motility (Fig S2C). While immobile in soft agar, motV and cheY-3 mutants have increased motility in liquid¹⁹. We employed a mucus penetration assay^39,40,41 to investigate how the lack of motV may impact motility within the intestine. Both ΔmotV and ΔcheY-3 mutants had increased motility, migrating further into a 1% mucus column than wild-type (WT; Fig S2D).

In infant mice, the number of ΔmotV CFU recovered from the SI exceeded the CFU recovered from animals infected with WT V. cholerae and was similar to the CFU found with a ΔcheY-3 mutant (Fig. 2A), which is known to increase colonization¹⁵. Further analyses found that the heightened colonization of the ΔmotV strain was particularly prominent in the proximal SI, where ΔmotV CFU exceeded those of the WT by ~13-fold (Fig. 2B). Ordinarily, ~10x more WT V. cholerae CFU are recovered from the distal SI than the proximal SI. However, similar CFU of the ΔmotV mutant strain were recovered from the two parts of the intestine. The elevated capacity of the ΔmotV mutant to colonize the proximal SI was reduced by the provision of pmotV (Fig S2E), suggesting that this ΔmotV phenotype is specifically caused by the deletion of motV. Moreover, the heightened colonization capacity of the ΔmotV strain was confirmed in competition assays, where lacZ⁺ ΔmotV outcompeted a lacZ^- WT strain 13-fold in the proximal SI and 4-fold in the distal SI (Fig. 2C).

**Fig. 2: Deletion of *motV* increased *V. cholerae* SI colonization, especially in the proximal SI and within the crypts.**

A 1:1 ratio of eGFP-tagged ΔmotV and tdTomato-tagged WT V. cholerae were co-inoculated into infant mice to determine if motV deletion changed the pathogen’s localization within the SI. Notably, previous work found that fluorophore expression does not measurably alter V. cholerae fitness¹⁴. There was a much greater abundance of the ΔmotV strain compared to the WT strain, particularly in the proximal SI, where it was difficult to detect the WT strain (Fig. 2D, E). Besides corroborating the competitive advantage of the ΔmotV mutant along the proximal to distal axis of the SI, the fluorescence microscopy also revealed a differential localization of the two strains along the crypt-villus axis. In all segments of the SI, the ΔmotV mutant had an enhanced capacity to access the crypts and form microcolonies (Fig. 2D, E; Fig S3), consistent with the increased motility of the ΔmotV mutant in mucus (Fig S2D). Thus, the absence of motV enhances colonization at least in part by facilitating the pathogen’s entry into the SI crypts, especially in the proximal SI.

The ΔmotV mutant localizes early to the SI crypts

To gain insight into how deletion of motV augments V. cholerae intestinal colonization, we compared the WT and mutant populations throughout the course of infection. There was a pronounced collapse in the size of the WT population over the first 3 h of infection, especially in the proximal SI (Fig. 3A), as previously described ref. ⁴². After the first 3 h, the WT population expanded in the proximal, medial, and distal SI. Notably, the size of the ΔmotV mutant population was much less constricted over the first 3 h, particularly in the proximal SI. Subsequently, the ΔmotV population remained more numerous than the WT population in the proximal SI from 3 to 24 hours, while the populations approached parity in the medial and distal SI at later timepoints.

Fig. 3: The Δ*motV* mutant localizes in the SI crypts earlier during infection than WT *V. cholerae.*

We hypothesized that the constitutive straight swimming motility phenotype of the ΔmotV mutant might contribute to the pathogen’s capacity to persist in the proximal SI. To test this hypothesis, we used fluorophore-labeled V. cholerae to measure the pathogen’s localization throughout the course of infection (Fig. 3B). Within the first hour, when the total burden was similar for both strains (Fig. 3A), it was already apparent that the ΔmotV mutant was more numerous in the intervillous crypts. We quantified the fraction of crypts per field that were occupied by at least one V. cholerae at each timepoint (Fig. 3C). Throughout the infection, a greater fraction of crypts in the proximal small intestine were occupied by ΔmotV than WT. Greater crypt occupancy by the ΔmotV mutant was also observed in the medial and distal SI for most of the infection, with wild-type reaching parity at 24-hours. The increased penetration of the ΔmotV mutant into the crypts may result from its constitutive straight swimming and suggests that the motility behavior of the wild-type strain is insufficient to efficiently reach the crypt niche, particularly early in infection.

The ΔmotV mutant circumvents the host bottleneck

We hypothesized that the failure of WT V. cholerae to reach the crypts and persist in the proximal SI during the first hours following inoculation may contribute to the initial collapse of the population and account for at least a portion of the WT colonization bottleneck. To directly characterize how deletion of motV impacts the V. cholerae colonization bottleneck, we used ‘STAMPR’, a lineage tracing method that tracks bacterial population dynamics during infection using otherwise genetically identical cells identified by unique DNA barcodes⁴³. Comparing the frequency and diversity of barcodes in the inoculum to samples from host tissues enables measurement of the number of unique bacterial cells (founding population) from which the observed population originated (Fig. 4A). The change in the number of cells from the inoculum to the founding population quantifies the infection bottleneck - the set of factors (e.g., physical barriers, immune processes, and the microbiota) that limit the capacity of a bacterial population to establish infection, with smaller founding populations reflecting tighter (more restrictive) infection bottlenecks.

