Abstract
The TIR-containing protein C (TcpC) of the uropathogenic Escherichia coli strain CFT073 modulates innate immunity by interfering with the Toll-like receptor and NALP3 inflammasome signaling cascade. During a urinary tract infection the pathogen encounters epithelial and innate immune cells and replicates by several orders of magnitude. We therefore analyzed whether these cell types and also the density of the pathogen would induce the recently defined promoter of the CFT073 tcpC gene to, in time, dampen innate immune responses. Using reporter constructs we found that the uroepithelial cell line T24/83 and the monocytic cell line THP-1 induced the tcpC promoter. Differentiation of monocytic THP-1 cells to macrophages increased their potential to switch on the promoter. Cell-associated CFT073 displayed the highest promoter activity. Since potassium represents the most abundant intracellular ion and is secreted to induce the NLRP3 inflammasome, we tested its ability to activate the tcpC promoter. Potassium induced the promoter with high efficiency. Sodium, which is enriched in the renal cortex generating an antibacterial hypersalinity, also induced the tcpC promoter. Finally, the bacterial density modulated the tcpC promoter activity. In the search for promoter-regulating proteins, we found that the DNA-binding protein H-NS dampens the promoter activity. Taken together, different cell types and salts, present in the kidney, are able to induce the tcpC promoter and might explain the mechanism of TcpC induction during a kidney infection with uropathogenic E. coli strains.
Highlights
The Escherichia coli strain CFT073 was isolated from a patient with acute pyelonephritis [1]
Using reporter constructs we found that the uroepithelial cell line T24/83 and the monocytic cell line THP-1 induced the tcpC promoter
We reported earlier that CFT073 causes kidney abscesses TIR-containing protein C (TcpC)-dependently in mice that contain an accumulation of neutrophils and bacteria [5,10]
Summary
The Escherichia coli strain CFT073 was isolated from a patient with acute pyelonephritis [1]. We used green fluorescence protein (gfpmut2)-reporter constructs which contain a 645 bp element starting at position 2200564 and ending at position 2201209 of the CFT073 genome, designated promoter 1 (P1), and a 240 bp element starting at position 2202535 and ending at position 2202775 of the chromosome of CFT073, designated P2 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 activated the plasmid-based reporter constructs transformed into CFT073 on plasmids and stimulated a chromosomal reporter strain where we replaced tcpC by gfpmut. We focused our analysis on P2 since it responded much more vigorously than P1
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