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

Abstract

Enterohemorrhagic <i>Escherichia coli</i> 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 <i>E. coli</i> K-12 growth <i>in vitro</i> and for EHEC growth <i>in vivo</i> in the infant rabbit colon. Surprisingly, many conserved loci contribute to EHEC’s but not to K-12’s growth <i>in vitro</i>. There was a restrictive bottleneck for EHEC colonization of the rabbit colon, which complicated identification of EHEC genes facilitating growth <i>in vivo</i>. 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 <i>in vivo</i> and revealed that only a few effectors are critical for <i>in vivo</i> fitness. Over 200 mutants not previously associated with EHEC survival/growth <i>in vivo</i> also appeared attenuated <i>in vivo</i>, and a subset of these putative <i>in vivo</i> fitness factors were validated. Some were found to contribute to efficient type-three secretion while others, including <i>tatABC, oxyR, envC, acrAB</i>, and <i>cvpA</i>, 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, <i>Vibrio cholerae</i> and <i>Vibrio parahaemolyticus</i> 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.