**Fig. 4: *V. cholerae* lacking *motV* evaded the colonization bottleneck.**

The increased burden of the ∆motV mutant in the SI compared to WT could be due to the strain undergoing a less restrictive bottleneck, leading to a larger founding population and/or more net replication. To distinguish between these possibilities and quantify the ∆motV bottleneck, we used STAMPR to compare the population dynamics of barcoded WT and ΔmotV bacteria in P5 mice. When administered similar-sized inoculums, the founding population of the ΔmotV strain in the SI was ~100-fold greater than that measured for the WT strain (Fig. 4B), indicating that the mutant experienced a much less restrictive bottleneck. Surprisingly, the ΔmotV strain only experienced a ~ 2-fold bottleneck, with half of the ΔmotV cells in the inoculum surviving to become the founding population (Fig. 4B). Thus, the absence of motV allows V. cholerae to evade the factors that underlie the infection bottleneck. We propose that the ability of the ΔmotV mutant to evade host bottlenecks is caused by its constitutive straight swimming phenotype, which allows cells to persist in the proximal SI and invade deeper into the intestinal crypts (Fig. 3), forming replicative microcolonies (Fig. 2D).

Deletion of motV increases V. cholerae virulence

We observed that the infant mouse bedding was more stained with diarrheal discharge in animals infected with the ΔmotV mutant than those infected with the WT strain (Fig S4), suggesting that the ΔmotV mutant induced more diarrhea in addition to increasing colonization. Indeed, the weight of the bedding, a measure of the diarrheal discharge, was greater in the ΔmotV group than WT (Fig. 5A). Furthermore, the mice infected with the mutant strain had more weight loss and a greater ratio of SI/body weight (a measure of fluid accumulation) than mice infected with the WT strain (Fig. 5A). Although the ΔcheY-3 mutant colonized at least as well as the ΔmotV mutant (Fig. 2A), it did not lead to increased diarrheal discharge or weight loss (Fig. 5A). Thus, the increased colonization of the ΔmotV strain likely does not entirely account for its elevated diarrheaogenicity. The elevated virulence of the ΔmotV strain was also apparent in survival studies^9,44, where pups were returned to dams, and morbidity was monitored until a predetermined 30-hour endpoint. The ΔmotV mutant resulted in mortality in infected animals more rapidly than either the WT or ΔcheY-3 strains (Fig. 5B).

**Fig. 5: Deletion of *motV* increased *V. cholerae* diarrheaogenicity.**

The principal cause of V. cholerae-associated diarrhea is cholera toxin⁵. To test whether deletion of motV increases diarrheaogenicity by causing cells to produce more toxin, we measured toxin production in laboratory conditions that induce cholera toxin synthesis (AKI)^45,46. The WT, ΔmotV, and ΔcheY-3 strains produced similar quantities of cholera toxin in these conditions (Fig S5). Since both the ΔcheY-3 and ΔmotV mutants exhibit defective motility characterized by an inability to tumble and swim in soft agar¹⁹ (Fig S2), increased burden during infection (Fig. 2A), and similar production of cholera toxin in culture, the cause of the increased virulence of the ΔmotV mutant remains unclear.

The ΔmotV mutant is hyper-transmissible

Many of the phenotypes of the ΔmotV mutant are associated with transmission, including bottleneck evasion, replication to high burdens in the intestine, and shedding into the environment in diarrhea. To experimentally test whether these characteristics feed-forward to increase spread between animals, we devised a method to quantify V. cholerae transmissibility (Fig. 6A). In this system, post-natal day 4 mice from several litters were randomized into new litters to minimize litter bias. The next day, ~1/3 of the mice in each new litter were infected with the indicated V. cholerae strain; these infected ‘seed’ mice were then mixed with approximately twice as many uninfected ‘contact’ mice (the remaining 2/3 of the new litter) and returned to foster dams in individual cages for 20 h (Fig. 6A). At that point, the number of CFU in the SI of seed and contact groups was quantified to determine the number of infected contacts and the robustness of their colonization.

**Fig. 6: Deletion of *motV* increased inter-animal transmission.**

Across three independent trials, seed animals infected with the ΔmotV mutant were more transmissive than WT (Fisher’s exact test P value 0.0002); 13/18 (72%) of contacts of ΔmotV seeds were infected, whereas 2/19 (11%) of contacts of WT seeds were infected (Fig. 6A). Additionally, contact animals that were infected by ΔmotV seed animals tended to have greater intestinal colonization than contacts of the WT seed animals (Fig. 6C). Thus, the ΔmotV mutant is ‘hyper-transmissible’, indicating that MotV impedes V. cholerae transmission as well as colonization. To our knowledge, the ΔmotV strain represents the first description of a hyper-transmissible V. cholerae mutant.

We leveraged the ΔcheY-3 mutant to test whether the hyper-transmissibility of the ΔmotV mutant is solely attributable to its increased intestinal colonization. Like motV mutants, strains lacking cheY-3 exhibit increased colonization (Fig. 2A and refs. ^14,15,16). However, the ΔcheY-3 mutant was not hyper-transmissible in this assay, transmitting to fewer contacts (4/19; 21%) than ΔmotV (Fig. 6A). These results suggest that the increased colonization of ΔmotV does not fully account for its enhanced transmission.

