Genetic analysis of Vibrio parahaemolyticus intestinal colonization Troy P. Hubbarda,b, Michael C. Chaoa,b, Sören Abelc,d, Carlos J. Blondela,b, Pia Abel zur Wieschc,d, Xiaohui Zhoue, Brigid M. Davisa,b,f, and Matthew K. Waldora,b,f,1 aDepartment of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115; bDivision of Infectious Diseases, Brigham & Women’s Hospital, Boston, MA 02115; cDepartment of Pharmacy, Faculty of Health Sciences, UiT-The Arctic University of Norway, 9037 Tromso, Norway; dCentre for Molecular Medicine Norway, Nordic EMBL Partnership for Molecular Medicine, 0318 Oslo, Norway; eDepartment of Pathobiology & Veterinary Science, University of Connecticut, Storrs, CT 06269; and fHoward Hughes Medical Institute, Boston, MA 02115 Edited by Margaret J. McFall-Ngai, University of Hawaii at Manoa, Honolulu, HI, and approved April 18, 2016 (received for review January 31, 2016) Vibrio parahaemolyticus is the most common cause of seafood-borne V. parahaemolyticus is presumed to use many bacterial factors, gastroenteritis worldwide and a blight on global aquaculture. This in addition to T3SS2, to survive and proliferate within the human organism requires a horizontally acquired type III secretion system gastrointestinal tract. Putative colonization factors have been (T3SS2) to infect the small intestine, but knowledge of additional reported using a murine orogastric model of V. parahaemolyticus V. parahaemolyticus factors that underlie pathogenicity is limited. infection; however, the relevance of these findings to human We used transposon-insertion sequencing to screen for genes that disease is unclear because T3SS2 is not required for colonization of the contribute to viability of V. parahaemolyticus in vitro and in the mouse intestine (10). Genome-wide analysis of V. parahaemolyticus mammalian intestine. Our analysis enumerated and controlled for the host infection bottleneck, enabling robust assessment of genetic colonization factors has not been performed in any host; however, for contributions to in vivo fitness. We identified genes that contribute other pathogens, passage of transposon-insertion libraries through to V. parahaemolyticus colonization of the intestine independent of infection models has enabled highly effective, largely unbiased, and known virulence mechanisms in addition to uncharacterized compo- genome-wide identification of factors required for fitness in vivo (11). nents of T3SS2. Our study revealed that toxR,anancestrallocusin We used transposon-insertion sequencing (TIS) to categorize Vibrio species, is required for V. parahaemolyticus fitnessinvivoand genetic loci based on contributions to V. parahaemolyticus viability for induction of T3SS2 gene expression. The regulatory mechanism in vitro and in vivo (11). We discovered genes and regulatory by which V. parahaemolyticus ToxR activates expression of T3SS2 networks that contribute to intestinal colonization independent of Vibrio cholerae resembles ToxR regulation of distinct virulence ele- the few known V. parahaemolyticus virulence factors. We identi- ments acquired via lateral gene transfer. Thus, disparate horizontally fied uncharacterized components of T3SS2 and found that ToxR, acquired virulence systems have been placed under the control of an ancestral Vibrio transcription factor previously thought to be this ancestral transcription factor across independently evolved human pathogens. dispensable for regulation of T3SS2 gene expression (9), is, in fact, critical for V. parahaemolyticus intestinal colonization and T3SS2 Vibrio parahaemolyticus | transposon-insertion sequencing | activity. ToxR is also an important regulator of horizontally acquired type III secretion | bacterial pathogenesis | pathogen evolution virulence genes in Vibrio cholerae, where it controls an unrelated set of virulence elements acquired via lateral gene transfer (12). Thus, he gram-negative γ-proteobacterium Vibrio parahaemolyticus pathogenic vibrios have independently linked ToxR, which is re- Tthrives in either pathogenic or symbiotic association with ma- sponsive to signals encountered in the intestine, to control of critical rine organisms and as a planktonic bacterium (1). This facultative virulence factors. human pathogen, abundant in aquatic environments, was first isolated following a food poisoning outbreak in 1952 and has Significance emerged as the leading cause of seafood-associated gastroenteritis worldwide and a blight on global aquaculture (2, 3). Sequencing of We conducted a genome-wide screen to identify bacterial factors the V. parahaemolyticus genome and the development of animal required for Vibrio parahaemolyticus, an important cause of sea- models of infection have demonstrated a critical