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

<div><p>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. <i>Chlamydia spp.</i> are believed to use T3S to infect their host cell, and the translocator proteins are an essential component of this system. <i>Chlamydia pneumoniae</i> 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 <i>C. pneumoniae</i> 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 <i>Chlamydia pneumoniae</i>.</p></div

LcrH_1 (<i>Cpn0811</i>) interacts with CopD at amino acids 120–125.

<p>Recombinant LcrH_1 interacted with amino acids 1–157 of CopD. CopD mutants were created through overlapping PCR to create ^P120ACopD_1–157, ^L122ACopD_1–157, and ^P125ACopD_1–157. Mutations at the conserved amino acids within the predicted chaperone binding domain disrupted the interaction between CopD_1–157 and the chaperone LcrH_1, but not other identified interactions.</p

Summary of proposed interactions between CopD and type III secretion proteins.

<p>Interactions identified through Glutathione-S-Transferase pull down assays mapped to a topographic overview of CopD.</p

Comparison of putative chaperone binding domains between <i>Chlamydiaceae</i> and other T3SS containing Gram-negative bacteria.

<p>Putative chaperone binding domains were identified within the N-terminal regions of orthologous proteins to CopD from <i>C. pneumoniae</i>. P1, P3, P6, represent positions 1, 3, and 6, respectively of the PxLxxP motif. Percent identity refers to amino acid sequence identity comparing CopD to full length sequences of orthologous proteins.</p

Topographic overview of structural prediction of CopD.

<p>Solid black regions represent transmembrane domains. Orange block represents a predicted coiled-coil domain in the C-terminus of the protein. Light blue depicts predicted Chaperone Binding Domain (CBD) located from amino acids 120–125.</p

Inhibition of <i>Chlamydia pneumoniae</i> with CopD antibodies.

<p>Panels A–D show inhibition assay results performed with either no antibody (A), CopD antibody (B), pre-immune sera (C), or control antibody (α-GST) (D). Panel E shows the degree of inhibition by of CopD antibodies compared to control antibodies. Chlamydial inclusions are stained green, while HeLa cells are stained red by Evan's blue counterstain. Panel F demonstrates reactivity of anti-CopD with (1) <i>C. pneumonia</i> infected HeLa cell lysate, (2) uninfected HeLa cell lysate, (3) recombinant GST-CopD_1–157 produced in <i>E. coli</i>, and (4) recombinant GST produced in <i>E. coli</i>. Experiments were performed in triplicate. Error bars represent 2 standard deviations. Images represent random fields of view. * = P<0.0001.</p