Since the hyper-transmissibility of the ΔmotV mutant is not solely attributable to increased colonization, we hypothesized that increased transmission could be caused by increased shedding of the pathogen in the more abundant diarrhea of mice infected by the mutant, a phenotype not shared with ΔcheY-3. To test this hypothesis, we used a ΔctxAB mutant that does not produce cholera toxin, the principal cause of V. cholerae-associated diarrhea⁵. As expected, mice infected with strains lacking ctxAB or lacking both ctxAB and motV had less diarrheal discharge and less fluid accumulate in their SI than mice infected with the ΔmotV mutant (Fig S6). There was no transmission from seed animals infected with a strain lacking ctxAB (0/18; 0%; Fig. 6A), suggesting that cholera toxin promotes the transmission of WT V. cholerae in this system. Furthermore, seed animals infected with the ΔmotV ΔctxAB double mutant were less transmissive than seed mice infected with V. cholerae lacking motV alone (Fig. 6A, C), suggesting that cholera toxin-driven diarrhea contributes to transmission of the ΔmotV mutant.

Nevertheless, cholera toxin was not required for the ΔmotV mutant to transmit, as the ΔmotV ΔctxAB double mutant transmitted to 6/18 (33%) of contacts (Fig. 6A). Notably, since seed animals infected with the ΔmotV ΔctxAB double mutant were more transmissive than seeds infected with a ΔctxAB single mutant, a portion of the ΔmotV ‘s hyper-transmissibility is independent of cholera toxin-induced diarrhea.

Discussion

A hallmark of infectious agents is their capacity for transmission between hosts. However, there is a relative paucity of experimental models to measure pathogen transmissibility, limiting our knowledge of the genes and processes involved in transmission. Here, we found that suckling mice infected with V. cholerae could transmit the pathogen to uninfected littermates, providing an experimental model of transmission. We leveraged this model to characterize how the motility-linked gene motV modifies V. cholerae transmission. The ΔmotV mutant localized to the intestinal crypts earlier and over a larger area of the small intestine than wild-type V. cholerae. This change in localization was associated with 3 phenotypes that likely drive the hyper-transmissibility of the ΔmotV mutant: (1) evasion of the host bottleneck, (2) elevated SI colonization, and (3) increased diarrheaogenicity.

Our findings suggest a model to describe how the constitutive straight swimming phenotype of the ΔmotV mutant may result in hyper-transmissibility (Fig. 7). While WT chemotaxis inefficiently localizes V. cholerae to the crypt niche during the first hours following inoculation (Fig. 3B-C), deletion of motV enables earlier and more efficient occupancy of the intestinal crypts, especially in the proximal SI. We propose that the aberrant pattern of localization of the ΔmotV mutant underlies many of its other phenotypes. Thus, early crypt occupancy may enable the ΔmotV mutant to escape peristaltic flow and linger longer in the proximal SI, potentially explaining why the ΔmotV mutant experiences a less restrictive bottleneck. Furthermore, occupying more of the SI likely enables the ΔmotV strain to reach a higher burden, and the higher burden combined with proximity to the intestinal epithelium likely contributes to greater intoxication of the host and increased pathogen shedding. Moreover, shed ΔmotV V. cholerae avoids the bottleneck and colonizes the next host at a lower dose. Collectively, aberrant motility leads to more cells from the inoculum initiating infection, elevated burden, and increased diarrhea, which together feed-forward to heighten transmission. Our findings illuminate the processes that are likely exploited by many enteric pathogens for their characteristic transmission: (1) evasion of host barriers, (2) replication to high burdens in infected tissues, and (3) dissemination in diarrhea, and demonstrate how intra-animal pathogenesis results in inter-animal transmission.

**Fig. 7: Model connecting the heightened inter-animal transmission of Δ*motV V. cholerae* to aberrant motility, bottleneck evasion, increased burden, and increased diarrheaogenicity.**

We observed that the ΔmotV mutant had an unusual property – it largely escaped the host infection bottleneck (Fig. 4). In general, we interpret bottlenecks as host barriers to infection, and that host and/or microbiota changes can modulate the bottleneck. For example, the low pH of the stomach and the presence of an intact microbiota both contribute to the bottleneck restricting enteric colonization by the murine pathogen Citrobacter rodentium^47,48. The motV mutant is a new example of an emerging paradigm^49,50 that pathogen factors contribute to the bottleneck.

We propose that the circumvention of the bottleneck by the ΔmotV mutant is a consequence of its aberrant swimming behavior and greater localization to the SI crypt niche. Paradoxically, by this logic, MotV activity in wild-type V. cholerae subjects the pathogen to more stringent population restriction. We observed that deletion of motV increased V. cholerae motility in mucus (Fig S2D). Previous work determined that mucus is a potent barrier to V. cholerae colonization of the infant mouse proximal SI¹⁴, the SI segment with the largest increase in colonization caused by deletion of motV. These results may reveal that a major underlying contributor to the V. cholerae bottleneck in CD1 infant mice is a failure of wild-type cells to penetrate the mucus in the proximal SI, resulting in the removal of bacteria by intestinal peristalsis.

