Commensal Pathogen Competition Impacts Host Viability

Commensal pathogen competition impacts host viability

David Fasta,1, Benjamin Kostiuka,1, Edan Foleya,2,3, and Stefan Pukatzkib,2,3

aDepartment of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB T6G 2S2, Canada; and bDepartment of Immunology & Microbiology, University of Colorado School of Medicine, Aurora, CO 80045

Edited by David S. Schneider, Stanford University, Stanford, CA, and accepted by Editorial Board Member Ralph R. Isberg May 25, 2018 (received for review February 5, 2018)

While the structure and regulatory networks that govern type-six the host in disease progression mediated by pathogen-commensal secretion system (T6SS) activity of Vibrio cholerae are becoming interactions is unclear. increasingly clear, we know less about the role of T6SS in disease. We used the Drosophila−Vibrio model to study the interplay Under laboratory conditions, V. cholerae uses T6SS to outcompete between T6SS and commensal microbes in the development of many Gram-negative species, including other V. cholerae strains and disease. This model has several advantages for this work. Flies human commensal bacteria. However, the role of these interactions succumb to Vibrio infection (14); the gut microbiome of flies is has not been resolved in an in vivo setting. We used the Drosophila manipulatable (15), and intestinal homeostasis is maintained by melanogaster model of cholera to define the contribution of T6SS to similar pathways in flies and in more complex vertebrates (16). We V. cholerae pathogenesis. Here, we demonstrate that interactions found that the T6SS-positive El Tor strain, C6706, establishes a between T6SS and host commensals impact pathogenesis. Inactiva- lethal cholera-like disease in adult flies. Inactivation of T6SS ac- tion of T6SS, or removal of commensal bacteria, attenuates disease tivity significantly impaired host colonization, reduced disease severity. Reintroduction of the commensal, Acetobacter pasteuria- symptoms, and extended host survival. T6SS-dependent killing of nus, into a germ-free host is sufficient to restore T6SS-dependent flies requires Drosophila to be associated with the Gram-negative pathogenesis in which T6SS and host immune responses regulate commensal, Acetobacter pasteurianus (Ap). Removal of commen- viability. Together, our data demonstrate that T6SS acts on com- sal bacteria abrogates T6SS-mediated killing of the host, and mensal bacteria to promote the pathogenesis of V. cholerae. MICROBIOLOGY reintroduction of Ap, either alone or in combination with addi- T6SS | microbiome | Drosophila | Vibrio cholerae tional commensals, fully restores T6SS-dependent lethality. Mu- tation of the Immune Deficiency (IMD) pathway relieves T6SS- dependent lethality, implicating innate defenses in T6SS-mediated he bacterium Vibrio cholerae is responsible for several million host death. Collectively, our work establishes that interactions Tcases of diarrheal disease and over 120,000 deaths annually V. cholerae between T6SS and commensal bacteria contribute to the pro- (1). Once ingested, pathogenic bacteria pass through Drosophila the gastric acid barrier, penetrate the mucin layer of the small gression of disease in . intestine, and adhere to the underlying epithelium. V. cholerae multiplies rapidly, secretes cholera toxin, and exits the human Significance host in immense numbers during diarrheal purges (2). Despite numerical inferiority upon