The Chlamydia pneumoniae effector SemD exploits its host’s endocytic machinery by structural and functional mimicry
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ABSTRACT:
SUBMITTER: Kocher F
PROVIDER: S-EPMC11344771 | biostudies-literature | 2024 Jan
REPOSITORIES: biostudies-literature
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Project description:Clathrin-mediated endocytosis is an essential process that forms vesicles from the plasma membrane. Although most of the protein components of the endocytic protein machinery have been thoroughly characterized, their organization at the endocytic site is poorly understood. We developed a fluorescence microscopy method to track the average positions of yeast endocytic proteins in relation to each other with a time precision below 1 s and with a spatial precision of ~10 nm. With these data, integrated with shapes of endocytic membrane intermediates and with superresolution imaging, we could visualize the dynamic architecture of the endocytic machinery. We showed how different coat proteins are distributed within the coat structure and how the assembly dynamics of N-BAR proteins relate to membrane shape changes. Moreover, we found that the region of actin polymerization is located at the base of the endocytic invagination, with the growing ends of filaments pointing toward the plasma membrane.
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Project description:We here describe four proteins of Chlamydia pneumoniae, which might play a role in host-pathogen interaction. The hypothetical bacterial proteins CPn0708 and CPn0712 were detected in Chlamydia pneumoniae-infected host cells by indirect immunofluorescence tests with polyclonal antisera raised against the respective proteins. While CPn0708 was localized within the inclusion body, CPn0712 was identified in the inclusion membrane and in the surrounding host cell cytosol. CPn0712 colocalizes with actin, indicating its possible interaction with components of the cytoskeleton. Investigations on CPn0809 and CPn1020, two Chlamydia pneumoniae proteins previously described to be secreted into the host cell cytosol, revealed colocalization with calnexin, a marker for the ER. Neither CPn0712, CPn0809 nor CPn1020 were able to inhibit host cell apoptosis. Furthermore, transient expression of CPn0712, CPn0809 and CPn1020 by the host cell itself had no effect on subsequent infection with Chlamydia pneumoniae. However, microarray analysis of CPn0712-expressing host cells revealed six host cell genes which were regulated as in host cells infected with Chlamydia pneumoniae, indicating the principal usefulness of heterologous expression to study the effect of Chlamydia pneumoniae proteins on host cell modulation.
| S-EPMC2525848 | biostudies-other
Project description:The actin cytoskeleton lies at the heart of many essential cellular processes. There are hundreds of proteins that cells use to control the size and shape of actin cytoskeletal networks. As such, various pathogens utilize different strategies to hijack the infected eukaryotic host actin dynamics for their benefit. These include the control of upstream signaling pathways that lead to actin assembly, control of eukaryotic actin assembly factors, encoding toxins that distort regular actin dynamics, or by encoding effectors that directly interact with and assemble actin filaments. The latter class of effectors is unique in that, quite often, they assemble actin in a straightforward manner using novel sequences, folds, and molecular mechanisms. The study of these mechanisms promises to provide major insights into the fundamental determinants of actin assembly, as well as a deeper understanding of host-pathogen interactions in general, and contribute to therapeutic development efforts targeting their respective pathogens. This review discusses mechanisms and highlights shared and unique features of actin assembly by pathogen effectors that directly bind and assemble actin, focusing on eukaryotic actin nucleator functional mimics Rickettsia Sca2 (formin mimic), Burkholderia BimA (Ena/VASP mimic), and Vibrio VopL (tandem WH2-motif mimic).
