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

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.

Chlamydia trachomatis is the most prevalent cause of preventable blindness worldwide and a major reason for infectious infertility in females. Several bacterial factors have been implicated in the pathogenesis of C. trachomatis. Combining structural and mutational analysis, we have shown that the proteolytic function of CT441 depends on a conserved Ser/Lys/Gln catalytic triad and a functional substrate-binding site within a flexible PDZ (postsynaptic density of 95 kDa, discs large, and zonula occludens) domain. Previously it has been suggested that CT441 is involved in modulating estrogen signaling responses of the host cell. Our results show that although in vitro CT441 exhibits proteolytic activity against SRAP1, a coactivator of estrogen receptor, CT441-mediated SRAP1 degradation is not observed during the intracellular developmental cycle before host cells are lysed and infectious Chlamydiae are released. Most compellingly, we have newly identified a chaperone activity of CT441, indicating a role of CT441 in prokaryotic protein quality control processes. The shed further light on protein-quality control processes in Chlamydiae, HtrA from S. negevensis (HtrASn) has been structurally and biochemically characterized. X-ray crystallographic structures show that PDZ domains of HtrASn enable self-compartmentalizing into 6-mers and 12-mers. Both crystal structures display a unique architecture among prokaryotic HtrA proteins which is mostly attributed to a different orientation and position of the PDZ2 domain. The 12-mer reveals two peptides bound to the PDZ1 domain and to the active site which show that, similar to other members of HtrA proteins, proteolytic activity of HtrASn is allosterically activated by substrate binding. The structures provide essential insights into stabilization of large oligomeric assemblies of chlamydial HtrAs and thereby represent a structural framework for rational development of future antichlamydial compounds.