arrival in the gut, V. cholerae over- Enteric pathogens including the causative agent of cholera, comes the natural barrier presented by commensal gut bacteria, Vibrio cholerae, use the type-six secretion system (T6SS) to kill through adaptive responses that permit aggressive expansion in commensal microbes in the host intestine. Eradicating compet- the host. V. cholerae uses a type-six secretion system (T6SS) to ing microbes allows pathogens to improve colonization. How- deliver toxic effectors into prokaryotic and eukaryotic prey. If the ever, it is not known whether commensal destruction has target cell lacks cognate immunity proteins, it rapidly succumbs to additional consequences on host viability. We used the Dro- the injected toxin, allowing V. cholerae to dominate a niche (3, 4). sophila model of cholera to determine the impacts of T6SS on fly T6SS selectively targets Gram-negative bacteria and eukaryotic health and longevity. We found that T6SS-dependent competi- phagocytes such as macrophages, providing V. cholerae a competition with the symbiotic Acetobacter pasteurianus intensified tive advantage (5). In contrast, Gram-positive bacteria are immune disease symptoms, and accelerated host death. Gnotobiotic flies to T6SS-mediated toxicity, potentially due to their thick peptido- without A. pasteurianus abolished T6SS-dependent death, and glycan layer (4, 6). Studies with other bacteria suggest that patho- reintroduction of A. pasteurianus alone was sufficient to restore gens use T6SS to overcome barriers presented by host commensals accelerated death. These observations implicate T6SS-dependent (7). For example, Salmonella enterica Serovar Typhimurium uses a interactions with commensal bacteria as a factor for the pro- T6SS to outcompete Gram-negative commensals and enhance gression of cholera. colonization of the adult mouse gut (7). Alternatively, the Cam- pylobacter jejuni T6SS is thought to act on eukaryotic cells to sup- Author contributions: D.F., B.K., E.F., and S.P. designed research; D.F. and B.K. performed − research; D.F., B.K., E.F., and S.P. analyzed data; and D.F., B.K., E.F., and S.P. wrote port persistent in vivo colonization of IL-10 deficient mice (8). the paper. Studies with the infant mouse and rabbit models showed that V. cholerae T6SS is active inside the host (9, 10), and contributes The authors declare no conflict of interest. to inflammation in the infant mouse model (11). Furthermore, This article is a PNAS Direct Submission. D.S.S. is a guest editor invited by the Editorial Board. gene expression data showed an up-regulation of V. cholerae T6SS genes in infected humans (12). Despite experimental sup- Published under the PNAS license. port for T6SS activation inside the host, evidence is only now 1D.F. and B.K. contributed equally to this work. beginning to emerge that T6SS acts on intestinal bacteria during 2E.F. and S.P. contributed equally to this work. infection. For example, T6SS contributes to the eradication of 3To whom correspondence may be addressed. Email: [email protected] or stefan. commensal Escherichia coli to promote host colonization by V. [email protected]. cholerae during infection of infant mice (13). However, the im- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. mediate impact of T6SS-dependent interactions with commensal 1073/pnas.1802165115/-/DCSupplemental. bacteria on host viability is not known. Furthermore, the role of Published online June 18, 2018.

