High-throughput fitness screening and transcriptomics identify a role for a type IV secretion system in the pathogenesis of Crohn's disease-associated Escherichia coli.

Adherent-invasive Escherichia coli (AIEC) are pathogenic bacteria frequently isolated from patients who have Crohn's disease (CD). Despite the phenotypic differences between AIEC and commensal E. coli, comparative genomic approaches have been unable to differentiate these two groups, making the identification of key virulence factors a challenge. Here, we conduct a high-resolution, in vivo genetic screen to map AIEC genes required for intestinal colonization of mice. In addition, we use in vivo RNA-sequencing to define the host-associated AIEC transcriptome. We identify diverse metabolic pathways required for efficient gut colonization by AIEC and show that a type IV secretion system (T4SS) is required to form biofilms on the surface of epithelial cells, thereby promoting AIEC persistence in the gut. E. coli isolated from CD patients are enriched for a T4SS, suggesting a possible connection to disease activity. Our findings establish the T4SS as a principal AIEC colonization factor and highlight the use of genome-wide screens in decoding the infection biology of CD-associated bacteria that otherwise lack a defined genetic signature.

Transposon-insertion sequencing screens unveil requirements for EHEC growth and intestinal colonization.

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is an important food-borne pathogen that colonizes the colon. Transposon-insertion sequencing (TIS) was used to identify genes required for EHEC and E. coli K-12 growth in vitro and for EHEC growth in vivo in the infant rabbit colon. Surprisingly, many conserved loci contribute to EHEC's but not to K-12's growth in vitro. There was a restrictive bottleneck for EHEC colonization of the rabbit colon, which complicated identification of EHEC genes facilitating growth in vivo. Both a refined version of an existing analytic framework as well as PCA-based analysis were used to compensate for the effects of the infection bottleneck. These analyses confirmed that the EHEC LEE-encoded type III secretion apparatus is required for growth in vivo and revealed that only a few effectors are critical for in vivo fitness. Over 200 mutants not previously associated with EHEC survival/growth in vivo also appeared attenuated in vivo, and a subset of these putative in vivo fitness factors were validated. Some were found to contribute to efficient type-three secretion while others, including tatABC, oxyR, envC, acrAB, and cvpA, promote EHEC resistance to host-derived stresses. cvpA is also required for intestinal growth of several other enteric pathogens, and proved to be required for EHEC, Vibrio cholerae and Vibrio parahaemolyticus resistance to the bile salt deoxycholate, highlighting the important role of this previously uncharacterized protein in pathogen survival. Collectively, our findings provide a comprehensive framework for understanding EHEC growth in the intestine.

Unsupervised Learning Approach for Comparing Multiple Transposon Insertion Sequencing Studies.

Transposon insertion sequencing (TIS) is a widely used technique for conducting genome-scale forward genetic screens in bacteria. However, few methods enable comparison of TIS data across multiple replicates of a screen or across independent screens, including screens performed in different organisms. Here, we introduce a post hoc analytic framework, comparative TIS (CompTIS), which utilizes unsupervised learning to enable meta-analysis of multiple TIS data sets. CompTIS first implements screen-level principal-component analysis (PCA) and clustering to identify variation between the TIS screens. This initial screen-level analysis facilitates the selection of related screens for additional analyses, reveals the relatedness of complex environments based on growth phenotypes measured by TIS, and provides a useful quality control step. Subsequently, PCA is performed on genes to identify loci whose corresponding mutants lead to concordant/discordant phenotypes across all or in a subset of screens. We used CompTIS to analyze published intestinal colonization TIS data sets from two vibrio species. Gene-level analyses identified both pan-vibrio genes required for intestinal colonization and conserved genes that displayed species-specific requirements. CompTIS is applicable to virtually any combination of TIS screens and can be implemented without regard to either the number of screens or the methods used for upstream data analysis.IMPORTANCE 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.

A live vaccine rapidly protects against cholera in an infant rabbit model.

