leupeptins and Chlamydia-Infections

The obligate intracellular bacterium Chlamydia trachomatis occupies a parasitophorous vacuole and employs a type III secretion mechanism to translocate host-interactive proteins. These proteins most likely contribute to pathogenesis through modulation of host cell mechanisms crucial for the establishment and maintenance of a permissive intracellular environment. Using a surrogate Yersinia type III secretion system (T3SS), we have identified the conserved gene product CT847 as a chlamydial T3SS substrate. Yeast two-hybrid studies using CT847 as bait to screen a HeLa cell cDNA library identified an interaction with mammalian Grap2 cyclin D-interacting protein (GCIP). Immunoblot analyses of C. trachomatis-infected HeLa cells showed that GCIP levels begin to decrease (as compared with mock-infected HeLa cells) between 8 h and 12 h post infection. GCIP was virtually undetectable in 24 h time point material. This decrease was inhibited by proteasome inhibitors lactacystin and MG-132, and the T3SS inhibitor Compound 1. CT847 was detectible in purified reticulate body but not elementary body lysates, and reverse transcription polymerase chain reaction (RT-PCR) expression analyses indicate a mid-cycle expression pattern. Both of these findings are consistent with CT847 contributing to the observed effect on GCIP. Given the established roles of GCIP, we believe that we have discovered a novel C. trachomatis antihost protein whose activity is relevant to chlamydial pathogenesis.

Topics: Acetylcysteine; Bacterial Proteins; Chlamydia Infections; Chlamydia trachomatis; Gene Library; HeLa Cells; Humans; Leupeptins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Binding; Protein Interaction Mapping; Transcription Factors; Yersinia

Members of the inhibitor of apoptosis protein family are important factors that regulate apoptotic cell death. As demonstrated both by RT-PCR and Western Blot analysis C. pneumoniae infection of the human monocytic cell line Mono Mac 6 induces the expression of mRNA and protein of the cellular inhibitor of apoptosis 2 (c-IAP2). Blocking NF-kappaB DNA-binding activity by the proteasome inhibitor MG-132 results in decrease of C. pneumoniae-induced c-IAP2 expression. Therefore, C. pneumoniae may exploit the NF-kappaB pathway to induce expression of an antiapoptotic host cell protein that may contribute to intracellular survival of the pathogen.

Topics: Apoptosis; Blotting, Western; Chlamydia Infections; Chlamydophila pneumoniae; Cysteine Proteinase Inhibitors; Electrophoretic Mobility Shift Assay; Gene Expression Regulation; Humans; Leupeptins; Monocytes; NF-kappa B; Protein Biosynthesis; Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger