Commensal pathogen competition impacts host viability

Significance

Enteric pathogens including the causative agent of cholera, Vibrio cholerae, use the type-six secretion system (T6SS) to kill commensal microbes in the host intestine. Eradicating competing microbes allows pathogens to improve colonization. However, it is not known whether commensal destruction has additional consequences on host viability. We used the Drosophila model of cholera to determine the impacts of T6SS on fly health and longevity. We found that T6SS-dependent competition with the symbiotic Acetobacter pasteurianus intensified disease symptoms, and accelerated host death. Gnotobiotic flies without A. pasteurianus abolished T6SS-dependent death, and reintroduction of A. pasteurianus alone was sufficient to restore accelerated death. These observations implicate T6SS-dependent interactions with commensal bacteria as a factor for the progression of cholera.

Abstract

While the structure and regulatory networks that govern type-six secretion system (T6SS) activity of Vibrio cholerae are becoming increasingly clear, we know less about the role of T6SS in disease. Under laboratory conditions, V. cholerae uses T6SS to outcompete many Gram-negative species, including other V. cholerae strains and human commensal bacteria. However, the role of these interactions has not been resolved in an in vivo setting. We used the Drosophila melanogaster model of cholera to define the contribution of T6SS to V. cholerae pathogenesis. Here, we demonstrate that interactions between T6SS and host commensals impact pathogenesis. Inactivation of T6SS, or removal of commensal bacteria, attenuates disease severity. Reintroduction of the commensal, Acetobacter pasteurianus, into a germ-free host is sufficient to restore T6SS-dependent pathogenesis in which T6SS and host immune responses regulate viability. Together, our data demonstrate that T6SS acts on commensal bacteria to promote the pathogenesis of V. cholerae.

The bacterium Vibrio cholerae is responsible for several million cases of diarrheal disease and over 120,000 deaths annually (1). Once ingested, pathogenic V. cholerae bacteria pass through the gastric acid barrier, penetrate the mucin layer of the small intestine, and adhere to the underlying epithelium. V. cholerae multiplies rapidly, secretes cholera toxin, and exits the human host in immense numbers during diarrheal purges (2). Despite numerical inferiority upon arrival in the gut, V. cholerae overcomes the natural barrier presented by commensal gut bacteria, through adaptive responses that permit aggressive expansion in the host. V. cholerae uses a type-six secretion system (T6SS) to deliver toxic effectors into prokaryotic and eukaryotic prey. If the target cell lacks cognate immunity proteins, it rapidly succumbs to the injected toxin, allowing V. cholerae to dominate a niche (3, 4). T6SS selectively targets Gram-negative bacteria and eukaryotic phagocytes such as macrophages, providing V. cholerae a competitive advantage (5). In contrast, Gram-positive bacteria are immune to T6SS-mediated toxicity, potentially due to their thick peptidoglycan layer (4, 6). Studies with other bacteria suggest that pathogens use T6SS to overcome barriers presented by host commensals (7). For example, Salmonella enterica Serovar Typhimurium uses a T6SS to outcompete Gram-negative commensals and enhance colonization of the adult mouse gut (7). Alternatively, the Campylobacter jejuni T6SS is thought to act on eukaryotic cells to support persistent in vivo colonization of IL-10−deficient mice (8).

Studies with the infant mouse and rabbit models showed that V. cholerae T6SS is active inside the host (9, 10), and contributes to inflammation in the infant mouse model (11). Furthermore, gene expression data showed an up-regulation of V. cholerae T6SS genes in infected humans (12). Despite experimental support for T6SS activation inside the host, evidence is only now beginning to emerge that T6SS acts on intestinal bacteria during infection. For example, T6SS contributes to the eradication of commensal Escherichia coli to promote host colonization by V. cholerae during infection of infant mice (13). However, the immediate impact of T6SS-dependent interactions with commensal bacteria on host viability is not known. Furthermore, the role of the host in disease progression mediated by pathogen-commensal interactions is unclear.

We used the Drosophila−Vibrio model to study the interplay between T6SS and commensal microbes in the development of disease. This model has several advantages for this work. Flies succumb to Vibrio infection (14); the gut microbiome of flies is manipulatable (15), and intestinal homeostasis is maintained by similar pathways in flies and in more complex vertebrates (16). We found that the T6SS-positive El Tor strain, C6706, establishes a lethal cholera-like disease in adult flies. Inactivation of T6SS activity significantly impaired host colonization, reduced disease symptoms, and extended host survival. T6SS-dependent killing of flies requires Drosophila to be associated with the Gram-negative commensal, Acetobacter pasteurianus (Ap). Removal of commensal bacteria abrogates T6SS-mediated killing of the host, and reintroduction of Ap, either alone or in combination with additional commensals, fully restores T6SS-dependent lethality. Mutation of the Immune Deficiency (IMD) pathway relieves T6SS-dependent lethality, implicating innate defenses in T6SS-mediated host death. Collectively, our work establishes that interactions between T6SS and commensal bacteria contribute to the progression of disease in Drosophila.