www.pnas.org/cgi/doi/10.1073/pnas.1802165115 PNAS | July 3, 2018 | vol. 115 | no. 27 | 7099–7104 Downloaded by guest on September 26, 2021 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 C6706ΔvasK 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. 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 C6706ΔvasK. 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 C6706ΔvasK (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). Fig. 1. T6SS contributes to the pathogenesis of V. cholerae in a commensal- We infected flies for 24 h, and placed them in chambers with 1118 dependent manner. (A) Survival curves of 5- to 6-d-old CR w flies infected filter paper on the surface. To determine the defecation frequency with the indicated V. cholerae strains. LB alone served as mock infection. (B of infected flies, we counted individual blue dots hourly for the and C) Survival curve of CR flies infected with T6SS functional (C6706) or Δ Δ next 4 h. As controls, we measured defecation by uninfected flies T6SS nonfunctional (C6706 vasK and C6707 vipA) mutants. (D) Survival that we raised on a solid fly culture medium with blue dye, or on 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 bacterial growth medium supplemented with the same dye. We shows percent survival, and x axis shows infection time. Tables show Long- observed no difference in defecation frequency between flies − χ raised on solid or liquid diets, confirming that the bacterial growth rank (Mantel Cox) tests. In A, 2 and P values are relative to mock infected A flies; in B–D, χ2 and P values are relative to wild-type C6706 infected flies; n = medium does not cause diarrhea (Fig. 2 ). Likewise, O395 had no 50 per group, for all experiments. 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). Results Similarly, we found an increase in the number of fecal marks ΔvasK T6SS Interacts with Commensal Bacteria to Influence Host Viability. per fly from flies infected with C6706 .However,thisin- As Drosophila is susceptible to infection with V. cholerae (14), we crease was less pronounced than that of flies infected with C6706. reasoned that the fly provides a platform to determine the in vivo To assess the contributions of T6SS to defecation frequency, we V. cholerae performed a linear regression analysis on the groups indicated in function of T6SS. Pandemic strains belong to two A biotypes of the O1 serogroup. The classical biotype responsible Fig. 2 . We noticed a significant increase in the number of fecal marks per fly over time from flies infected with C6706, but not for the first six pandemics carries multiple nonsense mutations from mock-infected flies. Furthermore, there was a significantly and deletions in T6SS genes, resulting in a disabled T6SS (17). lower increase in the number of fecal marks per fly from Conversely, the El Tor biotype, responsible for the seventh pan- C6706ΔvasK-infected flies, and a smaller portion of these fecal demic, has a functional T6SS that becomes active upon host entry ΔvasK A V. cholerae marks could be attributed to infection with C6706 (Fig. 2 ), (9, 10). To examine the impact of T6SS on patho- indicating that T6SS increases the severity of diarrheal symptoms genesis, we tracked the survival of flies that we infected orally with in infected flies. However, T6SS inactivation does not abate di- either a classical biotype, O395, or an El Tor biotype, C6706. arrheal symptoms likely due to other virulence factors. C6706 has been shown to be avirulent in the fly model, due to To measure T6SS effects on defecation volume, we calculated repression of quorum sensing by the regulator HapR (18). How- the surface area of each dot as a proxy for volume. We observed Drosophila ever, our laboratory isolate of C6706 kills adult ,dueto an increase in the area of fecal spots from mock-infected flies decreased hapR levels (18, 19). As controls, we measured the vi- raised on a liquid diet compared with flies raised on a solid diet ability of adult flies raised on lysogeny broth (LB). Infection with (Fig. 2B). Infection with O395 did not impact defecation volume O395 caused a moderate reduction in adult viability compared (Fig. 2B). In contrast, both C6706 and C6706ΔvasK significantly with controls (Fig. 1A). In contrast, the median viability of C6706- increased fecal volume relative to mock-infected controls (Fig. infected flies was a third of that observed for controls (50 h vs. 149 2B), confirming enhanced diarrheal disease in flies infected with h; Fig. 1A), and all C6706-infected flies perished within 72 h of either strain. Finally, as the shedding of V. cholerae in fecal infection (compared with 170 h for mock-infected flies). We then matter accompanies diarrhea, we quantified the number of V. asked whether disabling T6SS in C6706 affects pathogenesis. We cholerae bacteria excreted by flies that we challenged with the infected adult flies with wild-type C6706, or with C6706 carrying different strains of V. cholerae. Whereas we only detected V. an in-frame deletion of vasK, which encodes an inner membrane cholerae in the feces of a single fly infected with the O395 strain, protein essential for T6SS assembly (6). We found that disabling we found that 8 out of 10 flies infected with C6706 shed V.

