Evidence for the secretion of Chlamydia trachomatis CopN by a type III secretion mechanism

The medically significant, obligate intracellular pathogen Chlamydia tracho matis replicates within vacuoles termed inclusions. A developmental cycle i s initiated after entry into a host cell and is manifested by the transform ation of infectious elementary bodies (EBs) to larger, non-infectious retic ulate bodies (RBs). Analysis of the C. trachomatis genome has revealed that chlamydiae possess genes that may encode a type III secretion apparatus. I n other Gram-negative pathogens, the type III secretion mechanism is used t o target virulence factors directly to the host cell cytoplasm and is essen tial for full virulence. To evaluate the possibility of a functional type I II secretion mechanism in C. trachomatis, we initially focused on a locus c ontaining genes encoding products with similarity to chaperones (Scc1), sec retion pore components (Cds1 and Cds2) and secreted proteins (CopN) from ot her type III systems. Gene expression was tested by reverse transcriptase-p olymerase chain reaction (RT-PCR) of total RNA extracted from infected HeLa cell monolayers at 2, 6, 12 and 20 h after infection and normalized for th e number of C. trachomatis genomes present. Message was detected for Scc1 a t all times, whereas message for all other tested genes was detected in sig nificant amounts at 12 h and 20 h. Immunoblot analysis with Scc1- and CopN- specific antibodies revealed that CopN and Scc1 were present in EBs, RBs an d whole-culture extracts harvested 20 h after infection. CopN is homologous to the secreted protein YopN of Yersinia sp., and analysis of monolayers 2 0 h after infection via indirect immunofluorescence showed specific labelli ng of inclusion membranes when probed with CopN-specific antibodies but not with Scc1-specific antibodies. His-tagged CopN and a chlamydial cytoplasmi c control protein (NrdB) were expressed in Yersinia enterocolitica containi ng or lacking the virulence plasmid pYV. CopN, but not NrdB, was secreted b y Y. enterocolitica in a Ca2+- and pYV-dependent fashion. These data indica te that components of the putative type III apparatus of C. trachomatis are expressed and that at least one of these products is secreted by chlamydia e to the inclusion membrane. The observation that CopN is also secreted by the Yersinia type III apparatus provides support for the notion that chlamy diae secrete proteins via a type III mechanism.