Outbreaks of cholera, a rapidly fatal diarrheal disease, often spread explosively. The efficacy of reactive vaccination campaigns-deploying 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 and incorporating safeguards against vaccine reversion. We demonstrate that administration of HaitiV 24 hours before lethal challenge with wild-type 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.

Despite the advent of new techniques for genetic engineering of bacteria, allelic exchange through homologous recombination remains an important tool for genetic analysis. Currently, sacB-based vector systems are often used for allelic exchange, but counterselection escape, which prevents isolation of cells with the desired mutation, occasionally limits their utility. To circumvent this, we engineered a series of “pTOX” allelic-exchange vectors. Each plasmid encodes one of a set of inducible toxins, chosen for their potential utility in a wide range of medically important proteobacteria. A codon-optimized rhaS transcriptional activator with a strong synthetic ribosome-binding site enables tight toxin induction even in organisms lacking an endogenous rhamnose regulon. Expression of the gene encoding blue AmilCP or magenta TsPurple nonfluorescent chromoprotein facilitates monitoring of successful single- and double-crossover events using these vectors. The versatility of these vectors was demonstrated by deleting genes in Serratia marcescens, Escherichia coli O157:H7, Enterobacter cloacae, and Shigella flexneri. Finally, pTOX was used to characterize the impact of disruption of all combinations of the 3 paralogous S. marcescens peptidoglycan amidohydrolases on chromosomal ampC β-lactamase activity and the corresponding β-lactam antibiotic resistance. Mutation of multiple amidohydrolases was necessary for high-level ampC derepression and β-lactam resistance. These data suggest why β-lactam resistance may emerge during treatment less frequently in S. marcescens than in other AmpC-producing pathogens, like E. cloacae. Collectively, our findings suggest that the pTOX vectors should be broadly useful for genetic engineering of Gram-negative bacteria. IMPORTANCE Targeted modification of bacterial genomes is critical for genetic analysis of microorganisms. Allelic exchange is a technique that relies on homologous recombination to replace native loci with engineered sequences. However, current allelic-exchange vectors often enable only weak selection for successful homologous recombination. We developed a suite of new allelic-exchange vectors, pTOX, which were validated in several medically important proteobacteria. They encode visible nonfluorescent chromoproteins that enable easy identification of colonies bearing integrated vectors and permit stringent selection for the second step of homologous recombination. We demonstrate the utility of these vectors by using them to investigate the effect of inactivation of Serratia marcescens peptidoglycan amidohydrolases on β-lactam antibiotic resistance.

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.

31 Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is an important food-borne 32 pathogen that colonizes the colon. Transposon-insertion sequencing (TIS) was used to 33 identify genes required for EHEC and commensal E. coli K-12 growth in vitro and for 34 EHEC growth in vivo in the infant rabbit colon. Surprisingly, many conserved loci 35 contribute to EHEC's but not to K-12's growth in vitro, suggesting that gene acquisition 36 during EHEC evolution has heightened the pathogen's reliance on certain metabolic 37 processes that are dispensable for K-12. There was a restrictive bottleneck for EHEC 38 colonization of the rabbit colon, which complicated identification of EHEC genes 39 facilitating growth in vivo. Both a refined version of an existing analytic framework as 40 well as PCA-based analysis were used to compensate for the effects of the infection 41 bottleneck. These analyses confirmed that the EHEC LEE-encoded type III secretion 42 apparatus is required for growth in vivo and revealed that only a few effectors are critical 43 for in vivo fitness. Numerous mutants not previously associated with EHEC 44 survival/growth in vivo also appeared attenuated in vivo, and a subset of these putative 45 in vivo fitness factors were validated. Some were found to contribute to efficient type-46 three secretion while others, including tatABC, oxyR, envC, acrAB, and cvpA, promote 47 EHEC resistance to host-derived stresses encountered in vivo. cvpA, which is also 48 required for intestinal growth of several other enteric pathogens, proved to be required 49 for EHEC, Vibrio cholerae and Vibrio parahaemolyticus resistance to the bile salt 50 deoxycholate. Collectively, our findings provide a comprehensive framework for 51 understanding EHEC growth in the intestine. 523 53 Author Summary 54 Enterohemorrhagic E. coli (EHEC) are important food-borne pathogens that infect the 55 colon. We created a highly saturated EHEC transposon library and used transposon 56 insertion sequencing to identify the genes required for EHEC growth in vitro and in vivo 57 in the infant rabbit colon. We found that there is a large infection bottleneck in the rabbit 58 model of intestinal colonization, and refined two analytic approaches to facilitate 59 rigorous identification of new EHEC genes that promote fitness in vivo. Besides the 60 known type III secretion system, more than 200 additional genes were found to 61 contribute to EHEC survival and/or growth within the intestine. The requirement for 62 some of these new in vivo fitness factors was confirmed, and their contributions to 63 infection were investigated. This set of genes should be of considerable value for future 64 studies elucidating the processes that enable the pathogen to proliferate in vivo and for 65 design of new therapeutics. 66 4 67 Introduction 68 Enterohemorrhagic Escherichia coli (EHEC) is an important food-borne pathogen that 69 causes gastrointestinal (GI) infections worldwide. EHEC is a non-invasive pathogen that 70 colonizes the human colon and gives rise to sporadi...

