The tail-specific protease (Tsp) of Chlamydiae spp.: Mechanistic and structural characterization of its interactions with host-cell proteins

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Chlamydiae are obligate intracellular pathogens that use secreted effector proteins to modify signaling pathways of eukaryotic host cells. In the human pathogen Chlamydia trachomatis, the serine protease CT441 has been reported to interfere with the NF-kappaB pathway of the host inflammatory response by cleaving p65. In addition, CT441 specifically targets SRAP1, the protein component of steroid-receptor RNA activator 1 (SRA1) of estrogen receptor alpha. It has been suggested that this interaction retains SRAP1 in the host-cell cytoplasm, thereby preventing the expression of many estradiol-induced target genes which is important for chlamydial survival and development within the host cell. The largely uncharacterized CT441 shows sequence similarities to tail-specific proteases (Tsps) involved in bacterial protein-quality control in E. coli. The goal of this project is to contribute to the understanding of the modulation of host-cell pathways by Chlamydiae. We will focus on elucidating the detailed mechanisms of interactions between chlamydial Tsps with host-cell proteins. We aim to determine three-dimensional structures for CT441 and homologues from related species. Based on structural information, site-directed mutagenesis will be used to conduct a detailed mechanistic analysis. In addition, the interaction between CT441 and SRAP1 will be studied in vitro. Using full-length proteins or individual domains of CT441 and SRAP1 in biochemical interaction analyses, protease assays and X-ray crystallography will help to clarify the mode of the interaction. Ultimately, we aim to determine the molecular structure of a complex between SRAP1 and a chlamydial Tsp protein. A human fallopian-tube tissue model of chlamydial infections of the upper genital tract will be used to study the influence of CT441 on host cell SRAP1 levels in a physiological environment. The project is expected to reveal the molecular basis for chlamydial modulation of host-cell metabolism and infectivity by estrogen.