The Dual Function of the Chlamydia pneumoniae Cpn0572 Protein in Modulating the Host Actin Cytoskeleton

Chlamydiae are gram-negative obligate intracellular bacterial pathogens of humans and animals. C. pneumoniae is a common respiratory pathogen that has been associated with a variety of chronic diseases including asthma and atherosclerosis. Chlamydiae display a biphasic developmental cycle with an infectious, but metabolically inactive elementary body (EB) and a non-infectious metabolically active reticulate body (RB). Uptake of EBs is a crucial step for infection. Like other pathogenic bacteria, it is believed that Chlamydiae species deliver their effector proteins to trigger their own entry and survival within the host cell. Among the few chlamydial effector proteins identified, the C. trachomatis Tarp is the only identified protein translocated into the host cell where it colocalizes with actin pedestals underneath the attached EB (Clifton, Fields et al. 2004). In a follow up study, it has been shown that Tarp nucleates actin polymerization in vitro (Jewett, Fischer et al. 2006). Several studies analyzing Tarp investigated the biochemical properties of the protein in vitro, however the molecular mechanism of how Tarp interacts and modulates the host actin in vivo has not been shown. In this study, we analyzed the C. pneumoniae homologous protein Cpn0572. Both proteins share high homology in the central region but are markedly different in the N-terminal and the C-terminal regions. To investigate the role of Cpn0572 in modulating host cell processes, we used the yeast Saccharomyces cerevisiae as an eukaryotic model system. Therefore, we expressed Cpn0572 in yeast and performed a phenotypic analysis. Cpn0572-expressing yeast cells exhibited irreversible reduced growth and increased sensitivity against the actin destabilizing drug Latrunculin-A (Lat-A). Further studies revealed that cpn0572 interacts genetically with actin mutant alleles. Cpn0572 transformed the yeast wild type actin into clumps and the GFP-Cpn0572 fusion protein exclusively colocalized with these actin clumps and stabilized them against Lat-A. The actin phenotype induced by Cpn0572 and the colocalization between GFP-Cpn0572 and actin clumps were also observed in the transfected human HEK-239 cells. A deletion analysis identified a central domain of Cpn0572 (DUF) which is conserved in all homologues proteins of Cpn0572 from different Chlamydia species. We could show that this conserved domain is crucial for colocalization with and stabilization of filamentous actin (F-actin) structures. DUF alone could bind in vitro pre-assembled mammalian F-actin and pulled down actin from HEp-2 cells lysates. Using in vivo and in vitro experiments, mutations introduced in the conserved amino acids within DUF resulted in loss of Cpn0572 capability in actin clumping, colocalization with actin structures and binding F-actin. The combination of DUF with other domains of Cpn0572 protein yielded different actin and yeast growth phenotypes. The C-terminal region of the Cpn0572 protein increased the actin stabilization activity of DUF (which on its own is not lethal for yeast growth) and caused yeast lethality. On the other hand, the N-terminal region containing DUF induced actin clumping and lethality in yeast cells. Using the advantages of yeast, we showed that Cpn0572 stabilizes actin by competing with and/or displacing of the actin-depolymerizing factor cofilin from binding F-actin. These results have been extended and verified in vitro using mammalian actin and cofilin proteins. Competition with cofilin in binding F-actin has also been shown for DUF and the C-terminus of Cpn0572. Like Tarp, Cpn0572 induced actin nucleation in vitro independent from other cellular proteins and DUF required a region of 110 amino acid residues upstream of DUF to achieve this activity. Mutations within this fragment identified a crucial stretch of proline amino acids surrounding a central glutamic acid that is crucial for Cpn0572 actin clumping activity in yeast and human cells expressing Cpn0572, however this proline stretch is not required for Cpn0572-induced actin nucleation activity in vitro. Using antibodies raised against the recombinant Cpn0572 protein, the expression of Cpn0572 was confirmed by western blot analysis in both infectious EBs and human cells infected with C. pneumoniae. Microscopical analysis showed that Cpn0572 is translocated 10 minutes after infection underneath the attached EBs into the human cells. Collectively, our study suggests a model showing the possible dual function of Cpn0572 in stabilizing and nucleating actin in vivo during infection. Further experiments which are currently being carried out in yeast and human cells will advance our understanding of the role of Cpn0572 in C. pneumoniae infection.