B-301. Characterization of Inner Membrane YscU and C-Ring YscQ Proteins of the Type III Secretion System of Chlamydophila pneumoniae

R. K. Toor1, J. D. Turner1, D. L. Johnson1, J. B. Mahony1,2;
1McMaster Univ., Hamilton, ON, CANADA, 2St. Joseph's Healthcare, Hamilton, ON, CANADA.

Background: The Type III Secretion System (T3SS) is used by gram-negative bacteria to deliver toxins and effector proteins into host cells. T3SS “injectisomes” are structurally similar between bacterial species and consist of 20 - 25 proteins, of which roughly half are genetically conserved. Chlamydophila pneumoniae (Cpn) is a gram negative, obligate, intracellular bacterium that encodes a T3SS that may be required to infect host cells. Chlamydial YscQ may be part of a C-ring at the base of the “injectisome”, functioning in concentrating effector and structural proteins of the T3SS. YscU is a predicted inner membrane protein that may act to stabilize the “injectisome” within the inner membrane. Chlamydial YscU is predicted to have four transmembrane domains and a cytoplasmic domain that may be required for secretion of effector proteins. We therefore sought to determine if YscQ and YscU interact with other predicted chlamydial T3S proteins. Methods: Full length YscQ, YscU and YscD were cloned into pEX15 and pEX17 expression vectors using the Gateway system (Invitrogen) and expressed as GST and His- tagged proteins in E. coli BL-21 (DE3). Three fragments of the cytoplasmic C-terminal domain of YscU were cloned and expressed as recombinant proteins. Expression was induced with IPTG and proteins detected using Western Blot analysis with anti-His or anti-GST antibody. Recombinant proteins were purified on Ni-NTA agarose and hyper-immune serum was made to full length YscQ, YscD, and the cytoplasmic fragment of YscU. Interaction between YscQ and YscD was assessed using a co-immunoprecipitation assay. Results: The production of recombinant chlamydial T3S proteins YscQ, YscU and YscD in E. coli was shown by coomassie blue staining and Western blot. Antisera to YscQ and YscD stained Cpn inclusions. Co-immunoprecipitiation assays showed an interaction between YscQ and YscD. Conclusions: We have demonstrated an interaction between chlamydial YscQ and YscD, suggesting that these two proteins are bona fide T3S proteins. Studies are currently underway elucidating additional binding partners of YscQ, YscU and YscD in the chlamydial T3S apparatus.