Insights Into a Chlamydia pneumoniae-Specific Gene Cluster of Membrane Binding Proteins.

Chlamydia pneumoniae is an obligate intracellular pathogen that causes diseases of the upper and lower respiratory tract and is linked to a number of severe and chronic conditions. Here, we describe a large, C. pneumoniae-specific cluster of 13 genes (termed mbp1-13) that encode highly homologous chlamydial proteins sharing the capacity to bind to membranes. The gene cluster is localized on the chromosome between the highly diverse adhesin-encoding pmp genes pmp15 and pmp14. Comparison of human clinical isolates to the predicted ancestral koala isolate indicates that the cluster was acquired in the ancestor and was adapted / modified during evolution. SNPs and IN/DELs within the cluster are specific to isolates taken from different human tissues and show an ongoing adaptation. Most of the cluster proteins harbor one or two domains of unknown function (DUF575 and DUF562). During ectopic expression in human cells these DUF domains are crucial for the association of cluster proteins to the endo-membrane system. Especially DUF575 which harbors a predicted transmembrane domain is important for binding to the membrane, while presence of the DUF562 seems to be of regulatory function. For Mbp1, founding member of the cluster that exhibits a very limited sequence identity to the human Rab36 protein, we found a specific binding to vesicles carrying the early endosomal marker PtdIns(3)P and the endosomal Rab GTPases Rab11 and Rab14. This binding is dependent on a predicted transmembrane domain with an α-helical / ß-strand secondary structure, as the mutant version Mbp1mut, which lacks the ß-strand secondary structure, shows a reduced association to PtdIns(3)P-positive membranes carrying Rab11 and Rab14. Furthermore, we could not only show that Mbp1 associates with Rab36, but found this specific Rab protein to be recruited to the early C. pneumoniae inclusion. Detection of endogenous Mbp1 and Mbp4 reveal a colocalization to the chlamydial outer membrane protein Momp on EBs. The same colocalization pattern with Momp was observed when we ectopically expressed Mbp4 in C. trachomatis. Thus, we identified a C. pneumoniae-specific cluster of 13 membrane binding proteins (Mbps) localizing to the bacterial outer membrane system.

CPn0572, the C. pneumoniae ortholog of TarP, reorganizes the actin cytoskeleton via a newly identified F-actin binding domain and recruitment of vinculin.

Chlamydia pneumoniae is one of the two major species of the Chlamydiaceae family that have a profound effect on human health. C. pneumoniae is linked to a number of severe acute and chronic diseases of the upper and lower respiratory tract including pneumonia, asthma, bronchitis and infection by the pathogen might play a role in lung cancer. Following adhesion, Chlamydiae secrete effector proteins into the host cytoplasm that modulate the actin cytoskeleton facilitating internalization and infection. Members of the conserved TarP protein family comprise such effector proteins that polymerize actin, and in the case of the C. trachomatis TarP protein, has been shown to play a critical role in pathogenesis. In a previous study, we demonstrated that, upon bacterial invasion, the C. pneumoniae TarP family member CPn0572 is secreted into the host cytoplasm and recruits and associates with actin via an actin-binding domain conserved in TarP proteins. We have now extended our analysis of CPn0572 and found that the CPn0572 actin binding and modulating capability is more complex. With the help of the fission yeast system, a second actin modulating domain was identified independent of the actin binding domain. Microscopic analysis of HEp-2 cells expressing different CPn0572 deletion variants mapped this domain to the C-terminal part of the protein as CPn0572536-755 binds F-actin in vitro and colocalizes with aberrantly thickened actin cables in vivo. Finally, microscopic and bioinformatic analysis revealed the existence of a vinculin binding sequence in CPn0572. Our findings contribute to the understanding of the function of the TarP family and underscore the existence of several actin binding domains and a vinculin binding site for host actin modulation.

The Chlamydia pneumoniae Tarp Ortholog CPn0572 Stabilizes Host F-Actin by Displacement of Cofilin.

Pathogenic Chlamydia species force entry into human cells via specific adhesin-receptor interactions and subsequently secrete effector proteins into the host cytoplasm, which in turn modulate host-cell processes to promote infection. One such effector, the C. trachomatis Tarp factor, nucleates actin polymerization in vitro. Here we show that its C. pneumoniae ortholog, CPn0572, associates with actin patches upon bacterial invasion. GFP-CPn0572 ectopically expressed in yeast and human cells co-localizes with actin patches and distinctly aberrantly thickened and extended actin cables. A 59-aa DUF 1547 (DUF) domain, which overlaps with the minimal actin-binding and protein oligomerization fragment required for actin nucleation in other Tarp orthologs, is responsible for the aberrant actin phenotype in yeast. Interestingly, GFP-CPn0572 in human cells associated with and led to the formation of non-actin microfilaments. This phenotype is strongly enhanced in human cells expressing the GFP-tagged DUF deletion variant (GFP-ΔDUF). Finally ectopic CPn0572 expression in yeast and in-vitro actin filament binding assays, demonstrated that CPn0572 stabilizes pre-assembled F-actin by displacing and/or inhibiting binding of the actin-severing protein cofilin. Remarkably, the DUF domain suffices to displace cofilin from F actin. Thus, in addition to its actin-nucleating activities, the C. pneumoniae CPn0572 also stabilizes preformed host actin filaments.