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Project description:The ability of Legionella pneumophila to proliferate within various protozoa in the aquatic environment and in macrophages indicates a remarkable evolution and microbial exploitation of evolutionarily conserved eukaryotic processes. Ankyrin B (AnkB) of L. pneumophila is a non-canonical F-box-containing protein, and is the only known Dot/Icm-translocated effector of L. pneumophila essential for intra-vacuolar proliferation within both macrophages and protozoan hosts. We show that the F-box domain of AnkB and the (9)L(10)P conserved residues are essential for intracellular bacterial proliferation and for rapid acquisition of polyubiquitinated proteins by the Legionella-containing vacuole (LCV) within macrophages, Dictyostelium discoideum, and Acanthamoeba. Interestingly, translocation of AnkB and recruitment of polyubiquitinated proteins in macrophages and Acanthamoeba is rapidly triggered by extracellular bacteria within 5 min of bacterial attachment. Ectopically expressed AnkB within mammalian cells is localized to the periphery of the cell where it co-localizes with host SKP1 and recruits polyubiquitinated proteins, which results in restoration of intracellular growth to the ankB mutant similar to the parental strain. While an ectopically expressed AnkB-(9)L(10)P/AA variant is localized to the cell periphery, it does not recruit polyubiquitinated proteins and fails to trans-rescue the ankB mutant intracellular growth defect. Direct in vivo interaction of AnkB but not the AnkB-(9)L(10)P/AA variant with the host SKP1 is demonstrated. Importantly, RNAi-mediated silencing of expression of SKP1 renders the cells non-permissive for intracellular proliferation of L. pneumophila. The role of AnkB in exploitation of the polyubiquitination machinery is essential for intrapulmonary bacterial proliferation in the mouse model of Legionnaires' disease. Therefore, AnkB exhibits a novel molecular and functional mimicry of eukaryotic F-box proteins that exploits conserved polyubiquitination machinery for intracellular proliferation within evolutionarily distant hosts.
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Project description:Vasculogenic mimicry (VM) describes a process by which cancer cells establish an alternative perfusion pathway in an endothelial cell-free manner. Despite its strong correlation with reduced patient survival, controversy still surrounds the existence of an in vitro model of VM. Furthermore, many studies that claim to demonstrate VM fail to provide solid evidence of true hollow channels, raising concerns as to whether actual VM is actually being examined. Herein, we provide a standardized in vitro assay that recreates the formation of functional hollow channels using ovarian cancer cell lines, cancer spheres and primary cultures derived from ovarian cancer ascites. X-ray microtomography 3D-reconstruction, fluorescence confocal microscopy and dye microinjection conclusively confirm the existence of functional glycoprotein-rich lined tubular structures in vitro and demonstrate that many of structures reported in the literature may not represent VM. This assay may be useful to design and test future VM-blocking anticancer therapies.
| S-EPMC5539303 | biostudies-literature
Project description:Although the actin network is commonly hijacked by pathogens, there are few reports of parasites targeting microtubules. The proposed member of the LcrE protein family from some Chlamydia species (e.g. pCopN from C. pneumoniae) binds tubulin and inhibits microtubule assembly in vitro. From the pCopN structure and its similarity with that of MxiC from Shigella, we definitively confirm CopN as the Chlamydia homolog of the LcrE family of bacterial proteins involved in the regulation of type III secretion. We have also investigated the molecular basis for the pCopN effect on microtubules. We show that pCopN delays microtubule nucleation and acts as a pure tubulin-sequestering protein at steady state. It targets the β subunit interface involved in the tubulin longitudinal self-association in a way that inhibits nucleotide exchange. pCopN contains three repetitions of a helical motif flanked by disordered N- and C-terminal extensions. We have identified the pCopN minimal tubulin-binding region within the second and third repeats. Together with the intriguing observation that C. trachomatis CopN does not bind tubulin, our data support the notion that, in addition to the shared function of type III secretion regulation, these proteins have evolved different functions in the host cytosol. Our results provide a mechanistic framework for understanding the C. pneumoniae CopN-specific inhibition of microtubule assembly.
