A novel single-cell genomics approach to analyse host targets of bacterial effectors that drive intracellular replication of Chlamydia trachomatis in vivo.

• Applicant: Professor Dr. Thomas Rudel
• Subject Area: Medical Microbiology and Mycology, Hygiene, Molecular Infection Biology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 561044222

Chlamydia trachomatis (Ctr) is an obligate intracellular human pathogen, which causes a broad range of acute and chronic diseases. It is the leading cause of bacterial sexually transmitted diseases, with more than 130 million new cases world-wide annually. Infection of the urogenital tract by Chlamydia in women can lead to infertility, ectopic pregnancies and pelvic inflammatory disease. These long-term persistent infections also increase the risk of HIV infections and might contribute to the development of cervical and ovarian cancer. During infection, Ctr uses its type 3 secretion system (T3SS) to deliver more than 100 effectors including inclusion membrane (Inc) proteins into the host cell. The conventional T3SS (cT3SS) effectors constitute a second class of effectors that are delivered into the host cell where they target the cytosol, the cytoskeleton or cell organelles. Both classes of effector proteins, Incs and cT3SS, play central roles in host cell invasion, nutrient acquisition and immune evasion and are important for Ctr pathogenesis. Understanding their function will provide insight into the development of diseases connected to chlamydial infection. Advances in single-cell RNA-seq (scRNA-seq) have revolutionized our ability to classify distinct immune cell subsets during infection. Recent studies applying scRNA-seq describe cell type-specific states that represent their activation process and function in response to infection. We have recently developed scPAIR-seq, a highly flexible and sensitive approach that allows to follow multiple bacterial barcodes in parallel using scRNA-seq protocols. Using this approach, we now study infections with a pooled tagged bacterial mutant library and establish, at single cell resolution, both the identity of the infecting mutant and its impact on host transcriptome, providing understanding of the virulence-immune network on a global scale. An amazing number of secreted chlamydial effectors have been recently identified and only very few have been functionally investigated. Drawing on our complementary expertise, we will leverage on scPAIR-seq approach (Avraham lab) to a preclinical model of Ctr (Rudel lab), to bridge the current gap and gain mechanistic insights on Chlamydia type 3 secretion system effectors as drivers of in vivo infection. We propose to functionally investigate all so far identified secreted conventional and a selection of inclusion membrane resident type 3 secreted chlamydial effectors in vivo at a single cell resolution. This project will allow us to obtain (i) a comprehensive map of host cell subsets infected by Ctr in vivo, (ii) the role of secreted effectors in the survival and replication of Ctr in epithelial cells, and (iii) the function of individual chlamydial effectors in manipulating host defence pathways that interfere with development and replication of these pathogenic bacteria in vivo.

• DFG Programme: Research Grants
• International Connection: Israel
• Partner Organisation: The Israel Science Foundation
• Cooperation Partner: Professor Dr. Roi Avraham