In addition, CopD interacted with its putative chaperone, LcrH_1, first, by an essential chaperone binding motif of PxLxxP at amino acids 120�C 125, and second, via a predicted transmembrane domain at amino acids 138�C157. We also showed that LcrH_1 interacts with only monomeric CopD in a 1:1 ratio and not tetrameric or decameric CopD, as evidenced by size exclusion chromatography. Finally, we show that polyclonal antibodies directed against an N-terminal epitope of CopD inhibited chlamydial infection. Collectively, these findings are consistent with CopD functioning as a hydrophobic translocator of the C. pneumoniae T3S apparatus. Since T3S knock-outs cannot be made in Chlamydia it is not possible to unequivocally demonstrate the role of individual T3S components. We have used in vitro protein interactions to demonstrate interactions between CopD and three other T3S proteins. CdsN, the ATPase of the Chlamydia T3SS, plays a key role in substrate selection and mediates EF-24 effector-chaperone disassociation prior to secretion, allowing effectors to be translocated through the injectisome. We demonstrated that the Nterminal fragment of CopD interacted with CdsN. This suggests that CopD is delivered to the base of the needle apparatus, possibly associated with its putative chaperone, allowing CdsN to dissociate the effector-chaperone complex to initiate secretion. The filament protein, CdsF, and its orthologs in other bacteria, form the needle of the injectisome and is believed to play a role in facilitating the insertion of translocators into the host cell membrane. We have demonstrated that CopD interacts with CdsF, and have identified two specific regions of CopD, viz. CopD1�C157 and CopD158�C206 that facilitate this interaction. In Yersinia spp., the plug protein, YopN, has been shown to interact with YopD. We explored this interaction in C. pneumoniae using a GST pull down assay and confirmed the interaction between CopD158�C206 and CopN. A summary of the interactions identified in this study appear in Figure 6. It is interesting to note that all of the protein interactions that we have identified occur KRM-III within the N-terminus of CopD, and we have not identified any proteins that interact within the C-terminus of CopD.