Abstract
Uropathogenic Escherichia coli (UPEC) is the cause of ~75% of all urinary tract infections (UTIs) and is increasingly associated with multidrug resistance. This includes UPEC strains from the recently emerged and globally disseminated sequence type 131 (ST131), which is now the dominant fluoroquinolone-resistant UPEC clone worldwide. Most ST131 strains are motile and produce H4-type flagella. Here, we applied a combination of saturated Tn5 mutagenesis and transposon directed insertion site sequencing (TraDIS) as a high throughput genetic screen and identified 30 genes associated with enhanced motility of the reference ST131 strain EC958. This included 12 genes that repress motility of E. coli K-12, four of which (lrhA, ihfA, ydiV, lrp) were confirmed in EC958. Other genes represented novel factors that impact motility, and we focused our investigation on characterisation of the mprA, hemK and yjeA genes. Mutation of each of these genes in EC958 led to increased transcription of flagellar genes (flhD and fliC), increased expression of the FliC flagellin, enhanced flagella synthesis and a hyper-motile phenotype. Complementation restored all of these properties to wild-type level. We also identified Tn5 insertions in several intergenic regions (IGRs) on the EC958 chromosome that were associated with enhanced motility; this included flhDC and EC958_1546. In both of these cases, the Tn5 insertions were associated with increased transcription of the downstream gene(s), which resulted in enhanced motility. The EC958_1546 gene encodes a phage protein with similarity to esterase/deacetylase enzymes involved in the hydrolysis of sialic acid derivatives found in human mucus. We showed that over-expression of EC958_1546 led to enhanced motility of EC958 as well as the UPEC strains CFT073 and UTI89, demonstrating its activity affects the motility of different UPEC strains. Overall, this study has identified and characterised a number of novel factors associated with enhanced UPEC motility.
Highlights
Uropathogenic Escherichia coli (UPEC) are the most common cause of urinary tract infection (UTI), a disease of major significance to global human health [1,2,3]
We recently described the combined application of saturated Tn5 mutagenesis and transposon directed insertion site sequencing (TraDIS) to comprehensively define the complete set of genes associated with resistance to human serum in the UPEC sequence type 131 (ST131) strain EC958 [43]
Our TraDIS analysis identified 30 genes associated with the enhanced motility of EC958
Summary
Uropathogenic Escherichia coli (UPEC) are the most common cause of urinary tract infection (UTI), a disease of major significance to global human health [1,2,3]. The combined affect of genetic variation, redundancy and genomic diversity means that no single virulence factor is uniquely associated with the ability of UPEC to cause disease. This complex picture is further convoluted by increased resistance to antibiotics, which complicates the treatment of UTI and highlights the urgent need to better understand UPEC pathogenesis. Flagella-mediated motility is required for UPEC ascension to the upper urinary tract and subsequent dissemination to other sites [17] Complementing these studies, others have shown that flagella contribute to UPEC invasion of mouse renal epithelial collecting duct cells [5] and enhanced adhesion to and invasion of bladder epithelial cells [14]. Flagella are required for UPEC biofilm formation on abiotic surfaces [12]
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