7100 | www.pnas.org/cgi/doi/10.1073/pnas.1802165115 Fast et al. Downloaded by guest on September 26, 2021 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 C6706ΔvasK 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 pro- genitor 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.

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 Lb A

or (Fig. 4 ). We challenged the populations with C6706 or MICROBIOLOGY 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 1118 Fig. 2. T6SS contributes to cholera-like disease. (A) Fecal marks from w intestines, or intestines that exclusively carry Lb (Fig. 4 B and C). flies fed solid fly food or LB broth (mock) supplemented with O395, In each case, the numbers of C6706 and C6706ΔvasK increased C6706ΔvasK, or C6706 for 24 h. The table shows a linear regression analysis ’ over time and reached nearly identical levels at 24 h of infection of each group, and P values are the result of a Student s t test at 4 h. (B) Fecal (Fig. 4B). These data indicate that T6SS is dispensable for the mark area, in micrometers, of spots counted. Each point is the average area ’ colonization of a GF gut, or a gut that houses the Gram-positive of a given replicate. Statistics show Student s t tests for each group com- Lb pared with solid food. (C) V. cholerae shed per fly fed LB or infected with bacteria . In contrast, removal of T6SS significantly impaired Δ the ability of V. cholerae to colonize an adult intestine that we V. cholerae C6706, C6706 vasK, or O395 for 24 h. Each point is the number Ap of Vibrio isolated from fecal matter of a single fly. preassociated with the Gram-negative commensal, . In this scenario, C6706 titers increased significantly from 6 h to 24 h of infection in the intestines of Ap-colonized adults. In contrast, cholerae. Consistent with contributions of T6SS to disease severity, there was no increase in the load of C6706ΔvasK from 6 h to 24 h we only found 5 out of 10 C6706ΔvasK-infected flies shed the (Fig. 4D). By 24 h, we found an appreciable, although not stat- bacteria. In short, our results establish a role for T6SS in diarrheal ically significant, difference in CFU/Fly between C6706 and symptoms during a V. cholerae infection: Loss of T6SS reduces C6706ΔvasK (Fig. 4D). These data indicate that T6SS supports defecation frequency, and lowers shedding of V. cholerae in the colonization of intestines that exclusively carry Ap. feces of infected animals. As O395 has comparatively mild effects As T6SS assists colonization of a gut associated with Ap,we on host viability, and to specifically examine the influence of T6SS asked whether T6SS kills Ap in a standard competition assay (6). on disease progression, we chose to exclusively study the effects of For these in vitro assays, we used V52, a strain of V. cholerae that C6706 and C6706ΔvasK on flies in subsequent experiments. does not require in vivo stimulation to activate T6SS, and employs the same T6SS effector molecules as C6706 (4). Consistent T6SS Promotes Intestinal Epithelial Damage. During infection with with an earlier study (6), V. cholerae effectively killed the T6SS- V. cholerae, diarrhea is accompanied by ultrastructural changes to susceptible prey E. coli K12 strain MG1655 (Fig. 4E). Further- the host intestinal epithelium (23). Therefore, we used trans- more, we saw no evidence of T6SS-dependent killing of either Lp mission electron microscopy (TEM) to examine posterior midgut or Lb (Fig. 4E). This matches previous observations that Gram- (the small intestine analog) ultrastructure of mock-infected flies, or positive bacteria are naturally refractory to T6SS activity (6, 28). flies challenged with C6706 or C6706ΔvasK for 50 h. Intestines In contrast, we noticed substantial T6SS-dependent killing of Ap from mock-infected flies had a readily identifiable lumen, an epi- by V. cholerae (Fig. 4E). These data raise the possibility that thelium of evenly spaced columnar cells with extensive brush T6SS facilitates host colonization through eradication of Ap.To borders, and morphologically normal nuclei and mitochondria test this hypothesis, we measured total Ap numbers in the in- (Fig. 3 A–F). In contrast, we could not discern an intact intestine in testines of flies that we monoassociated with Ap and challenged flies challenged with C6706 (Fig. 3 G–I). The gut consisted of a with C6706 or C6706ΔvasK. We did not detect obvious impacts disorganized mass of cells that lacked apical brush borders, and of T6SS-positive C6706 on intestinal Ap numbers (Fig. 4F). As completely engulfed the lumen. We observed extensive shedding of colonization of the intestinal tract is hallmarked by fly-to-fly epithelial structures into the presumptive lumen (boxes, Fig. 3 G– variability (29, 30), it is possible that our assay failed to detect J), and high magnification images revealed characteristics of cell subtle changes in Ap numbers. However, we cannot exclude the death, such as nuclear decondensation, and swollen mitochondria possibility that infection with C6706 leads to relocalization of Ap (Fig. 3 K and L). Infection with C6706ΔvasK caused a phenotype within the intestine, thereby exacerbating disease. Nonetheless, that was intermediate between mock-infected controls and C6706- our data suggest that V. cholerae infection does not substantially infected adults. Guts infected with C6706ΔvasK retained elements alter total Ap numbers. As we did not detect a change in Ap

