Use of signature-tagged mutagenesis to identify genes associated with colonization of sheep by E. coli O157:H7

Abstract

Outbreaks of Escherichia coli O157:H7 in the United States due to contaminated foods are a public health issue and a continuing problem. The major reservoir for these organisms is the gastrointestinal tract of ruminants where they are a member of the resident microbiota. Several factors that contribute to the colonization of cattle have been identified, but a systematic screen of genes that might contribute to the colonization and persistence phenotype in mature ruminants has not been reported. Using a sheep model of persistence, signature tagged mutagenesis (STM) was used to screen 1326 mutants for a persistence-negative phenotype of E. coli O157:H7. We identified 9 genes by STM that appeared to be required for colonization and/or survival in sheep. Three of the genes had functions associated with central metabolism (thiK, ftrA and nrdB), one was involved with LPS formation (wbdP), one encodes a non-LEE encoded effector protein (nleB) and one was a methyltransferase encoded on a prophage (Z2389). The remaining three genes did not have homology with any known genes. Six sheep given ΔwbdP and 2 sheep each were given mutants (ΔthiK (Z1745), ΔftrA (Z2164) and Z2389). The ΔwbdP mutant was recovered from the feces of 4/6 sheep at 6 days pi with a mean number of 1.42logCFU/g feces compared to 4.6logCFU/g feces for the wild type strain. This difference was significant (P<0.001) over the time course of the experiment (days 6-23). Both ΔthiK and ΔftrA mutants were recovered from 1 of 2 sheep at 9 days PI by enrichment procedures (<50CFU/g feces) whereas mutant Z2389 was not recovered from either animal past 2 days pi. The roles of all of these gene products require further study to determine how the persistence phenotype of a given strain of E. coli O157:H7 interacts with host factors.

A Metagenome-Wide Association Study and Arrayed Mutant Library Confirm Acetobacter Lipopolysaccharide Genes Are Necessary for Association with Drosophila melanogaster

“…For example, a TnSeq experiment of Snodgrassella alvi colonization in honey bees also revealed that LPS is an important factor in colonization ( Powell et al 2016 ). In non-insect hosts, E. coli LPS mutants are hyper-susceptible to host immunity in C. elegans (( Kuo et al 2016 )), and LPS is important for E. coli colonization and persistence in sheep ( Cornick et al 2017 ). Vibrio cholerae LPS mutants had a 30 fold reduction in colonization of the mouse gut ( Nesper et al 2002 ).…”

Type III Secretion Effectors with Arginine N-Glycosyltransferase Activity

“…The Glu 253 residue, together with Tyr 219 were identified in a previous study as essential for enzymatic activity and virulence function of NleB1 [152]. Interestingly, the presence of nleB1 in EHEC and EPEC strains is associated with colonization of sheep [153] and cattle [154], with human disease [155][156][157][158][159], and with an increase in disease severity [160][161][162][163][164][165]. In contrast, the role of NleB2 in virulence is currently unclear.…”