By escaping the colonization bottleneck, bacteria lacking motV remain at a higher burden throughout the small intestine for the entire infection. The increased colonization of ΔmotV V. cholerae, particularly in the proximal SI, is similar to that observed with cheY-3 chemotactic mutants (Fig. 2A-C)^14,15,16. Although motV is not known to be linked to the output of V. cholerae’s complex chemoreceptors and chemosensory pathways, deletion of motV and cheY-3 cause similar motility phenotypes¹⁹. Cells lacking motV or cheY-3 are both only capable of counterclockwise flagellar rotation; they are unable to reverse the direction of flagellar rotation from counterclockwise to clockwise and consequently show increased smooth swimming in straight lines, decreased tumbling, and are unable to swim in soft agar^16,19. Studies using fluorescently labeled chemotactic deficient V. cholerae suggested that chemosensory pathways are not required for V. cholerae to penetrate into the intestinal crypts or for the localization of the pathogen along the crypt-villus axis¹⁴. The similar swimming behavior and colonization phenotypes of the cheY-3 and motV mutants suggest that their constitutive straight swimming phenotype may cause their shared capacity for heightened colonization of the proximal SI.

We propose that the aberrant swimming behavior and greater localization of the ΔmotV mutant to the small intestine crypts increases disease in infected hosts. The swimming behavior of ΔmotV may increase diarrheaogenicity by bringing the pathogen into a virulence-stimulating location and/or increasing the efficacy of toxin delivery by locating the pathogen closer to the epithelium. Moreover, the increased pathogen burden in animals infected with the ΔmotV mutant likely also increases the quantity of toxin production, although elevated pathogen burden alone cannot fully explain the heightened diarrhea as cheY-3 mutants have similar increased colonization without increasing diarrhea. While the molecular mechanism linking motV to intoxication remains to be described, the difference in diarrheaogenicity of the cheY-3 and motV mutants provided a tool to probe the role of toxigenic diarrhea in V. cholerae transmission.

The ΔmotV mutant’s increased colonization, virulence, evasiveness, and transmission could all be linked to its aberrant swimming behavior. Although motility is important in V. cholerae virulence, many pathogenic bacteria have lost their flagellum (e.g., Shigella spp., Mycobacterium tuberculosis, Klebsiella pneumoniae) or suppress expression of their flagellum during infection (e.g., Listeria monocytogenes)^{51,52,53,54,55}. However, among the pathogens that have retained the flagella, there is evidence that motility and chemotaxis play a role during infection¹³. As bacterial flagella are highly immunostimulatory, the lack of flagella in some pathogenic bacteria is generally believed to be an evolved mechanism for immune evasion. V. cholerae’s extreme motility during infection¹⁴ suggests that motility provides an evolutionary advantage to this pathogen that outweighs the downsides of stimulating the immune system. By linking increased motility to transmission, the phenotypes of the ΔmotV mutant highlight the potential evolutionary advantages of pathogen flagellation during enteric infection.

There is limited information regarding the role of V. cholerae motility and chemotaxis in the human intestine. In the feces of cholera patients it has been observed that V. cholerae are motile with reduced expression of several chemotaxis genes¹⁸. However, the microarray used in this study did not report information on motV or cheY-3. Likewise, there are limited studies of V. cholerae motility and chemotaxis in infant rabbits, which, like humans and infant mice, display Tcp-dependent V. cholerae colonization¹⁷. There have been several Tn-seq studies in infant rabbits, but the results disagree on the role of motV and cheY-3 during infection^27,36,37,38. Fu, Waldor, and Mekalanos 2013 were the only study to validate a chemotaxis-related hit; they found that transposon-disruption of several chemotaxis-related genes, including motV, elevate V. cholerae infant rabbit colonization, and validated that a vspR mutant had greater than a 10-fold advantage in colonizing infant rabbits compared to WT³⁶. However, other Tn-seq studies found that motV mutation has no impact^27,37 or is detrimental to infant rabbit colonization³⁸. Further work is required to understand the role of V. cholerae motility and chemotaxis in the human intestine.

Limitations of this study

The evolutionary benefits of motV are unclear. Presumably, V. cholerae motility and chemotaxis promote survival in the pathogen’s natural environments. By contrast, we find that MotV’s response to the stimuli encountered inside of the infant mouse SI is apparently maladaptive to survival/growth; the absence of motV promotes V. cholerae intestinal colonization and transmission between suckling mice, properties that seem beneficial for the propagation of the pathogen. These findings suggest a limitation of this otherwise valuable model. Possibly, the increased disease associated with the deletion of motV may increase mortality and be evolutionarily disadvantageous in humans, V. cholerae’s natural host. However, regardless of the evolutionary logic for a gene like motV, our findings with the ΔmotV mutant illustrate the connection between intra-animal pathogenesis and inter-animal transmission.