Results

T6SS Interacts with Commensal Bacteria to Influence Host Viability.

As Drosophila is susceptible to infection with V. cholerae (14), we reasoned that the fly provides a platform to determine the in vivo function of T6SS. Pandemic V. cholerae strains belong to two biotypes of the O1 serogroup. The classical biotype responsible for the first six pandemics carries multiple nonsense mutations and deletions in T6SS genes, resulting in a disabled T6SS (17). Conversely, the El Tor biotype, responsible for the seventh pandemic, has a functional T6SS that becomes active upon host entry (9, 10). To examine the impact of T6SS on V. cholerae pathogenesis, we tracked the survival of flies that we infected orally with either a classical biotype, O395, or an El Tor biotype, C6706. C6706 has been shown to be avirulent in the fly model, due to repression of quorum sensing by the regulator HapR (18). However, our laboratory isolate of C6706 kills adult Drosophila, due to decreased hapR levels (18, 19). As controls, we measured the viability of adult flies raised on lysogeny broth (LB). Infection with O395 caused a moderate reduction in adult viability compared with controls (Fig. 1A). In contrast, the median viability of C6706-infected flies was a third of that observed for controls (50 h vs. 149 h; Fig. 1A), and all C6706-infected flies perished within 72 h of infection (compared with 170 h for mock-infected flies). We then asked whether disabling T6SS in C6706 affects pathogenesis. We infected adult flies with wild-type C6706, or with C6706 carrying an in-frame deletion of vasK, which encodes an inner membrane protein essential for T6SS assembly (6). We found that disabling T6SS in C6706 significantly impaired pathogenesis (Fig. 1B). As variability in fly killing exists from experiment to experiment (SI Appendix, Fig. S1), likely due to subtle differences between individual cultures of flies, control experiments with C6706 and C6706∆vasK were repeated concurrently with each new experiment and plotted accordingly. On average, mutation of vasK extended median survival by 16% (SI Appendix, Fig. S1). Deletion of vipA, a protein that makes up the outer sheath of the T6SS infection machine (20), had near-identical attenuating effects on host killing (Fig. 1C). Combined, these results establish that T6SS contributes to V. cholerae pathogenesis in vivo. However, inactivation of T6SS does not abolish pathogenesis. This is consistent with earlier reports that V. cholerae employs additional virulence factors (14, 18, 21) to kill the host in a T6SS-independent manner.

Fig. 1.

T6SS contributes to the pathogenesis of V. cholerae in a commensal-dependent manner. (A) Survival curves of 5- to 6-d-old CR w¹¹¹⁸ flies infected with the indicated V. cholerae strains. LB alone served as mock infection. (B and C) Survival curve of CR flies infected with T6SS functional (C6706) or T6SS nonfunctional (C6706ΔvasK and C6707ΔvipA) mutants. (D) Survival curve of GF flies infected with C6706 or C6706ΔvasK. D was performed at the same time and infected with the same bacterial cultures as B. The y axis shows percent survival, and x axis shows infection time. Tables show Long-rank (Mantel−Cox) tests. In A, χ2 and P values are relative to mock infected flies; in B–D, χ2 and P values are relative to wild-type C6706 infected flies; n = 50 per group, for all experiments.

As T6SS targets eukaryotic and prokaryotic cells (5, 6, 11), we asked whether T6SS contributes to host killing either by direct effects on the host or by indirect effects on the intestinal microbiota. We examined survival rates of conventionally reared (CR) and germ-free (GF) flies that we challenged with C6706 or C6706∆vasK. If T6SS acts directly on the fly, we expect that removal of commensal bacteria will not affect T6SS-dependent killing of the host. Instead, we found that an absence of commensal bacteria impaired C6706-dependent killing to the point that it was no longer distinguishable from C6706∆vasK (Fig. 1D), indicating that T6SS-dependent killing of a fly host requires the presence of commensal bacteria.

T6SS Contributes to Disease.

