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

Abstract

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.

Adaptation of Arginine Synthesis among Uropathogenic Branches of the Escherichia coli Phylogeny Reveals Adjustment to the Urinary Tract Habitat

“…We therefore conclude that positive selection analysis is indeed a powerful and complementary technique for identifying genes important for virulence. The success rate of PSG validation (deletion of 10/29 PSGs has a fitness defect, during chronic infection [8 PSGs] and/or QIR formation [6 PSGs]) compares favorably to other methods for identifying UPEC virulence genes, such as gene presence/absence analysis, candidate gene analysis (mostly targeted at "traditional" virulence factors or pathogenicity islands), and transcriptomics analyses (38,40,(80)(81)(82)(83)(84). Of note, other mutant screens, such as Tn-seq, have very high validation rates.…”

DEG 15, an update of the Database of Essential Genes that includes built-in analysis tools

“…Another advantage of Tn-seq is that it identifies not only essential protein-coding genes, but also non-coding genomic elements. For instance, by using Tn-seq, a large number of non-coding genomic elements have been determined in Acinetobacter baumannii ( 24 ), Brevundimonas subvibrioides ( 25 ), Escherichia coli O157:H7 ( 26 ), Mycobacterium tuberculosis ( 27 ), Mycoplasma pneumonia ( 28 ), Salmonella entericaserovar Typhimurium ( 29 ), Sphingomonas wittichii ( 30 ) and Streptococcus pneumonia ( 31 ).…”

Shiga toxin remodels the intestinal epithelial transcriptional response to Enterohemorrhagic Escherichia coli

“…Bacterial strains were cultured in LB medium or on LB agar plates at 37˚C. A gentamicin-resistant mutant of E. coli O157:H7 strain EDL933 (ΔlacI::aacC1) [85] was used in all experiments in this study and gentamicin (Gm) was used at 10 μg/mL. The ΔΔstx mutant was constructed using lambda red recombineering [86] as described [87].…”