Outbreaks of cholera, a rapidly fatal diarrheal disease, often spread explosively. The efficacy of reactive vaccination campaigns-deploying Vibrio cholerae vaccines during epidemics-is partially limited by the time required for vaccine recipients to develop adaptive immunity. We created HaitiV, a live attenuated cholera vaccine candidate, by deleting diarrheagenic factors from a recent clinical isolate of V. cholerae and incorporating safeguards against vaccine reversion. We demonstrate that administration of HaitiV 24 hours before lethal challenge with wild-type V. cholerae reduced intestinal colonization by the wild-type strain, slowed disease progression, and reduced mortality in an infant rabbit model of cholera. HaitiV-mediated protection required viable vaccine, and rapid protection kinetics are not consistent with development of adaptive immunity. These features suggest that HaitiV mediates probiotic-like protection from cholera, a mechanism that is not known to be elicited by traditional vaccines. Mathematical modeling indicates that an intervention that works at the speed of HaitiV-mediated protection could improve the public health impact of reactive vaccination.

Time-Resolved Transposon Insertion Sequencing Reveals Genome-Wide Fitness Dynamics during Infection.

Transposon insertion sequencing (TIS) is a powerful high-throughput genetic technique that is transforming functional genomics in prokaryotes, because it enables genome-wide mapping of the determinants of fitness. However, current approaches for analyzing TIS data assume that selective pressures are constant over time and thus do not yield information regarding changes in the genetic requirements for growth in dynamic environments (e.g., during infection). Here, we describe structured analysis of TIS data collected as a time series, termed pattern analysis of conditional essentiality (PACE). From a temporal series of TIS data, PACE derives a quantitative assessment of each mutant's fitness over the course of an experiment and identifies mutants with related fitness profiles. In so doing, PACE circumvents major limitations of existing methodologies, specifically the need for artificial effect size thresholds and enumeration of bacterial population expansion. We used PACE to analyze TIS samples of Edwardsiella piscicida (a fish pathogen) collected over a 2-week infection period from a natural host (the flatfish turbot). PACE uncovered more genes that affect E. piscicida's fitness in vivo than were detected using a cutoff at a terminal sampling point, and it identified subpopulations of mutants with distinct fitness profiles, one of which informed the design of new live vaccine candidates. Overall, PACE enables efficient mining of time series TIS data and enhances the power and sensitivity of TIS-based analyses.IMPORTANCE Transposon insertion sequencing (TIS) enables genome-wide mapping of the genetic determinants of fitness, typically based on observations at a single sampling point. Here, we move beyond analysis of endpoint TIS data to create a framework for analysis of time series TIS data, termed pattern analysis of conditional essentiality (PACE). We applied PACE to identify genes that contribute to colonization of a natural host by the fish pathogen Edwardsiella piscicida. PACE uncovered more genes that affect E. piscicida's fitness in vivo than were detected using a terminal sampling point, and its clustering of mutants with related fitness profiles informed design of new live vaccine candidates. PACE yields insights into patterns of fitness dynamics and circumvents major limitations of existing methodologies. Finally, the PACE method should be applicable to additional "omic" time series data, including screens based on clustered regularly interspaced short palindromic repeats with Cas9 (CRISPR/Cas9).

The Nucleoid Binding Protein H-NS Biases Genome-Wide Transposon Insertion Landscapes.

