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 competi- tion 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 indi- vidual cultures of flies, control experiments with C6706 and C6706ΔvasK were repeated concurrently with each new experi- ment and plotted accordingly. On average, mutation of vasK ex- tended median survival by 16% (SI Appendix,Fig.S1). Deletion of vipA, a protein that makes up the outer sheath of the T6SS in- fection 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, in- activation 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 micro- biota. 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 re- moval of commensal bacteria will not affect T6SS-dependent killing of the host. Instead, we found that an absence of com- mensal 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 sup- plemented 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.