The principal focus of this study was the characterization of the many infection-related phenotypes of a V. cholerae motV mutant, and how these phenotypes contribute to inter-animal transmission. Strains lacking cheY-3 activity have previously been extensively characterized^{13,14,15,16,56,57,58}. The differences between cheY-3 and motV mutants are that only the deletion of motV increased virulence and inter-animal transmission. The molecular mechanisms accounting for the different infection phenotypes of these stains may result from differences in their respective intra-intestinal localization, virulence regulation, and/or colonization dynamics and represent an interesting area for future study. Regardless, the divergence of phenotypes between the motV and cheY-3 mutants provides a valuable tool for dissecting the mechanisms of V. cholerae transmission.

Methods

Biosafety statement

All work on V. cholerae was performed in Biosafety Level 2 (BSL2) facilities at the Brigham and Women’s Hospital according to protocols reviewed and approved by the Brigham and Women’s Hospital Institutional Biosafety Committee (protocol 2011B000082). All personnel working with bacteria were trained with relevant safety and protocol-specific procedures.

Risk-benefit analysis

This study was undertaken to enhance understanding of the cholera pathogen, which is an ongoing threat to global public health. This work included a transposon insertion site sequencing (Tn-seq) study in an infant mouse model to elucidate the pathogen factors that mediate enteric colonization. Such basic investigations are widely accepted to benefit public health by increasing our understanding of the mechanisms of pathogenesis.

Tn-seq screens study how disruption of bacterial genes by a transposon (knockout mutations) modify bacterial growth. Such loss-of-function (knockout) screens have been carried out in many different pathogens and models of infection and are generally accepted to be safe and low-risk because evolution has selected for the active form of genes. For V. cholerae, a pathogen that has co-evolved with its human host for millennia, loss-of-function (knockout) mutations generated within the lab are extremely unlikely to create a form of the bacteria that has not already been rejected by generations of evolutionary pressures. Loss-of-function (knockout) mutations are assumed safe because if they enhanced fitness of the pathogen in the natural host, then they would become more prevalent in wild populations. However, these mutations are not detectable in the wild population.

The vast majority of the Tn-Seq screens in animal models reported to date reveal genes on the right side of the volcano plot; deletion of these genes, like motV, increases colonization within the animal model. In general, the thinking in the field is that deletions that increase colonization in animal models are an artifact of adapting pathogens to non-natural hosts. Mice (and other experimental animals) are not natural hosts of V. cholerae. Thus, even though our findings suggest that deletion of motV causes increased colonization in the infant mouse model, this is not likely the case in humans. In general, although our work provides substantial new insight into the basic mechanisms by which motility contributes to pathogenicity, it does not provide a road map for the creation of a more pathogenic form of V. cholerae.

Bacterial strains and growth conditions

Unless otherwise noted, bacteria were grown at 37 °C in lysogeny broth (LB) in liquid culture shaking at 200 rpm or on solid media containing 1.5% agar (weight/vol.). For the induction of cholera toxin, V. cholerae was grown in AKI broth (0.5% sodium chloride, 0.3% sodium bicarbonate, 0.4% yeast extract, and 1.5% Bacto peptone) for 4 h static, followed by 4 h with rotation at 37 °C^46,59. All V. cholerae were derivatives of a spontaneous streptomycin (Sm) resistant mutant of a 2010 V. cholerae clinical isolate from Haiti²⁹. Selection was accomplished by supplementing media with 200 µg/mL Sm. If required, antibiotics or other supplements were used in the following concentrations: carbenicillin (Cb), 100 µg/mL; kanamycin (Km), 50 µg/mL; diaminopimelic acid (DAP), 0.3 mM; sucrose (Suc), 0.2%; 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal), 60 µg/mL. Bacterial stocks were stored at −80 °C in LB containing 25–35% (vol./vol.) glycerol. Strains used in this study are listed in Supplementary Data 2.

Strain and plasmid construction

V. cholerae strains with deletions of motV and cheY-3 were constructed using the vector pCVD442 and allelic exchange⁶⁰. 700 bp homology arms upstream and downstream of the target open reading frame were amplified from the V. cholerae genome using the primers in Supplementary Data 3. The amplified region was then cloned into pCVD442 and introduced into V. cholerae by conjugation. Integration of the non-replicative plasmid was selected on Sm+Cb and double cross-overs removing the antibiotic-resistance cassette was selected for by growth in sucrose. PCR and amplicon sequencing confirmed deletion of the entire open reading (from start to stop codon).

The ΔlacZ⁴⁴ and ΔctxAB⁹ mutants were from previous studies. V. cholerae lacZ::tdTomato was previously described in ref. ¹⁴, and the same method and vector (pJZ111) was used to introduce eGFP into the lacZ loci of the ΔmotV mutant to create ΔmotV lacZ::eGFP. pmotV was constructed by integrating the motV open reading frame into the pBR322 vector⁶¹ downstream of a ctxAB promoter using the primers listed in Supplementary Data 3.

Mice

Animal studies were conducted at the Brigham and Women’s Hospital in compliance with the Guide for the Care and Use of Laboratory Animals and according to protocols reviewed and approved by the Brigham and Women’s Hospital Institutional Animal Care and Use Committee (protocol 2016N000416).