As loss of T6SS impairs V. cholerae pathogenesis, we monitored how T6SS impacts the development of pathogen-laden diarrhea, the hallmark of cholera. We supplemented the infection culture with a nontoxic blue dye (22). We infected flies for 24 h, and placed them in chambers with filter paper on the surface. To determine the defecation frequency of infected flies, we counted individual blue dots hourly for the next 4 h. As controls, we measured defecation by uninfected flies that we raised on a solid fly culture medium with blue dye, or on bacterial growth medium supplemented with the same dye. We observed no difference in defecation frequency between flies raised on solid or liquid diets, confirming that the bacterial growth medium does not cause diarrhea (Fig. 2A). Likewise, O395 had no measurable effects on defecation frequency (Fig. 2A). In contrast, we found that C6706 caused an increase in the number of fecal marks per fly (Fig. 2A).

Fig. 2.

T6SS contributes to cholera-like disease. (A) Fecal marks from w¹¹¹⁸ flies fed solid fly food or LB broth (mock) supplemented with O395, C6706ΔvasK, or C6706 for 24 h. The table shows a linear regression analysis of each group, and P values are the result of a Student’s t test at 4 h. (B) Fecal mark area, in micrometers, of spots counted. Each point is the average area of a given replicate. Statistics show Student’s t tests for each group compared with solid food. (C) V. cholerae shed per fly fed LB or infected with V. cholerae C6706, C6706ΔvasK, or O395 for 24 h. Each point is the number of Vibrio isolated from fecal matter of a single fly.

Similarly, we found an increase in the number of fecal marks per fly from flies infected with C6706∆vasK. However, this increase was less pronounced than that of flies infected with C6706. To assess the contributions of T6SS to defecation frequency, we performed a linear regression analysis on the groups indicated in Fig. 2A. We noticed a significant increase in the number of fecal marks per fly over time from flies infected with C6706, but not from mock-infected flies. Furthermore, there was a significantly lower increase in the number of fecal marks per fly from C6706ΔvasK-infected flies, and a smaller portion of these fecal marks could be attributed to infection with C6706∆vasK (Fig. 2A), indicating that T6SS increases the severity of diarrheal symptoms in infected flies. However, T6SS inactivation does not abate diarrheal symptoms likely due to other virulence factors.

To measure T6SS effects on defecation volume, we calculated the surface area of each dot as a proxy for volume. We observed an increase in the area of fecal spots from mock-infected flies raised on a liquid diet compared with flies raised on a solid diet (Fig. 2B). Infection with O395 did not impact defecation volume (Fig. 2B). In contrast, both C6706 and C6706ΔvasK significantly increased fecal volume relative to mock-infected controls (Fig. 2B), confirming enhanced diarrheal disease in flies infected with either strain. Finally, as the shedding of V. cholerae in fecal matter accompanies diarrhea, we quantified the number of V. cholerae bacteria excreted by flies that we challenged with the different strains of V. cholerae. Whereas we only detected V. cholerae in the feces of a single fly infected with the O395 strain, we found that 8 out of 10 flies infected with C6706 shed V. cholerae. Consistent with contributions of T6SS to disease severity, we only found 5 out of 10 C6706ΔvasK-infected flies shed the bacteria. In short, our results establish a role for T6SS in diarrheal symptoms during a V. cholerae infection: Loss of T6SS reduces defecation frequency, and lowers shedding of V. cholerae in the feces of infected animals. As O395 has comparatively mild effects on host viability, and to specifically examine the influence of T6SS on disease progression, we chose to exclusively study the effects of C6706 and C6706∆vasK on flies in subsequent experiments.

T6SS Promotes Intestinal Epithelial Damage.

