Regulation of Expression of the TIR-Containing Protein C Gene of the Uropathogenic Escherichia coli Strain CFT073

Abstract

The uropathogenic Escherichia coli strain CFT073 causes kidney abscesses in mice Toll/interleukin-1 receptor domain-containing protein C (TcpC) dependently and the corresponding gene is present in around 40% of E. coli isolates of pyelonephritis patients. It impairs the Toll-like receptor (TLR) signaling chain and the NACHT leucin-rich repeat PYD protein 3 inflammasome (NLRP3) by binding to TLR4 and myeloid differentiation factor 88 as well as to NLRP3 and caspase-1, respectively. Overexpression of the tcpC gene stopped replication of CFT073. Overexpression of several tcpC-truncation constructs revealed a transmembrane region, while its TIR domain induced filamentous bacteria. Based on these observations, we hypothesized that tcpC expression is presumably tightly controlled. We tested two putative promoters designated P1 and P2 located at 5′ of the gene c2397 and 5′ of the tcpC gene (c2398), respectively, which may form an operon. High pH and increasing glucose concentrations stimulated a P2 reporter construct that was considerably stronger than a P1 reporter construct, while increasing FeSO4 concentrations suppressed their activity. Human urine activated P2, demonstrating that tcpC might be induced in the urinary tract of infected patients. We conclude that P2, consisting of a 240 bp region 5′ of the tcpC gene, represents the major regulator of tcpC expression.

The Promoter of the Immune-Modulating Gene TIR-Containing Protein C of the Uropathogenic Escherichia coli Strain CFT073 Reacts to the Pathogen’s Environment

“…The phenotype of the CFT073 ∆hns strain was weaker after 24 h of culture (Figure 8B). As we reported earlier, high pH values stimulated the P2 promoter [12]. We therefore compared the P2 promoter activity of the three different gene-deficient P2 reporter strains upon stimulation with increasing pH.…”

“…We repeated the experiment with the reporter construct pPc2397-Pc2398:gfpmut:KAN containing, in addition to the promoter P2, the promoter P1, which may generate a polycistronic mRNA encompassing c2397 and tcpC (c2398). While we described earlier the transcription start of P2 in the 5 UTR of the gene c2397 [12], we now determined the transcription start of P1 using a 5 RACE PCR approach and found two different transcription start sites, one 57 and the other one 13 bp before a putative start codon of c2397 (Figure S4). Increasing potassium chloride concentration also stimulated the pPc2397-Pc2398:gfpmut:KAN (P1 + P2) reporter construct (Figure 5C,D) with no real difference to pPc2398:gfpmut:KAN (P2).…”

“…We therefore tested the ability of the uroepithelial cell line T24/83 to activate the tcpC promoter. We transformed CFT073 with the reporter plasmid Pc2398:gfpmut2 containing the P2 promoter fused to the green fluorescent protein mut2 (gfpmut2), described above, to generate the plasmid-based reporter strain CFT073 pPc2398:gfpmut2:KAN [12]. We then co-incubated the CFT073 pPc2398:gfpmut2:KAN reporter strain for 4 and 24 h with titrated amounts of T24/83 cells in McCoy medium and measured its fluorescence intensity by flow cytometry.…”

“…The former is located in the 5 UTR of the gene c2397, the latter in the 5 UTR of c2398, which encodes TcpC. We found that P2 responded much more strongly than P1 to stimuli such as high pH, glucose concentration of 3 mmol/l or more, and urine [12]. These stimuli not only activated the plasmid-based reporter constructs transformed into CFT073 on plasmids but also stimulated a chromosomal reporter strain where we replaced tcpC by gfpmut2.…”

“…These stimuli not only activated the plasmid-based reporter constructs transformed into CFT073 on plasmids but also stimulated a chromosomal reporter strain where we replaced tcpC by gfpmut2. We mapped the transcription start of P2 44 bp 5 of a putative start codon of the c2398 RNA [12].…”