CD1 (Crl:CD1(ICR)) dams with litters (mixed sex) were purchased from Charles River Laboratories (strain #022). Mice were housed in a biosafety level 2 (BSL2) facility under specific pathogen free conditions at 68–75 °F, with 30–50% humidity, and a 12 h light/dark cycle. CD1 infant mice at postnatal day 3-5 were intragastrically inoculated⁴² with the indicated dose and strain of V. cholerae in 50 µL of LB. Except for survival and transmission experiments, the infant mice were housed separately from the dams in tissue-lined boxes during infection. For survival and transmission experiments, pups were returned to dams where they remained until morbidity or the predetermined end point of the experiment. Infant mice were euthanized by isoflurane inhalation followed by decapitation. Adult mice were euthanized at the end of the study by isoflurane inhalation followed by cervical dislocation.

Infant mouse colonization

For colonization assays, infant mice were separated from their dams and intragastrically inoculated with the indicated dose and strain. Dose (colony forming units; CFU) was determined by retrospective serial dilution and plating on selective media containing Sm. At the indicated times (<24-hours post inoculation), pups were euthanized, and the SI was removed. Colonization burden was determined in either the whole SI, or the SI divided into 2 or 3 parts of equal length (proximal, medial, and distal). Tissue segments were further divided in half for experiments that combined measurements of bacterial localization (microscopy) and bacterial burden. To disassociate the bacteria from the tissue, organs were homogenized in LB using a bead beater (BioSpec Product, Inc) and 2 stainless-steel 3.2 mm beads. SI CFU was determined by serial dilution and plating on selective media containing Sm.

Competitive infections were performed the same as other colonization experiments, except that the inoculum was a ~ 1:1 mixture of the indicated strains. Blue/white CFU were determined by plating organ homogenates on LB + Sm/X-gal. Competetitive index was calculated by dividing the blue:white ratio from the SI by the blue:white ratio from the inoculum.

To measure diarrheaogenicity (diarrheal discharge, SI fluid accumulation, and weight loss), infant mice were individually housed for 18 h following inoculation. The mice were weighed immediately following inoculation and immediately before euthanasia to determine weight loss. Following infection, the weight of the pup was compared to the weight of the entire resected SI to determine SI fluid accumulation. The bedding was weighed at the start and end of the experiment to determine diarrheal discharge.

Tn-seq

Experiments were performed with a WT Himar transposon library³⁸. The library was expanded as a control on solid LB+Sm agar (LB library). For animal experiments, ~5 × 10⁷ CFU of the library was inoculated into infant mice. 18 h later, V. cholerae from the SIs of individual pups (Pup 1–3) or the pooled SIs of 10 mice from the same litter (Pooled litter) were outgrown overnight on LB+Sm agar. After outgrowth, cells were scraped off plates and frozen at -80 °C until processing.

Library preparation and sequencing were performed according to established protocols^30,62. gDNA was harvested using the GeneJet gDNA Isolation Kit. gDNA was fragmented to 400 bp using an ultrasonicator. Fragment ends were repaired with New England Biolabs Quick Blunting Kit and A-tailing was performed with Taq DNA-polymerase. Adaptor sequences were ligated onto fragments with T4 DNA ligase and used to perform PCR to enrich fragments containing the transposon. 300–500 bp fragments were isolated using gel extraction. Libraries were then sequenced on an Illumina NextSeq.

Sequencing data was processed using CLC Genomics Workbench (version 12.0.2; Qiagen) and the R-based RTISAn pipeline, according to established protocols^27,30. Trimming parameters (version 2.3): 3′ sequence – ACCACGAC; 5′ sequence – CAACCTGT; mismatch cost = 1; gap cost = 1; minimum internal score = 7; minimum end score = 4; discard reads <10 nt. Mapping parameters (version 1.6): reference – Haiti WT genome⁶³; mismatch cost = 1; insertion cost = 3; deletion cost = 3; length fraction = 0.95; similarity fraction = 0.95; reads mapped to multiple locations were mapped randomly; global alignment. RTISAn was used to convert mapping files to a positional tally (TAtally) exclusively counting insertions at TA dinucleotides. To account for the stochastic loss of mutants caused by infection bottlenecks, the LB library TAtally (input) was resampled 100 times to approximate the diversity of the animal TAtally (output). The simulations of the input were compared to the output to derive an average fold-change and P value (two-sided Mann–Whitney test).

To visualize the number of unique insertion sites per sample across a range of sampling depths, the TAtally was subjected to multinomial resampling beginning with the maximum read count of each sample and subsampling in two-fold lower increments. The number of unique insertions was calculated at each sampling depth. Prior to resampling, the TAtally was filtered by removing the bottom 1% of all reads to remove noise resulting from Illumina index sequence hopping.

Microscopy

For microscopy, SI segments were retrieved as described for mouse colonization and then processed for cryosectioning⁶⁴. Tissue samples (~2 cm) were fixed in PBS with 4% paraformaldehyde at 4 °C for 2–4 h, placed in PBS with 30% sucrose overnight at 4 °C, and then mounted in a medium containing a 1:2.5 ratio of 30% Sucrose: Optical Tissue Clearing medium (OCT). Mounted tissue pieces were snap-frozen in liquid nitrogen, stored at −20 °C for 1 h, then transferred to a −80 °C freezer until processing.

