Characterising the functional roles of the three promoters in region one of the Escherichia coli Group 2 capsule gene cluster

The expression of capsular polysaccharide is a common feature found in extra-intestinal isolates causing neonatal meningitis, septicaemia and urinary tract infections. Group 2 capsular polysaccharides such as K1 antigen is expressed by various uropathogenic strains of E. coli (UPEC) that cause urinary tract infections. The capsule is considered to be an essential virulence factor conferring resistance to host defences. Moreover, capsule displays phase variable expression that adds to the virulence by providing fitness during the infection process. The expression of Group 2 capsular polysaccharide is complex as it involves three tandem promoters in PR1 region (PR1-1, PR1-2 and PR1-3) which drive the transcription of genes essential for polysaccharide export and a convergent promoter, PR3, which drive the transcription to capsule-synthesis genes. The Group 2 capsule is temperature regulated at transcriptional level with the capsule being expressed at 37°C but not at 20°C. Regulatory proteins H-NS and BipA play an essential role in temperature regulation. Additionally, 5' untranslated regions of PR1 and PR3 are involved in the regulation of transcription by interacting with other regulatory proteins such as IHF and SlyA. In this study, I dissect the regulation of the three functional promoters in PR1 region at a chromosomal level by mutating the -10 functional elements of each promoter in the chromosome of a UPEC isolate (strain UTI89). The effect of such mutations on transcription exiting PR1 was measured in PR1-lacZ transcriptional fusion and the effect of such mutations on capsule expression was investigated in UTI89 background. This study revealed that PR1-1 is the master promoter that activates the transcription from PR1-2 and PR1-3 when the bacteria were grown in LB, probably by transcription-coupled DNA supercoiling. Additionally, the data presented in this study suggests that PR1-2 and PR1-3 are coupled promoters that also depend on each other. Interestingly, mutating either PR1- 2 or PR1-3 resulted in an increase in the phase variable expression of the capsule indicating that PR1-1 is capable of driving expression of the full capsule in some of the cells even in the absence of the other promoters. The role of regulatory proteins (IHF, SlyA and H-NS) on PR1 promoters was investigated at 37 °C by measuring the transcription from PR1-lacZ possessing mutations in the desired promoter with mutations in the genes that encode these regulatory proteins. The examined regulatory proteins had variable effects on transcription exiting PR1 according to the growth phase. IHF activates PR1-1 at the exponential phase while represses PR1-2 and/or PR1-3 at the stationary phase. H-NS represses PR1-1 at mid-exponential phase while activates it at stationary phase. SlyA was required for maximum transcription from PR1-1 at early exponential phase. To examine the role of the three promoters in PR1 region in an intracellular environment, gentamicin protection assays were performed using different PR1 mutants in UTI89 to infect PD07i bladder cells. The unencapsulated bacteria were the initial colonizers which preferentially adhere to and invade bladder cells and once the bacteria were internalized, some of them expressed the capsule. This study showed that the interplay between the three promoters in LB is not applicable in the intracellular environment of bladder cells. Interestingly, PR1-3 was responsible for the encapsulation inside the bladder cells as this promoter was capable of acting independently of PR1-1 or PR1-2 inside the bladder cell, while both PR1-1 and PR1-2 were dispensable. The role of PR1 promoters in the carriage of UTI89 in the large intestine was investigated in the murine model. Unexpectedly, the capsule showed no effect in UTI89 carriage during the followed period. This study adds to the complex pattern of the regulatory network at PR1 as it suggests that the regulation varies according to the encountered environment that may trigger a specific regulatory protein to regulate the transcription from a particular promoter in PR1 region.