Identification of Effector Proteins in Chlamydia trachomatis Elementary Bodies Through Functional Genomics and Proteomics

JOE DAN DUNN; HECTOR ALEX SAKA; RAPHAEL VALDIVIA

Los Angeles, California, USA

Encuentro; 5th Biennial Meeting of the Chlamydia Basic Research Society; 2011

Chlamydia Basic Research Society

Chlamydia trachomatis translocates effector proteins into its host cell to create an environment suitable for replication. Elementary bodies (EBs) possess a type 3 secretion (T3S) system and likely employ a large cohort of effectors that are critical for establishing an infection, but few have been identified. We used a combination of functional genomics and proteomic approaches to identify potential effectors that are translocated into host cells during EB entry. We created a mammalian expression library that comprises ~130 Chlamydia ORF-EGFP fusions, monitored the localizations of these fusion proteins in transiently transfected HeLa cells, and observed that a subset display tropism for eukaryotic organelles. Because the primary function of the metabolically inert EBs is to infect cells, we hypothesized that they are pre-packaged with the effectors required to initiate infection and that these effectors are more abundant than proteins with housekeeping functions. Therefore, we performed quantitative tandem mass spectroscopy on purified EB lysates to identify the most abundant proteins. In addition to Tarp and CT694, known T3S substrates, we identified over 30 additional potential effectors. A significant subset of these abundant EB proteins are predicted T3S substrates and/or target specific mammalian organelles, including the plasma membrane, the ER, 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. We are using heterologous T3S assays, immunofluorescence microscopy, and cell biology approaches to confirm that the predicted effectors are bona fide.