8–10 µm thick sections were cut on a Leica CM1860 cryostat and mounted on MAS adhesive slides (Matsunami Glass). Once samples were adhered to the slide, to preserve native fluorescence the samples were processed according to⁶⁴. After sections were cut, tissue pieces were delineated with a PAP-pen, then slides were dried in the dark for 20 min at room-temperature. Ice-cold 4% PFA was then added for 8 min while slides were stored in a humid chamber. PFA was removed and slides were then washed with PBS and 3% BSA-PBS. Then, a 1:1000 dilution of Alexa Fluor 405-conjugated phalloidin (ThermoFisher) was added to each segment for 15 min, in a dark humid chamber. After 15 minutes, slides were washed with PBS and 3% BSA-PBS again and mounted with VectaShield Plus non-hardening antifade mounting medium (vector laboratories). Slides were stored for 1-hour flat at room-temperature, then sealed with clear nail polish and stored at 4 °C until imaging. Slides were imaged with a Nikon Ti2 Eclipse spinning disk confocal microscope using a 40x oil immersion lens with a numerical aperture of 1.40 and an Andor Zyla 4.2 Plus sCMOS monochrome camera. Image analysis was performed on the ImageJ (FIJI (2.14.0)) software using custom macros.

For quantification of the localization of ∆motV:WT V. cholerae ratios in competitive infection, the number of WT (red) and ∆motV (green) V. cholerae were counted in 50–60 100x100 micron images per location (proximal/medial/distal; crypt/top of villus), taken from 20-30 larger (244.16x244.16 micron) fields. When 0 WT V. cholerae were observed, the ∆motV:WT ratio was set to 35 and labeled as “ND” (not detected), as the highest ratio of ∆motV:WT V. cholerae with WT organisms detected was 32:1.

For quantification of crypt occupancy, the total number of crypts in each of 20 244.16 x 244.16 micron fields per time point, infection, and location was recorded, as was the number of crypts in each field containing ≥1 V. cholerae. Crypt occupancy was determined by dividing the number of occupied crypts by the total number of crypts per field.

Soft agar motility

1 µl of an overnight culture of the indicated strains were injected into semisolid LB agar (0.3%; weight/vol.). Plates were cultured at 37 °C for 6-hours and the colony diameter was measured. A WT control was included on every plate.

Mucus penetration

Mucus columns were prepared by adding 800 µL of 1% porcine mucus (Sigma) to a sterile syringe. V. cholerae were cultured overnight, diluted in saline (0.45% NaCl), and 50 µl were layered on top of the column. Columns were incubated for 30 minutes at 37 °C. To quantify penetration, 100 µl fractions were collected and plated to determine CFU.

Bottleneck quantification

Barcoded V. cholerae libraries were created with the plasmid donor library pSM1⁶⁵. The pSM1 donor library contains ~70,000 unique plasmids each with a random ~25 bp barcode carried within a Tn7 site-specific transposon. Barcodes were introduced into the V. cholerae Tn7-integration site by triparental mating of the recipient V. cholerae strain with the pSM1 donor library and a helper plasmid (pJMP1039) that expresses the Tn7 transposase. Transconjugants were then selected on LB Sm Km plates. After outgrowth, transconjugates were harvested from selective plates in LB glycerol (25-35%; weight/vol.) and stored in aliquots at -80 °C.

STAMP libraries were inoculated and SIs were retrieved as described for other infant mouse colonization assays. V. cholerae burden (CFU) was quantified by serial dilution and plating on selective media. V. cholerae from the remaining sample was outgrown on LB Sm and bacterial samples were harvested and stored at −80 °C in LB glycerol (25–35%; weight/vol.) until processing. Samples were processed for barcode sequencing and STAMPR analysis as described in Hullahalli et al. 2021⁴³. Bacteria were boiled to release DNA and PCR was performed to amplify the barcode region and add sequencing adapters. Samples were sequenced on a NextSeq (Illumina). R and the STAMPR analysis pipeline⁴³ was used to demultiplex sequencing reads, trim, and map to the donor library pSM1. Founding population (Nr) was determined by STAMPR by comparing the number and frequency of barcodes recovered from the SI to a control sample outgrown from the animal inoculum. STAMPR scripts are available online at [https://github.com/hullahalli/stampr_rtisan].

Infant mouse survival

Infant mouse challenge assays were performed as described in Sit et al., 2019⁹. Infant mice were orally inoculated with 10⁶ CFU of the indicated V. cholerae strain and returned to singly housed dams for maternal care. Survival was scored based on time from inoculation to morbidity. Body condition, diarrheal discharge, and temperature of infected mice were monitored every 4–6 h to determine the onset of symptoms. Symptomatic mice were monitored every 30-minutes until reaching morbidity, at which point the pups were immediately removed and euthanized. At the predetermined endpoint (30 h post inoculation), the remaining animals were scored as surviving and euthanized.

Cholera toxin ELISA

V. cholerae strains were cultured in LB or AKI conditions. LB-culture: 8-hours in LB, with rotation, at 37 °C. AKI-culture^46,59: 4 h static, followed by 4 h with rotation at 37 °C in AKI broth (0.5% sodium chloride, 0.3% sodium bicarbonate, 0.4% yeast extract, and 1.5% Bacto peptone).

