Influence of the TIR domain-containing protein C on the cellular infiltrate and kidney epithelium during pyelonephritis caused by the UPEC strain CFT073

• Applicant: Professor Dr. Thomas Miethke
• Subject Area: Medical Microbiology and Mycology, Hygiene, Molecular Infection Biology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 545030661

The uropathogenic Escherichia coli strain CFT073 modulates the innate immune system via its virulence factor TIR-containing protein C (TcpC). TcpC binds to critical components of pattern recognition receptors (PRRs) and modifies their function. In particular it interacts with Toll-like receptor 4 (TLR4) and its adaptor molecule myeloid differentiation factor (MyD88) but also with the inflammasome components NACHT leucine-rich repeat PYD protein 3 (NLRP3) and caspase-1. These PRRs are highly relevant for the recognition of pathogens and the induction of an immunological defense response. CFT073 causes kidney abscesses TcpC dependently, which consist of polymorphonuclear neutrophils and bacteria. However, a detailed analysis of the pathophysiological mechanisms during the kidney infection is missing. As a first objective, the proposal, therefore, suggests to localize CFT073 within the kidney and to explore the activity of its tcpC promoter. On the host side, we then explore the influence of TcpC on the composition of the infiltrating immune cells using multicolor immunofluorescence microscopy and flow cytometry. We also investigate the cytokine response by immune cells but also kidney epithelial cells performing intracellular stainings and explore whether TcpC modifies cytokine responses of the different cells. As second objective, we analyze the influence of the TcpC-targeted PRRs TLR4 and NLRP3 and their corresponding signaling chain members MyD88 and caspase-1 on the composition of the cellular infiltrate and the local cytokine response during kidney infection with CFT073. We infect the urinary tract of the corresponding gene-deficient mice to explore this issue. Third, we propose analyzing the transcriptomes of the host and the pathogen by dual RNA seq. On the host side, we focus on the expression of immune response genes while on the pathogens side we will explore the expression of virulence genes. TcpC was demonstrated recently to function as a NAD+ consuming enzyme and NAD+ is involved profoundly in the cellular metabolism of eukaryotic and prokaryotic cells. Thus, it is relevant and important to explore the influence of TcpC on both transcriptomes. As forth objective, we analyze the human inflammatory response during an infection of a kidney-epithelial cell line with CFT073 and the influence of TcpC on this response. We complement the analysis of the human inflammatory response by exploring the immune response in tumor nephrectomies with an accompanying infection.

• DFG Programme: Research Grants
• Co-Investigator: Dr. Ying Chen