B-300. Characterization of the Chlamydophilia pneumoniae Type III Secretion ATPase

C. B. Stone, D. L. Johnson, J. B. Mahony;
McMaster Univ. St. Joseph's Healthcare, Hamilton, ON, CANADA.

Background: Type III Secretion (T3S) is utilized by a wide range of Gram-negative pathogenic bacteria to deliver effector proteins into the host cell cytoplasm. The distinguishing feature of T3S is the presence of a needle complex which is projected from the bacterial outer membrane and pierces the host cell cytoplasmic membrane. The genome of C. pneumoniae contains genes coding for the T3S system but a systematic study of the T3S apparatus has not yet been undertaken. The objective of this work was to characterize the T3S ATPase of C. pneumoniae (YscN homolog Cpn707). Methods: Cpn707 was cloned as a GST-tagged fusion protein and over-expressed in E. coli. The protein was purified on glutathione beads under native conditions and was used to raise hyper-immune guinea pig antiserum. ATPase activity was measured using the Malachite Green Assay (R & D Systems) which is based on the ability of an ATPase to release inorganic phosphate from ATP. Native gels were used to determine whether Cpn707 forms high molecular weight oligomers. Immunofluorescence (IF) was used to visualize the localization of Cpn707 in HeLa cells 55 hours post infection. Results: Preliminary results indicate that GST-Cpn707 has ATPase activity. Recombinant ATPase generated absorbance levels at 610 nm that were 5 times above background obtained with heat-inactivated ATPase. The peak ATPase activity was 74.07 nmoles Pi liberated/min/ug protein. Native gels indicate that Cpn707 is able to oligomerize into high molecular weight forms. IF staining revealed that Cpn707 is localized within chlamydial inclusion bodies in infected HeLa cells. Conclusion: We have demonstrated that recombinant Cpn707 has ATPase activity and is able to form high molecular weight oligomers consistent with the behavior of other T3S system ATPases. Antibody to Cpn707 stained Chlamydial inclusions in infected cells. Collectively this data provides strong evidence that Cpn707 is the ATPase of C. pneumoniae T3S.