Fast et al. PNAS | July 3, 2018 | vol. 115 | no. 27 | 7101 Downloaded by guest on September 26, 2021 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.

numbers, we tested the alternate possibility that T6SS-mediated negative commensal Ap (Fig. 4E). These observations led us to interactions with a subset of intestinal Ap induce secondary re- ask whether interactions between T6SS and Ap are a prerequisite sponses in the host that accelerate death. For example, mutations for T6SS-mediated killing of the host. To test this hypothesis, we in the IMD antibacterial pathway attenuate V. cholerae-dependent examined host viability in adult flies that we associated exclu- killing of the host (ref. 31 and Fig. 4G). IMD contributes to anti- sively with Ap,orLb, and subsequently infected with C6706 or bacterial responses in the fly gut (32), and is similar to the mam- C6706ΔvasK. For each study, we ran a parallel infection study on malian TNF pathway, a regulator of intestinal inflammation in CR flies with the same cultures of V. cholerae. Loss of T6SS mammals (33, 34). To determine whether T6SS-mediated inter- significantly impaired pathogenesis in each test with control, CR actions with the host involve pathological activation of immune flies (Fig. 5 D–F). However, loss of T6SS did not diminish Vibrio responses, we infected wild-type and imd mutant flies with C6706 pathogenesis in adult flies that we associated exclusively with Lb or C6706ΔvasK. Mutation of either vasK or imd prolonged host (Fig. 5A). As Lb also fails to block host colonization by a T6SS- viability to near-equal extents (Fig. 4G). Ablation of T6SS in defective C6706 strain (Fig. 4C), our data suggest that interac- combination with an imd mutation extended host viability further tions between T6SS and Lb have minimal relevance for host vi- (Fig. 4G). These data suggest that additive effects from the T6SS of ability. In contrast, we detected significant involvement of T6SS V. cholerae and the IMD pathway of Drosophila synergistically in the extermination of adults that we monoassociated with Ap control host viability. (Fig. 5B), indicating that Ap is sufficient for T6SS-mediated killing of the host. We then asked whether Gram-positive com- The Microbiome Directly Influences T6SS-Dependent Pathogenesis. mensals can protect Drosophila from T6SS-dependent killing of T6SS contributes to Drosophila killing by V. cholerae (Fig. 1 B Ap-associated flies. Here, we associated adult Drosophila with a and C), T6SS-assisted killing of Drosophila requires an intestinal 1:1:1 mixture of Ap, Lb, and Lp. We then challenged the flies microbiome (Fig. 1D), and T6SS specifically targets the Gram- with C6706 or C6706ΔvasK, and measured survival rates. In this

7102 | www.pnas.org/cgi/doi/10.1073/pnas.1802165115 Fast et al. Downloaded by guest on September 26, 2021 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 replace- ment 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 MICROBIOLOGY

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 V52ΔvasK 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 (ΔvasK). (F)CFU/FlyofAp 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 C6706ΔvasK. Tables show Long-rank (Mantel−Cox) test; χ2andP values are relative to w1118 infected flies.

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. Discussion Fig. 5. Composition of commensal microbes impacts T6SS virulence contributions in vivo. (A) Survival curves for adult flies monoassociated with Lb.(B) Commensal bacteria form a protective barrier that shields the Survival curves for adult flies monoassociated with Ap.(C) Survival curves for host from microbial invaders (35). Here, we used the fly cholera adult flies polyassociated with Lb, Ap, and Lp.InA–C, flies were infected as model to ask whether T6SS-mediated pathogen-commensal in- indicated. (D–F) Survival curves for parallel infection studies performed on teractions influence host death. We found that T6SS contributes CR flies. The y axis represents percent survival, and the x axis represents to V. cholerae pathogenesis, and that T6SS accelerates host death infection time in hours. Tables show Log-rank (Mantel−Cox) test.