During infection with V. cholerae, diarrhea is accompanied by ultrastructural changes to the host intestinal epithelium (23). Therefore, we used transmission electron microscopy (TEM) to examine posterior midgut (the small intestine analog) ultrastructure of mock-infected flies, or flies challenged with C6706 or C6706∆vasK for 50 h. Intestines from mock-infected flies had a readily identifiable lumen, an epithelium of evenly spaced columnar cells with extensive brush borders, and morphologically normal nuclei and mitochondria (Fig. 3 A–F). In contrast, we could not discern an intact intestine in flies challenged with C6706 (Fig. 3 G–I). The gut consisted of a disorganized mass of cells that lacked apical brush borders, and completely engulfed the lumen. We observed extensive shedding of epithelial structures into the presumptive lumen (boxes, Fig. 3 G–J), and high magnification images revealed characteristics of cell death, such as nuclear decondensation, and swollen mitochondria (Fig. 3 K and L). Infection with C6706∆vasK caused a phenotype that was intermediate between mock-infected controls and C6706-infected adults. Guts infected with C6706∆vasK retained elements of intestinal organization, such as identifiable epithelial cells with brush borders, a recognizable lumen (Fig. 3 M–P), and intact nuclear and mitochondrial organization (Fig. 3 Q and R). However, we noticed that infection with C6706∆vasK caused an extrusion of epithelial cell matter into the lumen (boxes, Fig. 3M), a phenotype consistent with pathogen-mediated destruction of the host epithelium (24). As guts infected with C6706Δvask were less damaged than C6706-infected counterparts, we quantified intestinal progenitor cells in the posterior midguts of flies infected with C6706 or C6706Δvask (25). Progenitors undergo cell division to repair damage to the fly gut. Consistent with our TEM analysis, we found that guts infected with C6706Δvask had greater numbers of progenitors per area than C6706-infected guts (SI Appendix, Fig. S2). In summary, these results uncover a role for T6SS in the severity of disease in adult Drosophila. Inactivation of T6SS diminishes damage to the intestinal epithelium, lowers the severity of diarrhea, and extends host mortality times.

Fig. 3.

T6SS contributes to V. cholerae intestinal pathogenesis. TEM of the posterior midguts of flies, (A−F) mock-infected or (G−L) infected with C6706 or (M−R) C6706ΔvasK after 50 h of infection. Cells protruding into the lumen are indicated with boxes. (Large scale bars, 10 μm; small scale bars, 5 μm.) Epithelial cells, epc; microvilli, mv; visceral muscle, vm.

T6SS Influences Pathogen-Commensal Interactions in the Intestine.

As T6SS-assisted pathogenesis requires a microbiota (Fig. 1D), we asked whether intestinal bacteria influence colonization by V. cholerae. The gut microbiota of laboratory-reared Drosophila typically shows low diversity (15, 26). In our laboratory, fly intestines are dominated by the Gram-negative Ap, and the Gram-positive Lactobacillus species L. brevis (Lb) and L. plantarum (Lp) (27). To determine whether Ap or Lactobacilli influence host colonization by V. cholerae, we established populations of GF adult flies, and adults that we associated exclusively with Ap or Lb (Fig. 4A). We challenged the populations with C6706 or C6706ΔvasK, and measured the colony-forming units per fly (CFU/Fly) of V. cholerae as a function of time. We found that C6706 and C6706ΔvasK were equally effective at colonizing GF intestines, or intestines that exclusively carry Lb (Fig. 4 B and C). In each case, the numbers of C6706 and C6706ΔvasK increased over time and reached nearly identical levels at 24 h of infection (Fig. 4B). These data indicate that T6SS is dispensable for the colonization of a GF gut, or a gut that houses the Gram-positive bacteria Lb. In contrast, removal of T6SS significantly impaired the ability of V. cholerae to colonize an adult intestine that we preassociated with the Gram-negative commensal, Ap. In this scenario, C6706 titers increased significantly from 6 h to 24 h of infection in the intestines of Ap-colonized adults. In contrast, there was no increase in the load of C6706ΔvasK from 6 h to 24 h (Fig. 4D). By 24 h, we found an appreciable, although not statically significant, difference in CFU/Fly between C6706 and C6706ΔvasK (Fig. 4D). These data indicate that T6SS supports colonization of intestines that exclusively carry Ap.

Fig. 4.

Composition of the microbiome determines T6SS-mediated gut infection. (A) Generation of monoassociated flies. (B−D) CFU/Fly of V. cholerae strains C6706 and C6706ΔvasK of surface-sterilized (B) GF, (C) Lb monoassociated flies, and (D) Ap monoassociated flies at indicated times. Each point represents a replicate of five randomly selected flies. P values are the result of Student’s t tests. (E) An in vitro competitive assay between V. cholerae V52 and V52∆vasK against E. coli as a positive control and Ap, Lb, and Lp. Bacteria were coincubated for 4 h at 37 °C. Surviving prey bacteria in the presence of T6SS were divided by the surviving prey in the absence of T6SS (∆vasK). (F) CFU/Fly of Ap from flies infected with C6706 or C6706ΔvasK. Each point represents a biological replicate of five flies. (G) Survival of 5- to 6-d-old female CR w¹¹¹⁸ or imd flies infected with C6706 or C6706∆vasK. Tables show Long-rank (Mantel−Cox) test; χ2 and P values are relative to w¹¹¹⁸ infected flies.

