Characterization of the chlamydial type III secretion translocator proteins CopB and CopD

Abstract

Chlamydiae spp. are a significant cause of disease worldwide. Chlamydia pneumoniae is a respiratory pathogen which is believed to cause approximately 10% of community-acquired pneumoniae. Furthermore, C. pneumoniae infections have been associated with the development of atherosclerosis, exacerbation of asthma, multiple sclerosis and Alzheimer’s. Chlamydia trachomatis, the causative agent of trachoma, is responsible for over 6 million cases of preventable blindness each year. Furthermore, C. trachomatis is the leading bacterial sexually transmitted infection in North America. Yet, given how prevalent chlamydial infections are, there is still limited information regarding the pathogenic mechanisms by which Chlamydia infects its host. Type III secretion is a conserved pathogenic virulence factor used by gram-negative bacteria. Type III secretion has been extensively studied in other bacterial genera, but given the difficulty of working with Chlamydia, there have been few advances in our understanding of the role of type III secretion in chlamydial pathogenesis. A set of proteins, called translocators, are believed to be indispensable for infection; however, a comprehensive biochemical characterization of them has not been undertaken. Using GST-pulldown assays, interactions between Chlamydia outer protein (Cop) B and D from C. pneumoniae and other components of the type III secretion system are characterized. Furthermore, site-directed mutagenesis was used to elucidate the critical amino acids involved in interaction between the putative chaperone, LcrH_1, and both CopB and CopD. We also demonstrate that CopD is capable of forming higher ordered structures, and that CopD and LcrH_1 interact in a 1:1 ratio. Using polyclonal antibodies directed towards the translocator proteins, we demonstrate that CopB and CopD may be surface exposed prior to host cell contact and critical to infection. A synthetic peptide consisting of the chaperone binding domain was capable of blocking the interaction between LcrH_1 and the translocator proteins, as well as inhibiting infection in vitro. Lastly, an animal model of Chlamydia infection was developed to explore whether the use of type III secretion system proteins could be used to protect against Chlamydia infection and Chlamydia induced pathology.