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

Abstract

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...

An oral inoculation infant rabbit model forShigellainfection

“…We attempted to use transposon-insertion sequencing (TIS) to identify genetic loci contributing to S. flexneri colonization and pathogenesis, as we have done with V. cholerae (53, 54), V. parahaemolyticus (55), and EHEC (56). Initially, a high-density transposon mutant library in S. flexneri was created using a mariner -based transposon that inserts at TA dinucleotide sites in the genome.…”

A global data-driven census of Salmonella small proteins and their potential functions in bacterial virulence

“…For example, high-resolution transcriptomics, transposon mutagenesis, and Grad-seq data exist for Streptococcus pneumoniae (Aprianto et al, 2016(Aprianto et al, , 2018Hor et al, 2020;Rowe et al, 2019;van Opijnen and Camilli, 2012;Warrier et al, 2018). More generally, various transposonsequencing approaches have been applied to bacterial pathogens under virulence conditions (Karlinsey et al, 2019;Rendón et al, 2020;Warr et al, 2019), and dual RNAseq has become the gold-standard to chart the transcriptional landscape of pathogens during infection (Montoya et al, 2019;Pisu et al, 2020;Ritchie and Evans, 2019;Westermann et al, 2017). Re-inspection of these data may provide invaluable information on potentially new biological roles carried out by small proteins in bacterial pathogenesis.…”

Genetic analyses link the conserved inner membrane protein CvpA to the σEextracytoplasmic stress response

“…CvpA bears structural similarity to the inner membrane mechanosensitive ion channels MscS and MscL (HHPred, Phyre2) (22), and whole-proteome localization analysis in E. coli assigned CvpA to the inner membrane (32). Furthermore, several protein modeling algorithms (PSLPred, HHPred, Phobius, Phyre2, OCTOPUS) (22) predict that CvpA is a 4-pass inner membrane protein with a periplasmic C-terminus ( Fig 1A). To experimentally verify these assignments, we generated two plasmids that inducibly express sfGFP-CvpA fusions, with sfGFP on either the N-or C-terminus, enabling visualization of the localization of CvpA in EHEC.…”

“…The gene cvpA was identified in screens for genes required for intestinal colonization in several enteric pathogens, including Vibrio cholerae, Vibrio parahaemolyticus, and Salmonella Typhimurium (19)(20)(21), but the mechanism(s) that account for the colonization defects have not been characterized. We recently found that CvpA is required for the foodborne intestinal pathogen enterohemorrhagic Escherichia coli O157:H7 (EHEC) to optimally colonize the colon of infant rabbits; moreover, EHEC cvpA deletion mutants were highly sensitive to bile, and in particular the bile salt deoxycholate (DOC) (22). EHEC causes hemorrhagic colitis and in some cases the severe complication hemolytic uremic syndrome, a condition in which damage to blood vessels of the kidney microvasculature leads to renal failure.…”