Functional Genomics and Proteomics Identify Effector Proteins in Chlamydia trachomatis Elementary Bodies

J. D. DUNN; H. A. SAKA; RAPHAEL VALDIVIA

San Diego, California, USA

Congreso; 110th American Society for Microbiology General Meeting 2010; 2010

The sexually transmitted obligate intracellular Gram-negative pathogen Chlamydia trachomatis translocates effector proteins into its host cell to facilitate entry and to establish an environment suitable for replication. Elementary bodies (EBs), the infectious form of Chlamydia, likely employ a large cohort of effectors that are critical for establishing an infection. The lack of both a system for genetic manipulation and a cell-free culture system suitable for biochemical approaches has hindered the identification of these effector proteins. Moreover, C. trachomatis is of a highly divergent lineage of ancient pathogens, and many of its genes have homologs only in other Chlamydia species. To overcome these difficulties, we used a combination of functional genomics and proteomic approaches to identify potential effectors that are translocated during EB attachment and entry. We created a mammalian expression library comprising ~130 Chlamydia-specific ORFs fused to EGFP, and monitored the subcellular localization of these fusion proteins in transiently transfected HeLa cells. A distinct subset of these fusion proteins displayed tropism for specific host organelles. Because effector proteins secreted during the initial stages of infection are likely pre-packaged into the metabolically inert EBs, we used two-dimensional liquid chromatography of trypsin-digested EB proteins followed by quantitative tandem mass spectroscopy to identify the most abundant Chlamydia-specific proteins in EBs. In addition to TARP, a previously characterized effector that nucleates actin, we identified over 25 new potential effectors. A significant subset of these abundant EB proteins are predicted to be substrates of type 3 secretion and/or target specific mammalian organelles, including the plasma membrane, the Golgi apparatus, the nuclear envelope, and the nucleolus, when expressed as EGFP fusions. These observations suggest that numerous host cell modifications occur during the earliest stages of Chlamydia infection.