Chlamydia pneumoniae CopD Translocator Protein Plays a Critical Role in Type III Secretion (T3S) and Infection

Abstract

Pathogenic Gram-negative bacteria use type III secretion (T3S) to inject effector proteins into the host cell to create appropriate conditions for infection and intracellular replication. Chlamydia spp. are believed to use T3S to infect their host cell, and the translocator proteins are an essential component of this system. Chlamydia pneumoniae contains genes encoding two sets of translocator proteins; CopB and CopD, and CopB2 and CopD2. In this study, we identified novel interactions between CopD and three type III secretion proteins; namely, CopN, CdsN, and CdsF. We identified a CopD putative chaperone binding motif, PxLxxP, within the N-terminal region (CopD amino acids 120–125), which was necessary for interaction with its putative chaperone LcrH_1. Using size exclusion chromatography, we showed that CopD and LcrH_1 formed higher order structures in solution with CopD and LcrH_1 binding in a ratio of 1∶1, which is unique for T3SS translocator proteins. Lastly, we showed that antibodies to CopD reduced C. pneumoniae infectivity by >95%. Collectively, this data suggests that CopD plays a critical role in pathogenesis and likely functions as a hydrophobic translocator of the type III secretion system in Chlamydia pneumoniae.

A working model for the type III secretion mechanism in Chlamydia

“…Current evidence indicates that secreted CopB and CopD would form the invasion-related translocon enabling translocation of subsequently secreted effectors across the host membrane. Both CopB and CopD are present in EBs [56] and have structural characteristics consistent with translocator proteins [16, 17]. Consistent with translocator function, CopB accumulates in the inclusion membrane [30] and partitions as an integral membrane protein [21].…”

Immunization with chlamydial type III secretion antigens reduces vaginal shedding and prevents fallopian tube pathology following live C. muridarum challenge

“…We have previously shown that rabbits immunized with T3S proteins CopB, CopD and Cpn0803 from Chlamydia pneumoniae were able to produce neutralizing antibodies that blocked infection in vitro by 98% [15]. We now show that immunization with a novel multi-component vaccine (BD584) consisting of the N-terminal 100 amino acids of C. trachomatis CopB, CopD, and full length CT584 reduced both bacterial shedding from the vagina and upper genital tract pathology following intra-vaginal challenge in mice.…”

“…are obligate intracellular pathogens and use a T3SS to infect host cells. Despite the amino acid sequence differences seen between orthologous T3S proteins of other bacteria, the amino acid sequence of T3S components is highly conserved between chlamydial species, and between serovars of C. trachomatis [15,24,25]. Our BD584 antigen was selected because it is highly conserved between C. trachomatis serovars and C. muridarum.…”

“…To ensure that our immunization strategy was successful, sera from immunized mice were tested for the presence of antigen specific serum IgG. Previously, we have shown that antibodies raised against the translocators, CopB and CopD, can inhibit infection suggesting that antibodies directed towards these proteins block an essential aspect of T3S during infection [15,29]. Serum collected from mice immunized with BD584 reduced infectivity by 77% when compared to control serum.…”

“…We have previously shown that antibodies generated against N-terminal peptides of CopB and CopD were capable of neutralizing Chlamydia spp. [15,29]. Mice infected with C. muridarum produce serum antibodies against BD584 (Fig.…”