Proteomics of Chlamydia trachomatis Infection

J. WILL THOMPSON; LAURA G. DUBOIS; HECTOR A. SAKA; YADUNANDA KUMAR; M. ARTHUR MOSELEY; RAPHAEL VALDIVIA

San Diego, California, USA

Conferencia; US HUPO 2009 Annual Conference; 2009

US HUPO (United States Human Proteome Organization)

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that infects ocular and genital epithelial surfaces in humans, and can lead ultimately to blindness, pelvic inflammatory disease, and infertility (Schachter, 1999). Raphael Valdivia in the Duke Department of Molecular Genetics and Microbiology and the Duke Proteomics Core Facility have collaborated to investigate the proteomics of Chlamydia infection. Using HeLa cells and C. trachomatis serovar LGV-L2 as a model of infection, three Chlamydia proteomics projects are currently ongoing in the Proteomics Core Facility looking at differential expression and modification of both human and Chlamydia proteins. Differential expression analysis is being performed using bottom-up LC-MS/MS and LC-MSE with Waters nanoAcquity and Synapt HDMS instrumentation. Database searching for protein ID is being performed using Mascot and IdentityE algorithms, and quantitative analysis is being performed in Rosetta Elucidator. Two projects address protein expression as a function Chlamydia infection in lipid droplets, plasma membrane and membranes of the parasitic vacuole (‖inclusion‖). Another project is focused on Chlamydia protein expression in the two bacterial developmental forms: elementary bodies and reticulate bodies. From initial data, 684 proteins have been confidently identified and quantified across these studies, including 585 human proteins and 99 Chlamydia proteins. 128 of these proteins were identified in at least two of the three studies. Peptide-level expression data obtained has allowed for the verification of data from a recent publication (Kumar, Valdivia 2008) showing that the bacterial protease CPAF cleaves the n-terminal domains of the human membrane proteins vimentin, cytoskeletal keratin 8 and cytoskeletal keratin 18. In addition, proteomic data has revealed two additional bacterial protease substrates, cytoskeletal keratins 7 and 17. Cleavage of the n-terminus of these substrates has been confirmed by the apparent downregulation of n-terminal peptides in infected cells, and upregulation of n-terminal peptides with semi-tryptic specificity. Time-course experiments can help to reveal primary versus secondary Chlamydial protease substrates, and semi-tryptic peptides are being investigated as heretofore unknown cleavage sites of these proteases.   Schachter, J. (1999) Infection and disease epidemiology. In Chlamydia: Intracellular Biology, Pathogenesis and Immunity, R.S. Stephens, ed. (Washington, DC: ASM), pp. 31. Kumar, Y and Valdivia, R (2008) Actin and Intermediate Filaments Stabilize the Chlamydia trachomatis Vacuole by Forming Dynamic Structural Scaffolds. Cell Host and Microbe, 4, 159-169.