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Chlamydia pneumoniae Inclusion Membrane Protein Cpn0585 Interacts with Multiple Rab GTPases

Cortes, Claudio, Rzomp, Kimberly A., Tvinnereim, Amy, Scidmore, Marci A., Wizel, Benjamin
Infection and immunity 2007 v.75 no.12 pp. 5586-5596
Chlamydophila pneumoniae, antibodies, bacteria, cytosol, glutathione transferase, green fluorescent protein, guanosine triphosphate, guanosinetriphosphatase, human cell lines, membrane proteins, recruitment, two hybrid system techniques, vacuoles
Chlamydiae are intracellular bacteria that develop within a membrane-bound vacuole called an inclusion. To ensure that the inclusion is a safe niche for chlamydial replication, chlamydiae exploit a number of host cell processes, including membrane-trafficking pathways. Recently, several Rab GTPases were found to associate with the inclusions of various chlamydial species. Here we report that Cpn0585, a Chlamydia pneumoniae inclusion membrane protein (Inc), interacts with multiple Rab GTPases. The results from yeast two-hybrid experiments revealed that an amino-terminally truncated form of Cpn0585 (Cpn0585₁₀₂₋₆₅₁) interacts with Rab1, Rab10, and Rab11 but not with Rab4 or Rab6. Cpn0585-Rab GTPase interactions are direct and GTP dependent as shown in glutathione S-transferase pull-down assays using native and recombinant Cpn0585. In C. pneumoniae-infected HEp-2 cells transfected with enhanced green fluorescent protein (EGFP)-tagged Rab GTPases, the colocalization with Cpn0585 at the inclusion membrane was partial for EGFP-Rab1 and EGFP-Rab10, but extensive for wild-type EGFP-Rab11A and the constitutively active GTPase-deficient EGFP-Rab11AQ70L. Moreover, Cpn0585 colocalized with EGFP-Rab11AQ70L as early as 2 h postinfection. Upon delivery into live C. pneumoniae-infected cells, Cpn0585₆₂₈₋₆₅₁-specific antibodies bound to the inclusion membrane, demonstrating that the Rab GTPase-interacting domain of Cpn0585 faces the host cell cytosol. Finally, ectopic expression of Cpn0585₁₀₂₋₆₅₁ partially inhibited the development of C. pneumoniae inclusions in EGFP. but not in EGFP-Rab11AQ70L-expressing HEp-2 cells. Collectively, these data suggest that Cpn0585 is involved in the recruitment of Rab GTPases to the inclusion membrane and that interfering with this function may adversely impact the fitness of the C. pneumoniae inclusion for chlamydial replication.