GM1 ELISA was used to quantify the concentration of cholera toxin in cell-free supernatant samples according to established protocols⁶⁶. Equal volumes of LB or AKI supernatants were serially diluted and known concentrations of purified cholera toxin were used as the standard. 96-well polystyrene microtiter plates were coated with GM1 ganglioside overnight, and 4 µg/ml fatty acid-free bovine serum albumin (BSA) was used to block the GM1-coated plates for 1 h at room temperature. Next, 260 μl of the supernatants were added to the wells in duplicate and incubated for 1 h at room temperature. Subsequently, a rabbit anti-CT polyclonal antibody (1:10,000) and then an HRP-linked goat ani-rabbit IgG antibody (1:5,000) were added to the wells and incubated for 1 h at room temperature each. For the development of the cholera toxin-antibody complex, tetramethylbenzidine (TMB) substrate solution (Thermo Fisher Scientific) was used according to the manufacturer’s protocol. The color intensity in each well was measured at 485 nm in a plate reader. The absolute quantity of cholera toxin in the samples was estimated by comparison to the standard curve.

Infant mouse transmission

Infant mice were randomly re-assorted to prevent litter bias. ~1/3 of each litter were inoculated with the indicated V. cholerae strain (seeds) and returned to foster-dams for maternal care with naïve littermates (contacts). Seeds and contacts remained with dams for 20-hours, at which point seeds and contacts were removed and euthanized. Transmission to contacts was determined by enumerating CFU in the SI. Contacts with 0 CFU in the SI were determined to be uninfected and contacts with ≥1 CFU were determined to be infected.

Software and statistics

Data analysis was performed using CLC Genomics Workbench version 12.0.2 (Trim reads 2.3 and Read mapping 1.6 functions), R version 4.4.2 (RTISAn Tn-seq analysis³⁰), GraphPad Prism, and Excel. Information regarding the number of samples and statistical tests are described in the figure legends. Geometric means, geometric standard deviations, and non-parametric tests were used for analyzing Tn-seq data, bacterial burden, bacterial competition, crypt occupancy, and founding population. Means, standard deviations, and parametric tests were used for comparisons of bacterial motility, diarrheal discharge, SI fluid accumulation, animal weight loss, and cholera toxin. Graphics and figures were prepared with BioRender, GraphPad Prism, and PowerPoint.

Biological materials

Genetically modified strains of Vibrio cholerae were created for this study. Copies of these strains will be made available upon request from the corresponding author, Matthew K. Waldor.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

Sequencing reads from Tn-seq are deposited in the Sequencing Read Archive (SRA) under accession no. PRJNA1247499. Source data are provided with this paper as a source data file. Source data are provided with this paper.

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Acknowledgements

We thank members of the Waldor lab for helpful discussions and feedback on the manuscript. We acknowledge our funding: Howard Hughes Medical Institute (M.W.). NIH grants P30 DK034854 (I.C.) and R01 AI042347 (M.W.). Fellowships T32 DK007477-37 (I.C.) and Zingl−2024HHMI (F.Z.). This article is subject to HHMI’s Open Access to Publications policy. HHMI lab heads have previously granted a nonexclusive CC BY 4.0 license to the public and a sublicensable license to HHMI in their research articles. Pursuant to those licenses, the author-accepted manuscript of this article can be made freely available under a CC BY 4.0 license immediately upon publication. This manuscript is the result of funding in whole or in part by the National Institutes of Health (NIH). It is subject to the NIH Public Access Policy. Through acceptance of this federal funding, NIH has been given a right to make this manuscript publicly available in PubMed Central upon the Official Date of Publication, as defined by NIH.

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These authors contributed equally: Ian W. Campbell, Ruchika Dehinwal.

Authors and Affiliations

Division of Infectious Diseases at Brigham & Women’s Hospital, and Department of Microbiology at Harvard Medical School, Boston, MA, USA
Ian W. Campbell, Ruchika Dehinwal, Alexander A. Morano, Katherine G. Dailey, Franz G. Zingl & Matthew K. Waldor
Howard Hughes Medical Institute, Boston, MA, USA
Matthew K. Waldor

Authors

Ian W. Campbell
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Ruchika Dehinwal
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Alexander A. Morano
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Katherine G. Dailey
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Franz G. Zingl
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Matthew K. Waldor
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Contributions

Conceptualization: I.C., R.D., M.W. Methodology: I.C., R.D., A.M., F.Z. Investigation: I.C., R.D., A.M., K.D., F.Z. Visualization: I.C., K.D. Writing the original draft: I.C., R.D., M.W. Reviewing and editing the manuscript: I.C., R.D., A.M., K.D., F.Z., M.W.

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Correspondence to Matthew K. Waldor.

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Campbell, I.W., Dehinwal, R., Morano, A.A. et al. Vibrio cholerae motility is associated with inter-animal transmission. Nat Commun 16, 7989 (2025). https://doi.org/10.1038/s41467-025-62984-4

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Received: 07 March 2025
Accepted: 04 August 2025
Published: 27 August 2025
DOI: https://doi.org/10.1038/s41467-025-62984-4