Application of a C. trachomatis expression system to identify C. pneumoniae proteins translocated into host cells

Chlamydia pneumoniae is a Gram-negative, obligate intracellular pathogen that causes community-acquired respiratory infections. C. pneumoniae uses a cell contact-dependent type-III secretion (T3S) system to translocate pathogen effector proteins that manipulate host cellular functions. While several C. pneumoniae T3S effectors have been proposed, few have been experimentally confirmed in Chlamydia In this study, we expressed 382 C. pneumoniae genes in C. trachomatis as fusion proteins to an epitope tag derived from glycogen synthase kinase 3β (GSK3β) which is the target of phosphorylation by mammalian kinases. Based on the detection of the tagged C. pneumoniae protein with anti-phospho GSK3β antibodies, we identified 49 novel C. pneumoniae-specific proteins that are translocated by C. trachomatis into the host cytoplasm and thus likely play a role as modifiers of host cellular functions. In this manner, we identified and characterized a new C. pneumoniae effector CPj0678 that recruits the host cell protein PACSIN2 to the plasma membrane. These findings indicate that C. trachomatis provides a powerful screening system to detect candidate effector proteins encoded by other pathogenic and endosymbiotic Chlamydia species.Importance Chlamydia injects numerous effector proteins into host cells to manipulate cellular functions important for bacterial survival. Based on findings in C. trachomatis, it has been proposed that between 5-10% of the C. pneumoniae genome, a related respiratory pathogen, encodes secreted effectors. However only a few C. pneumoniae effectors have been identified and experimentally validated. With the development of methods for the stable genetic transformation of C. trachomatis, it is now possible to use C. trachomatis shuttle plasmids to express and explore the function of proteins from other Chlamydia in a more relevant bacterial system. In this study, we experimentally determined that 49 C. pneumoniae-specific proteins are translocated into the host cytoplasm by Chlamydia secretion systems, and identify a novel effector required to recruit the host factor PACSIN2 to the plasma membrane during infection.