The function of cvpA, a bacterial gene predicted to encode an inner membrane protein, is largely unknown. Early studies in E. coli linked cvpA to Colicin V secretion and recent work revealed that it is required for robust intestinal colonization by diverse enteric pathogens. In enterohemorrhagic E. coli (EHEC), cvpA is required for resistance to the bile salt deoxycholate (DOC). Here, we carried out genome-scale transposon-insertion mutagenesis and spontaneous suppressor analysis to uncover cvpA’s genetic interactions and identify common pathways that rescue the sensitivity of a ΔcvpA EHEC mutant to DOC. These screens demonstrated that mutations predicted to activate the σE-mediated extracytoplasmic stress response bypass the ΔcvpA mutant’s susceptibility to DOC. Consistent with this idea, we found that deletions in rseA and msbB and direct overexpression of rpoE restored DOC resistance to the ΔcvpA mutant. Analysis of the distribution of CvpA homologs revealed that this inner membrane protein is conserved across diverse bacterial phyla, in both enteric and non-enteric bacteria that are not exposed to bile. Together, our findings suggest that CvpA plays a role in cell envelope homeostasis in response to DOC and similar stress stimuli in diverse bacterial species. IMPORTANCE Several enteric pathogens, including Enterohemorrhagic E. coli (EHEC), require CvpA to robustly colonize the intestine. This inner membrane protein is also important for secretion of a colicin and EHEC resistance to the bile salt deoxycholate (DOC), but its function is unknown. Genetic analyses carried out here showed that activation of the σE-mediated extracytoplasmic stress response restored the resistance of a cvpA mutant to DOC, suggesting that CvpA plays a role in cell envelope homeostasis. The conservation of CvpA across diverse bacterial phyla suggests that this membrane protein facilitates cell envelope homeostasis in response to varied cell envelope perturbations.

In Escherichia coli, after DNA damage, the SOS response increases the transcription (and protein levels) of approximately 50 genes. As DNA repair ensues, the level of transcription returns to homeostatic levels. ClpXP and other proteases return the high levels of several SOS proteins to homeostasis. When all SOS genes are constitutively expressed and many SOS proteins are stabilized by the removal of ClpXP, microscopic analysis shows that cells filament, produce mini-cells and have branching protrusions along their length. The only SOS gene required (of 19 tested) for the cell length phenotype is recN. ClpXP or recN4174 (A552S, A553V), a mutant not recognized by ClpXP, produce filamentous cells with nucleoid partitioning defects. It is hypothesized that when produced at high levels during the SOS response, RecN interferes with nucleoid partitioning and Z-Ring function by holding together sections of the nucleoid, or sister nucleoids, providing another way to inhibit cell division. RecN is a member of the Structural Maintenance of Chromosome (SMC) class of proteins. It can hold pieces of DNA together and is important for doublestrand break repair (DSBR). RecN is degraded by ClpXP. Overexpression of recN + in the absence of

Adaptation to shifting temperatures is crucial for the survival of the bacterial pathogen Vibrio cholerae. Here, we show that colony rugosity, a biofilm-associated phenotype, is regulated by temperature in V. cholerae strains that naturally lack the master biofilm transcriptional regulator HapR. Using transposon-insertion mutagenesis, we found the V. cholerae ortholog of BipA, a conserved ribosome-associated GTPase, is critical for this temperature-dependent phenomenon. Proteomic analyses revealed that loss of BipA alters the synthesis of >300 proteins in V. cholerae at 22°C, increasing the production of biofilm-related proteins including the key transcriptional activators VpsR and VpsT, as well as proteins important for diverse cellular processes. At low temperatures, BipA protein levels increase and are required for optimal ribosome assembly in V. cholerae, suggesting that control of BipA abundance is a mechanism by which bacteria can remodel their proteomes. Our study reveals a remarkable new facet of V. cholerae’s complex biofilm regulatory network.

Enterohemorrhagic Escherichia coli (EHEC) is a food-borne pathogen that causes diarrheal disease and the potentially lethal hemolytic uremic syndrome. We used an infant rabbit model of EHEC infection that recapitulates many aspects of human intestinal disease to comprehensively assess colonic transcriptional responses to this pathogen. Cellular compartment-specific RNA-sequencing of intestinal tissue from animals infected with EHEC strains containing or lacking Shiga toxins (Stx) revealed that EHEC infection elicits a robust response that is dramatically shaped by Stx, particularly in epithelial cells. Many of the differences in the transcriptional responses elicited by these strains were in genes involved in immune signaling pathways, such as IL23A, and coagulation, including F3, the gene encoding Tissue Factor. RNA FISH confirmed that these elevated transcripts were found almost exclusively in epithelial cells. Collectively, these findings suggest that Stx potently remodels the host innate immune response to EHEC.