UNLABELLED: Transposon insertion sequencing (TIS; also known as TnSeq) is a potent approach commonly used to comprehensively define the genetic loci that contribute to bacterial fitness in diverse environments. A key presumption underlying analyses of TIS datasets is that loci with a low frequency of transposon insertions contribute to fitness. However, it is not known whether factors such as nucleoid binding proteins can alter the frequency of transposon insertion and thus whether TIS output may systematically reflect factors that are independent of the role of the loci in fitness. Here, we investigated whether the histone-like nucleoid structuring (H-NS) protein, which preferentially associates with AT-rich sequences, modulates the frequency of Mariner transposon insertion in the Vibrio cholerae genome, using comparative analysis of TIS results from wild-type (wt) and Δhns V. cholerae strains. These analyses were overlaid on gene classification based on GC content as well as on extant genome-wide identification of H-NS binding loci. Our analyses revealed a significant dearth of insertions within AT-rich loci in wt V. cholerae that was not apparent in the Δhns insertion library. Additionally, we observed a striking correlation between genetic loci that are overrepresented in the Δhns insertion library relative to their insertion frequency in wt V. cholerae and loci previously found to physically interact with H-NS. Collectively, our findings reveal that factors other than genetic fitness can systematically modulate the frequency of transposon insertions in TIS studies and add a cautionary note to interpretation of TIS data, particularly for AT-rich sequences. IMPORTANCE: Transposon insertion sequencing (TIS) is often used to assess the relative frequency with which genetic loci can be disrupted, which is taken as an indicator of their importance for bacterial fitness. Here, we report that biological factors other than the relative levels of fitness of insertion mutants can influence TIS output. We found that the presence of the DNA binding protein H-NS, which preferentially recognizes AT-rich sequences, is linked to significant underrepresentation of mutations within AT-rich loci in transposon insertion libraries. Furthermore, there is a marked correspondence between loci bound by H-NS and loci with an increased frequency of disruption in a Δhns insertion library relative to a wt library. Our data suggest that factors other than genetic fitness (e.g., DNA binding proteins such as H-NS) can systematically modulate the frequency of transposon insertions in TIS studies and add a note of caution for interpretation of TIS data.

CRISPR/Cas9 Screens Reveal Requirements for Host Cell Sulfation and Fucosylation in Bacterial Type III Secretion System-Mediated Cytotoxicity.

Type III secretion systems (T3SSs) inject bacterial effector proteins into host cells and underlie the virulence of many gram-negative pathogens. Studies have illuminated bacterial factors required for T3SS function, but the required host processes remain largely undefined. We coupled CRISPR/Cas9 genome editing technology with the cytotoxicity of two Vibrio parahaemolyticus T3SSs (T3SS1 and T3SS2) to identify human genome disruptions conferring resistance to T3SS-dependent cytotoxicity. We identity non-overlapping genes required for T3SS1- and T3SS2-mediated cytotoxicity. Genetic ablation of cell surface sulfation reduces bacterial adhesion and thereby alters the kinetics of T3SS1-mediated cytotoxicity. Cell surface fucosylation is required for T3SS2-dependent killing, and genetic inhibition of fucosylation prevents membrane insertion of the T3SS2 translocon complex. These findings reveal the importance of ubiquitous surface modifications for T3SS function, potentially explaining the broad tropism of V. parahaemolyticus, and highlight the utility of genome-wide CRISPR/Cas9 screens to discover processes underlying host-pathogen interactions.

Genetic analysis of Vibrio parahaemolyticus intestinal colonization.

Vibrio parahaemolyticus is the most common cause of seafood-borne gastroenteritis worldwide and a blight on global aquaculture. This organism requires a horizontally acquired type III secretion system (T3SS2) to infect the small intestine, but knowledge of additional factors that underlie V. parahaemolyticus pathogenicity is limited. We used transposon-insertion sequencing to screen for genes that contribute to viability of V. parahaemolyticus in vitro and in the mammalian intestine. Our analysis enumerated and controlled for the host infection bottleneck, enabling robust assessment of genetic contributions to in vivo fitness. We identified genes that contribute to V. parahaemolyticus colonization of the intestine independent of known virulence mechanisms in addition to uncharacterized components of T3SS2. Our study revealed that toxR, an ancestral locus in Vibrio species, is required for V. parahaemolyticus fitness in vivo and for induction of T3SS2 gene expression. The regulatory mechanism by which V. parahaemolyticus ToxR activates expression of T3SS2 resembles Vibrio cholerae ToxR regulation of distinct virulence elements acquired via lateral gene transfer. Thus, disparate horizontally acquired virulence systems have been placed under the control of this ancestral transcription factor across independently evolved human pathogens.