Unique Insights into Disease Progression using Quantitative Bottom-Up Proteomics: A Phenotypic Characterization of Chlamydia Infection using Gel-Free Label-Free LC/MS/MS

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

Philadelphia

Conferencia; 57th ASMS Conference on Mass Spectrometry; 2009

American Society for Mass Spectrometry

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). Chlamydia infection remains the most frequently reported STD in the US, and is usually asymptomatic.  To understand how this pathogen co-opts the architecture of cells, as well as the biochemical mechanisms of its virulence, we have performed an ensemble of quantitative proteomic experiments looking at differential expression and modification of both human and Chlamydia proteins, using HeLa cells and C. trachomatis serovar LGV-L2 as the model of infection.  Initial experiments characterized differential protein expression as a function of the progression of Chlamydia infection in three sub-cellular compartments – lipid droplets, plasma membranes, and parasitic vacuole membranes. In addition, we have characterized differential protein expression in the two Chlamydia developmental forms: elementary bodies and reticulate bodies. Differential expression analysis has been performed using bottom-up LC-MS/MS and LC-MSE with Waters nanoAcquity and Synapt HDMS instrumentation. Database searching for protein ID has been accomplished using Mascot and IdentityE algorithms, and quantitative analysis has been performed using Rosetta Elucidator. To-date, 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. Notably, the importance of using a bottom-up approach with a peptide-based data interpretation was clearly highlighted as these proteomic datasets have verified recently published data (Kumar, Valdivia 2008) showing that the Chlamydia protease CPAF cleaves the n-terminal domains of the human membrane proteins vimentin, cytoskeletal keratin 8 and cytoskeletal keratin 18. Importantly, this proteomic data identified two additional Chlamydia protease substrates, cytoskeletal keratins 7 and 17. Cleavage of the n-terminus of these proteins has been confirmed by the downregulation of n-terminal peptides in infected cells, and upregulation of n-terminal peptides exhibiting semi-tryptic specificity, (tryptic specificity on one end of the peptide and CPAF cleavage of the opposite end). 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.  These host protein cellular remodeling events are directly relevant to the virulence of the pathogen, as Chlamydia replicates with the within the vacuole inclusion body. Chlamydia ‘co-opts’ the function of host structural proteins via CPAF protease processing, stabilizing the inclusion body and minimizing the exposure of the Chlamydia in the inclusion body to host immune-surveillance proteins. 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.