As T6SS assists colonization of a gut associated with Ap, we asked whether T6SS kills Ap in a standard competition assay (6). For these in vitro assays, we used V52, a strain of V. cholerae that does not require in vivo stimulation to activate T6SS, and employs the same T6SS effector molecules as C6706 (4). Consistent with an earlier study (6), V. cholerae effectively killed the T6SS-susceptible prey E. coli K12 strain MG1655 (Fig. 4E). Furthermore, we saw no evidence of T6SS-dependent killing of either Lp or Lb (Fig. 4E). This matches previous observations that Gram-positive bacteria are naturally refractory to T6SS activity (6, 28). In contrast, we noticed substantial T6SS-dependent killing of Ap by V. cholerae (Fig. 4E). These data raise the possibility that T6SS facilitates host colonization through eradication of Ap. To test this hypothesis, we measured total Ap numbers in the intestines of flies that we monoassociated with Ap and challenged with C6706 or C6706ΔvasK. We did not detect obvious impacts of T6SS-positive C6706 on intestinal Ap numbers (Fig. 4F). As colonization of the intestinal tract is hallmarked by fly-to-fly variability (29, 30), it is possible that our assay failed to detect subtle changes in Ap numbers. However, we cannot exclude the possibility that infection with C6706 leads to relocalization of Ap within the intestine, thereby exacerbating disease. Nonetheless, our data suggest that V. cholerae infection does not substantially alter total Ap numbers. As we did not detect a change in Ap numbers, we tested the alternate possibility that T6SS-mediated interactions with a subset of intestinal Ap induce secondary responses in the host that accelerate death. For example, mutations in the IMD antibacterial pathway attenuate V. cholerae-dependent killing of the host (ref. 31 and Fig. 4G). IMD contributes to antibacterial responses in the fly gut (32), and is similar to the mammalian TNF pathway, a regulator of intestinal inflammation in mammals (33, 34). To determine whether T6SS-mediated interactions with the host involve pathological activation of immune responses, we infected wild-type and imd mutant flies with C6706 or C6706ΔvasK. Mutation of either vasK or imd prolonged host viability to near-equal extents (Fig. 4G). Ablation of T6SS in combination with an imd mutation extended host viability further (Fig. 4G). These data suggest that additive effects from the T6SS of V. cholerae and the IMD pathway of Drosophila synergistically control host viability.

The Microbiome Directly Influences T6SS-Dependent Pathogenesis.

T6SS contributes to Drosophila killing by V. cholerae (Fig. 1 B and C), T6SS-assisted killing of Drosophila requires an intestinal microbiome (Fig. 1D), and T6SS specifically targets the Gram-negative commensal Ap (Fig. 4E). These observations led us to ask whether interactions between T6SS and Ap are a prerequisite for T6SS-mediated killing of the host. To test this hypothesis, we examined host viability in adult flies that we associated exclusively with Ap, or Lb, and subsequently infected with C6706 or C6706∆vasK. For each study, we ran a parallel infection study on CR flies with the same cultures of V. cholerae. Loss of T6SS significantly impaired pathogenesis in each test with control, CR flies (Fig. 5 D–F). However, loss of T6SS did not diminish Vibrio pathogenesis in adult flies that we associated exclusively with Lb (Fig. 5A). As Lb also fails to block host colonization by a T6SS-defective C6706 strain (Fig. 4C), our data suggest that interactions between T6SS and Lb have minimal relevance for host viability. In contrast, we detected significant involvement of T6SS in the extermination of adults that we monoassociated with Ap (Fig. 5B), indicating that Ap is sufficient for T6SS-mediated killing of the host. We then asked whether Gram-positive commensals can protect Drosophila from T6SS-dependent killing of Ap-associated flies. Here, we associated adult Drosophila with a 1:1:1 mixture of Ap, Lb, and Lp. We then challenged the flies with C6706 or C6706∆vasK, and measured survival rates. In this experiment, we found that Gram-positive commensals do not impact T6SS-dependent killing of the host, suggesting that the presence of the common fly commensal Ap renders Drosophila sensitive to T6SS-dependent killing of the host irrespective of the presence of additional commensals.

Fig. 5.

Composition of commensal microbes impacts T6SS virulence contributions in vivo. (A) Survival curves for adult flies monoassociated with Lb. (B) Survival curves for adult flies monoassociated with Ap. (C) Survival curves for adult flies polyassociated with Lb, Ap, and Lp. In A–C, flies were infected as indicated. (D–F) Survival curves for parallel infection studies performed on CR flies. The y axis represents percent survival, and the x axis represents infection time in hours. Tables show Log-rank (Mantel−Cox) test.

