Forward genetic screens are powerful tools for functional genomics. The comparison of similar forward genetic screens performed in different organisms enables the identification of genes with similar or different phenotypes across organisms. Transposon insertion sequencing is a widely used method for conducting genome-scale forward genetic screens in bacteria, yet few bioinformatic approaches have been developed to compare the results of screen replicates and different screens conducted across species or strains. Here, we used principal-component analysis (PCA) and hierarchical clustering, two unsupervised learning approaches, to analyze the relatedness of multiple in vivo screens of pathogenic vibrios. This analytic framework reveals both shared pan-vibrio requirements for intestinal colonization and strain-specific dependencies. Our findings suggest that PCA-based analytics will be a straightforward widely applicable approach for comparing diverse transposon insertion sequencing screens.

M proteins are surface-anchored virulence factors in group A streptococci, human pathogens. Here, we identified an M-like protein, SzM, and its positive regulator, SezV, in Streptococcus equi subspecies zooepidemicus (SEZ), an important group of pathogens for domesticated animals, including horses and pigs. SzM and SezV homologues were found in the genomes of all SEZ and S. equi subspecies equi and M18 group A streptococcal strains analyzed but not in other streptococci. Mutant SEZ strains lacking either sezV or szM were highly attenuated in a mouse model of infection. Collectively, our findings suggest that SezV-related regulators and the linked SzM family of M-like proteins define a new subset of virulent streptococci.

Commensal bacteria on skin may limit the ability of pathogenic bacteria to cause clinically significant infections. The bacteria on healing acute wounds, which might provide such a protective effect, have not been described using culture-independent approaches in the absence of antibiotics.

Shigella species cause diarrheal disease globally. Shigellosis is typically characterized by bloody stools and colitis with mucosal damage and is the leading bacterial cause of diarrheal death worldwide. After the pathogen is orally ingested, it invades and replicates within the colonic epithelium through mechanisms that rely on its type III secretion system (T3SS). Currently, oral infection-based small animal models to study the pathogenesis of shigellosis are lacking. Here, we found that orogastric inoculation of infant rabbits with Shigella flexneri resulted in diarrhea and colonic pathology resembling that found in human shigellosis. Fasting animals prior to S. flexneri inoculation increased the frequency of disease. The pathogen colonized the colon, where both luminal and intraepithelial foci were observed. The intraepithelial foci likely arise through S. flexneri spreading from cell to cell. Robust S. flexneri intestinal colonization, invasion of the colonic epithelium, and epithelial sloughing all required the T3SS as well as IcsA, a factor required for bacterial spreading and adhesion in vitro. Expression of the proinflammatory chemokine interleukin 8 (IL-8), detected with in situ mRNA labeling, was higher in animals infected with wild-type S. flexneri versus mutant strains deficient in icsA or T3SS, suggesting that epithelial invasion promotes expression of this chemokine. Collectively, our findings suggest that oral infection of infant rabbits offers a useful experimental model for studies of the pathogenesis of shigellosis and for testing of new therapeutics. IMPORTANCE Shigella species are the leading bacterial cause of diarrheal death globally. The pathogen causes bacillary dysentery, a bloody diarrheal disease characterized by damage to the colonic mucosa and is usually spread through the fecal-oral route. Small animal models of shigellosis that rely on the oral route of infection are lacking. Here, we found that orogastric inoculation of infant rabbits with S. flexneri led to a diarrheal disease and colonic pathology reminiscent of human shigellosis. Diarrhea, intestinal colonization, and pathology in this model were dependent on the S. flexneri type III secretion system and IcsA, canonical Shigella virulence factors. Thus, oral infection of infant rabbits offers a feasible model to study the pathogenesis of shigellosis and to develop and test new therapeutics.

250 words max) 18 Shigella species cause diarrheal disease globally. Shigellosis is typically characterized by 19 bloody stools and colitis with mucosal damage and is the leading bacterial cause of diarrheal 20 death worldwide. Following oral ingestion, the pathogen invades and replicates within the 21 colonic epithelium through mechanisms that rely on its type III secretion system (T3SS). 22 Currently, oral infection-based small animal models to study the pathogenesis of shigellosis are 23 lacking. Here, we found that oro-gastric inoculation of infant rabbits with S. flexneri resulted in 24 diarrhea and colonic pathology resembling that found in human shigellosis. Fasting animals 25 prior to S. flexneri inoculation increased the frequency of disease. The pathogen colonized the 26 colon, where both luminal and intraepithelial foci were observed. The intraepithelial foci likely 27 arise through S. flexneri spreading from cell-to-cell. Robust S. flexneri intestinal colonization, 28 invasion of the colonic epithelium, and epithelial sloughing all required the T3SS as well as IcsA, 29 a factor required for bacterial spreading and adhesion in vitro. Expression of the 30 proinflammatory chemokine IL-8, detected with in situ mRNA labeling, was higher in animals 31 infected with wild-type S. flexneri versus mutant strains deficient in icsA or T3SS, suggesting 32 that epithelial invasion promotes expression of this chemokine. Collectively, our findings 33 suggest that oral infection of infant rabbits offers a useful experimental model for studies of the 34 pathogenesis of shigellosis and for testing of new therapeutics. 35 42 dependent on the S. flexneri type III secretion system and IcsA, canonical Shigella virulence 43 factors. Thus, oral infection of infant rabbits offers a feasible model to study the pathogenesis 44 of shigellosis and to develop and test new therapeutics. 45 48 life-threatening (1). This enteric pathogen, which is spread by the fecal-oral route between 49 humans, does not have an animal reservoir or vector (1). Annually, Shigella infections cause 50 tens of millions of diarrhea cases and ~200,000 deaths (2, 3). It is likely the leading cause of 51 diarrheal mortality worldwide in individuals older than 5 years (2, 3). Most Shigella infections 52 are attributable to S. flexneri, one of the four Shigella species, although in developed nations 53the prevalence of S. sonnei is higher (4-7). 54 The pathogen primarily causes colonic pathology that usually includes mucosal 55 ulceration and erosion due to sloughing of epithelial cells, and is typically characterized by 56 acute inflammation, with recruitment of neutrophils and plasma cells, congestion of blood 57 vessels, distorted crypt architecture, and hemorrhage (8, 9). While inflammatory responses to 58 Shigella invasion of colonic epithelial cells were thought to be the underlying cause of epithelial 59 cell destruction and hemorrhage, recent evidence suggests that pathogen-mediated 60 destruction of epithelial cells also plays a role in...

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