| S-EPMC4155683 | biostudies-literature
Project description:Ankyrin B (AnkB/LegAU13) is a translocated F box effector essential for the intracellular replication of the pathogen Legionella pneumophila. AnkB co-opts a host ubiquitin ligase to decorate the pathogen-containing vacuole with K48-linked polyubiquitinated proteins and degrade host proteins as a source of energy. Here, we report that AnkB commandeers the host ubiquitin-proteasome system through mimicry of two eukaryotic protein domains. Using X-ray crystallography, we determined the 3D structure of AnkB in complex with Skp1, a component of the human SCF ubiquitination ligase. The structure confirms that AnkB contains an N-terminal F box similar to Skp2 and a C-terminal substrate-binding domain similar to eukaryotic ankyrin repeats. We identified crucial amino acids in the substrate-binding domain of AnkB and showed them to be essential for the function of AnkB in L. pneumophila intracellular proliferation. The study reveals how Legionella uses molecular mimicry to manipulate the host ubiquitination pathway and proliferate intracellularly.
| S-EPMC6528793 | biostudies-literature
Project description:Type III secretion (T3S) is an essential virulence factor used by gram-negative pathogenic bacteria to deliver effector proteins into the host cell to establish and maintain an intracellular infection. Chlamydia is known to use T3S to facilitate invasion of host cells but many proteins in the system remain uncharacterized. The C. trachomatis protein CT584 has previously been implicated in T3S. Thus, we analyzed the CT584 ortholog in C. pneumoniae (Cpn0803) and found that it associates with known T3S proteins including the needle-filament protein (CdsF), the ATPase (CdsN), and the C-ring protein (CdsQ). Using membrane lipid strips, Cpn0803 interacted with phosphatidic acid and phosphatidylinositol, suggesting that Cpn0803 may associate with host cells. Crystallographic analysis revealed a unique structure of Cpn0803 with a hydrophobic pocket buried within the dimerization interface that may be important for binding small molecules. Also, the binding domains on Cpn0803 for CdsN, CdsQ, and CdsF were identified using Pepscan epitope mapping. Collectively, these data suggest that Cpn0803 plays a role in T3S.
| S-EPMC3260263 | biostudies-literature
Project description:The obligate intracellular human pathogen Chlamydia pneumoniae was subjected to dRNA-Seq to gain insights into the transcriptome. The two distinct life cycle forms elementary bodies (EB) and reticulate bodies (RB) were isolated from human Hep2 cell line by differential gradient centrifugation.
2011-08-18 | GSE24999 | GEO
Project description:BackgroundPathogenic chlamydiae are obligate intracellular pathogens and have adapted successfully to human cells, causing sexually transmitted diseases or pneumonia. Chlamydial outer protein N (CopN) is likely a critical effector protein secreted by the type III secretion system in chlamydiae, which manipulates host cells. However, the mechanisms of its action remain to be clarified. In this work, we aimed to identify previously unidentified CopN effector target in host cells.ResultsWe first performed a pull-down assay with recombinant glutathione S-transferase (GST) fusion CopN proteins (GST-CpCopN: Chlamydia pneumoniae TW183, GST-CtCopN: Chlamydia trachomatis D/UW-3/CX) as "bait" and soluble lysates obtained from human immortal epithelial HEp-2 cells as "prey", followed by SDS-PAGE with mass spectroscopy (MS). We found that a host cell protein specifically bound to GST-CpCopN, but not GST-CtCopN. MS revealed the host protein to be fructose bisphosphate aldolase A (aldolase A), which plays a key role in glycolytic metabolism. We also confirmed the role of aldolase A in chlamydia-infected HEp-2 cells by using two distinct experiments for gene knockdown with an siRNA specific to aldolase A transcripts, and for assessment of glycolytic enzyme gene expression levels. As a result, both the numbers of chlamydial inclusion-forming units and RpoD transcripts were increased in the chlamydia-infected aldolase A knockdown cells, as compared with the wild-type HEp-2 cells. Meanwhile, chlamydial infection tended to enhance expression of aldolase A.ConclusionsWe discovered that one of the C. pneumoniae CopN targets is the glycolytic enzyme aldolase A. Sequestering aldolase A may be beneficial to bacterial growth in infected host cells.
| S-EPMC4302594 | biostudies-literature