role for type III food-borne gastroenteritis, to survive in vitro and colonize the secretion in V. parahaemolyticus virulence (4, 5). mammalian intestine. Our analysis revealed uncharacterized com- Pathogenic isolates of V. parahaemolyticus encode two type III ponents of a horizontally acquired type III secretion system linked secretion systems (T3SSs), which are multiprotein structures that to virulence (T3SS2) and hundreds of genes that likely contribute to MICROBIOLOGY mediate the translocation of bacterial effector proteins directly colonization independent of T3SS2. Our work revealed that toxR,a into eukaryotic cells (4, 6). All V. parahaemolyticus strains encode a conserved gene in vibrios that governs expression of horizontally T3SS on the large chromosome (T3SS1), and the vast majority of acquired virulence factors in Vibrio cholerae, was critical for ex- clinical isolates, but few environmental isolates possess a horizontally pression of T3SS2. Thus, expression of disparate virulence-linked acquired pathogenicity island (VPaI-7) encoding a second T3SS elements, acquired via lateral gene transfer in independently (T3SS2) and one or more pore-forming toxins (TDH) (7). Studies evolved pathogenic vibrios, is controlled by a common ancestral using the infant rabbit model of V. parahaemolyticus infection, transcription factor. which recapitulates manifestations of human gastrointestinal disease (e.g., profuse diarrhea, enteritis, epithelial disruption), revealed that Author contributions: T.P.H., M.C.C., S.A., and M.K.W. designed research; T.P.H., C.J.B., and X.Z. performed research; T.P.H., S.A., and P.A.z.W. contributed new reagents/analytic although T3SS1 and TDH are dispensable for intestinal colonization tools; T.P.H., M.C.C., S.A., P.A.z.W., B.M.D., and M.K.W. analyzed data; and T.P.H., B.M.D., and pathogenesis, colonization and pathology are dependent on and M.K.W. wrote the paper. T3SS2, consistent with the epidemiological association between The authors declare no conflict of interest. T3SS2 and pathogenicity (5). Furthermore, T3SS2 gene expression This article is a PNAS Direct Submission. is induced during intestinal colonization, likely in response to bile, 1To whom correspondence should be addressed. Email: [email protected]. V. parahaemolyticus which promotes production of T3SS2 regulator This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. B (VtrB) (8, 9). 1073/pnas.1601718113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1601718113 PNAS | May 31, 2016 | vol. 113 | no. 22 | 6283–6288 Downloaded by guest on September 24, 2021 coli V. cholerae A 900 and , organisms in which the genes required for vi- ability have been extensively characterized (Fig. 2B and Dataset 800 Gene Classifications S2C). Many nonneutral V. parahaemolyticus genes had homologs UR (405) 700 among the 287 protein-coding genes absent from the Keio collection 600 (a proxy for the E. coli essential gene set), and most are reportedly 500 R (160) essential or domain-essential in V. cholerae (15). Still, a notable 400 fraction of the 565 genes had homologs that were individually 300 N (3,898) dispensable for E. coli or V. cholerae viability in vitro or lacked 200 homologs in these organisms. A small subset of E. coli essential Number of Genes Total = 4,458 V. parahaemolyticus D 100 genes (34 genes) was neutral in (Dataset S2 ) and contained genes similar to those from a comparable analysis of V. cholerae, which may be indicative of Vibrio-specific adaptations 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 in the associated processes (14). The 565 nonneutral genes likely Percentage of TA Sites Disrupted (binned) encompassed all or almost all genes required for V. parahaemolyticus B under-represented regional neutral growth in vitro. Notably, this set of genes contained loci that have 25 been deleted in previous studies, suggesting that the distribution of 0 insertion mutants within a transposon-insertion library can reflect Reads TA sites multiple factors, not solely the viability of each insertion mutant, and that our analysis likely overestimates the number of non- dnaA - VP0011 pbgA - VP2902 tamAt A - VPVP03070307 neutral loci in vitro. Fig. 1. EL-ARTIST gene classifications. (A) Distribution of percentage disruption Identification of Genes Required for Intestinal Colonization. + We used for genes with 10 TA sites. Genes classified as underrepresented (UR), re- V. parahaemolyticus R gional (R), and neutral (N) are represented within each bin and in aggregate. the Sm library to identify genes required (B) Transposon-insertion profiles of representative underrepresented, regional, for colonization of the mammalian gastrointestinal tract. Infant 9 and neutral genes. The vertical axis