Fast et al. PNAS | July 3, 2018 | vol. 115 | no. 27 | 7103 Downloaded by guest on September 26, 2021 Lactobacilli, was sufficient to restore T6SS-dependent killing of the plate where each well had been lined with filter paper soaked in PBS + host. These observations are in line with a model where T6SS- 5% sucrose. After 4 h, the filter paper was vortexed, and serial dilutions mediated killing of a proportion of intestinal Ap initiates sec- were made on LB + Streptomycin. CFUs were counted the next day. ondary events that enhance host destruction by V. cholerae.We consider the IMD pathway a possible mediator of such an effect. Generation of Monoassociated Drosophila. Virgin females were raised on se- Our work shows that additive effects between IMD and T6SS- lective medium for 5 d at 29 °C. After 5 d of antibiotic treatment, flies were dependent interactions with Ap program the intestinal environ- starved in sterile empty vials for 2 h before bacterial association. Flies were V. cholerae ment in a manner that supports pathogenesis. This then fed bacterial cultures (OD600 of 50) resuspended in 5% sucrose in PBS. model is supported by recent work in the infant mouse, demon- Flies were fed the bacteria sucrose suspension for 16 h at 29 °C and then kept V. cholerae strating that the T6SS of activates the immune system on autoclaved food for 5 d before infection. to a greater extent when commensals are present (13). The system described in this report presents a simple in vivo model to define Colony Forming Units per Fly. At indicated time points, 25 flies per infection − − the host microbe pathogen interactions that determine T6SS- group were collected and surface-sterilized to remove bacteria on the surface mediated death. of the fly. The flies were mechanically homogenized, and the homogenate Materials and Methods was plated to select for indicated bacteria. Extended materials and methods can be found in SI Appendix. Competition Assays. V. cholerae V52 or V52ΔvasK was mixed with commensal bacteria at a 10:1 ratio and coincubated. After a 2‐h incubation at Bacterial Strains. All Drosophila commensal bacteria strains used were iso- 37 °C, bacteria were harvested, serially diluted, and plated to enumerate lated from wild-type laboratory flies from the Foley laboratory at the Uni- surviving commensals. CFUs were counted the next day. versity of Alberta. V. cholerae C6706 was obtained from John Mekalanos (Harvard Medical School, Boston). TEM. Flies were washed with 95% ethanol and dissected into PBS. Posterior Fly Husbandry. All experiments were performed with virgin female flies; w1118 midguts were immediately excised and placed into fixative (3% para- flies were used as wild type. The imd−/− (imdEY08573) and w, esg-GAL4, UAS- formaldehyde + 3% glutaraldehyde). Flies were fixed, prepared, contrast- GFP, tubGAL80ts flies have previously been described (25, 36). sectioned, sectioned, and visualized.

Oral Infection with V. cholerae. Virgin female flies were fed Bloomington food ACKNOWLEDGMENTS. The imd mutants and esg transgenic flies were pro- for 5 d at 29 °C without flipping. Flies were starved for 2 h before being fed a vided by Dr. Bruno Lemaitre and Dr. Bruce Edgar. We thank Dr. Maya Shmulevitz

soaked cotton plug soaked with V. cholerae (OD600 of 0.125). Dead flies for her helpful discussions and supervision of B.K. We acknowledge microscopy were counted every 8 h. support from Dr. Stephen Ogg and Woo Jung Cho and the Faculty of Medicine and Dentistry core imaging service at the Cell Imaging Centre, University of V. cholerae Shedding Assay. Flies were infected as in Oral Infection with V. Alberta. The research was funded by Canadian Institute of Health Research cholerae. After a 24-h infection, individual flies were placed in a 96-well Grants MOP 7746 (to E.F.) and MOP 137106 (to S.P.).

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