Discussion

Commensal bacteria form a protective barrier that shields the host from microbial invaders (35). Here, we used the fly cholera model to ask whether T6SS-mediated pathogen-commensal interactions influence host death. We found that T6SS contributes to V. cholerae pathogenesis, and that T6SS accelerates host death by interactions with Ap. Removal of either T6SS or Ap extends the viability of infected adults, and inoculation of GF adult flies with Ap is sufficient to restore T6SS-dependent killing of the host. These results demonstrate an in vivo contribution of T6SS to V. cholerae pathogenesis.

Removal of all intestinal bacteria does not enhance host killing by T6SS-deficient V. cholerae, arguing against a simple replacement reaction where V. cholerae expands into a vacant niche left behind after T6SS-mediated killing of Ap. Furthermore, we did not see a substantial drop in Ap titers in flies challenged with V. cholerae, indicating that Ap persists during infection. This indicates that complete eradication of commensals is not a critical step in T6SS-mediated pathogenesis of V. cholerae. Instead, removal of commensal bacteria attenuated host killing by wild-type V. cholerae, suggesting that the presence of commensal bacteria is essential for T6SS-dependent killing of the host. Finally, we found that inoculation of GF adults with Ap, either alone or in combination with Lactobacilli, was sufficient to restore T6SS-dependent killing of the host. These observations are in line with a model where T6SS-mediated killing of a proportion of intestinal Ap initiates secondary events that enhance host destruction by V. cholerae. We consider the IMD pathway a possible mediator of such an effect. Our work shows that additive effects between IMD and T6SS-dependent interactions with Ap program the intestinal environment in a manner that supports V. cholerae pathogenesis. This model is supported by recent work in the infant mouse, demonstrating that the T6SS of V. cholerae activates the immune system to a greater extent when commensals are present (13). The system described in this report presents a simple in vivo model to define the host−microbe−pathogen interactions that determine T6SS-mediated death.

Materials and Methods

Extended materials and methods can be found in SI Appendix.

Bacterial Strains.

All Drosophila commensal bacteria strains used were isolated from wild-type laboratory flies from the Foley laboratory at the University of Alberta. V. cholerae C6706 was obtained from John Mekalanos (Harvard Medical School, Boston).

Fly Husbandry.

All experiments were performed with virgin female flies; w¹¹¹⁸ flies were used as wild type. The imd^−/− (imd^EY08573) and w, esg-GAL4, UAS-GFP, tubGAL80^ts flies have previously been described (25, 36).

Oral Infection with V. cholerae.

Virgin female flies were fed Bloomington food for 5 d at 29 °C without flipping. Flies were starved for 2 h before being fed a soaked cotton plug soaked with V. cholerae (OD₆₀₀ of 0.125). Dead flies were counted every 8 h.

V. cholerae Shedding Assay.

Flies were infected as in Oral Infection with V. cholerae. After a 24-h infection, individual flies were placed in a 96-well plate where each well had been lined with filter paper soaked in PBS + 5% sucrose. After 4 h, the filter paper was vortexed, and serial dilutions were made on LB + Streptomycin. CFUs were counted the next day.

Generation of Monoassociated Drosophila.

Virgin females were raised on selective medium for 5 d at 29 °C. After 5 d of antibiotic treatment, flies were starved in sterile empty vials for 2 h before bacterial association. Flies were then fed bacterial cultures (OD₆₀₀ of 50) resuspended in 5% sucrose in PBS. Flies were fed the bacteria sucrose suspension for 16 h at 29 °C and then kept on autoclaved food for 5 d before infection.

Colony Forming Units per Fly.

At indicated time points, 25 flies per infection group were collected and surface-sterilized to remove bacteria on the surface of the fly. The flies were mechanically homogenized, and the homogenate was plated to select for indicated bacteria.

Competition Assays.

V. cholerae V52 or V52∆vasK was mixed with commensal bacteria at a 10:1 ratio and coincubated. After a 2‐h incubation at 37 °C, bacteria were harvested, serially diluted, and plated to enumerate surviving commensals. CFUs were counted the next day.

TEM.

Flies were washed with 95% ethanol and dissected into PBS. Posterior midguts were immediately excised and placed into fixative (3% paraformaldehyde + 3% glutaraldehyde). Flies were fixed, prepared, contrast-sectioned, sectioned, and visualized.