TcpC secreting uropathogenic E. coli promoted kidney cells to secrete MIP-2 via p38 MAPK pathway

Abstract

Pyelonephritis is an infection of the upper urinary tract with characteristic histological change to neutrophil infiltration in the kidney. The majority of pyelonephritis is caused by uropathogenic Escherichia (E.) coli (UPEC) bearing distinct virulence factors. Toll/interleukin‑1 receptor domain‑containing protein C (TcpC) encoded by E. coli is an important virulence factor in the majority of strains of UPEC and inhibits macrophage‑mediated innate immunity, which serves an essential role in the pathogenesis of pyelonephritis. In the present study, it was demonstrated that TcpC induced kidney cells to produce macrophage inflammatory protein‑2 (MIP‑2; also known as C‑X‑C motif chemokine 2). MIP‑2 concentration in kidney homogenates from TcpC‑secreting UPEC CFT073 (TcpCwt) murine pyelonephritis models was significantly higher compared with that in kidney homogenates from tcpC knockout CFT073 (TcpC‑/‑) models. In vitro, TcpCwt dose‑dependently promoted MIP‑2 secretion in HEK‑293 cells. The concentration of MIP‑2 in culture supernatants of HEK‑293 co‑cultured with TcpCwt was profoundly higher compared with that of HEK‑293 co‑cultured with TcpC‑/‑. In the presence of anti‑TcpC antibody, the enhancement effect of TcpCwt on MIP‑2 production was completely abrogated, suggesting that the enhanced production of MIP‑2 was mediated by secreted TcpC. Furthermore, it was demonstrated that TcpC‑/‑ treatment had no effect on the p38 mitogen activated protein kinase (MAPK) signaling pathway, while TcpCwt treatment resulted in the activation of p38 MAPK in HEK‑293 cells, as indicated by a simultaneous increase in p38 and phosphorylated‑p38. In addition, inhibition of p38 MAPK with SB203580 significantly decreased MIP‑2 concentration and neutrophil recruitment activity in the supernatants of HEK‑293 cells co‑cultured with TcpCwt. This indicates that TcpC may promote MIP‑2 production in kidney cells through the p38 MAPK signaling pathway. Taken together, the data of the present study demonstrated that TcpC can induce MIP‑2 production, which may contribute to the characteristic histological change associated with pyelonephritis. This data has provided novel evidence to further clarify the pathogenesis of pyelonephritis and novel directions on the pathogenicity of TcpC‑secreting UPEC.

TcpC inhibits toll-like receptor signaling pathway by serving as an E3 ubiquitin ligase that promotes degradation of myeloid differentiation factor 88

“…Disease Control and Prevention, China). CFT073 Δtcpc was constructed in our laboratory as described previously [8]. The strains were cultured in LB medium at 37˚C.…”

“…Female C57BL/6 mice, 6-8 weeks of age, were purchased from SLC Laboratory Animal Co. Ltd. (Shanghai, China) and were housed in specific pathogen-free conditions. Mouse pyelonephritis models were prepared as previously described [8]. In briefly, mice were anesthetized with Avertin (40 mg of 2,2,2-tribromoethanol (Sigma, USA) dissolved in 1 ml of tertamyl alcohol (0.01 ml/g body weight intraperitoneally)) and infected by transurethral instillation of 10 8 CFU of CFT073 wt or CFT073 Δtcpc using a flexible polyethylene catheter.…”

“…Mice treated with normal saline served as the control group. The mice were sacrificed 3 days later and the kidneys were obtained for pathological examination as described previously [8].…”

“…Proteins from lysates of following three different treatment macrophages were prepared as described in our previous reports [8,52]. 1) K-macrophages isolated from murine models with pyelonephritis; 2) To detect the influence of TcpC on protein levels of MyD88 and NF-κB, 5×10 5 cells of BMDM, J774A.1 or RAW264.7 were separately co-cultured in transwell with CFT073 wt or CFT073 Δtcpc at MOI = 100 for the indicated time; 3) To detect the influence of rTcpC or rTcpC mutant on the protein levels of MyD88, 5×10 5 cells of J774A.1, RAW264.7, THP-1 or BMDM were cultured in 6-well culture plates (Corning, USA) overnight.…”

“…The disease is affecting 150 million people worldwide each year and the medical expenses exceed 6 billion dollars [4][5][6]. Uropathogenic Escherichia coli (UPEC) can specifically adhere to and infect epithelial cells of urinary tract mucosa [7,8]. UPEC is the most common pathogen of UTI, about 80% of UTI are caused by UPEC [9].…”