Dataset Information

Defining the interaction of the protease CpaA with its type II secretion chaperone CpaB and its contribution to virulence in Acinetobacter species.

ABSTRACT: Acinetobacter baumannii, Acinetobacter nosocomialis, and Acinetobacter pittii are a frequent cause of multidrug-resistant, healthcare-associated infections. Our previous work demonstrated that A. nosocomialis M2 possesses a functional type II secretion system (T2SS) that is required for full virulence. Further, we identified the metallo-endopeptidase CpaA, which has been shown previously to cleave human Factor V and deregulate blood coagulation, as the most abundant type II secreted effector protein. We also demonstrated that its secretion is dependent on CpaB, a membrane-bound chaperone. In this study, we show that CpaA expression and secretion are conserved across several medically relevant Acinetobacter species. Additionally, we demonstrate that deletion of cpaA results in attenuation of A. nosocomialis M2 virulence in moth and mouse models. The virulence defects resulting from the deletion of cpaA were comparable with those observed upon abrogation of T2SS activity. The virulence defects resulting from the deletion of cpaA are comparable with those observed upon abrogation of T2SS activity. We also show that CpaA and CpaB strongly interact, forming a complex in a 1:1 ratio. Interestingly, deletion of the N-terminal transmembrane domain of CpaB results in robust secretion of CpaA and CpaB, indicating that the transmembrane domain is dispensable for CpaA secretion and likely functions to retain CpaB inside the cell. Limited proteolysis of spheroplasts revealed that the C-terminal domain of CpaB is exposed to the periplasm, suggesting that this is the site where CpaA and CpaB interact in vivo Last, we show that CpaB does not abolish the proteolytic activity of CpaA against human Factor V. We conclude that CpaA is, to the best of our knowledge, the first characterized, bona fide virulence factor secreted by Acinetobacter species.

SUBMITTER: Kinsella RL

PROVIDER: S-EPMC5712607 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Defining the interaction of the protease CpaA with its type II secretion chaperone CpaB and its contribution to virulence in Acinetobacter species.

Kinsella Rachel L RL Lopez Juvenal J Palmer Lauren D LD Salinas Nichole D ND Skaar Eric P EP Tolia Niraj H NH Feldman Mario F MF

The Journal of biological chemistry 20171005 48

Acinetobacter baumannii, Acinetobacter nosocomialis, and Acinetobacter pittii are a frequent cause of multidrug-resistant, healthcare-associated infections. Our previous work demonstrated that A. nosocomialis M2 possesses a functional type II secretion system (T2SS) that is required for full virulence. Further, we identified the metallo-endopeptidase CpaA, which has been shown previously to cleave human Factor V and deregulate blood coagulation, as the most abundant t ...[more]

PMID: 28982978

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Project description:UnlabelledMycobacterium tuberculosis is a leading cause of death worldwide. The M. tuberculosis TAT (twin-arginine translocation) protein secretion system is present at the cytoplasmic membrane of mycobacteria and is known to transport folded proteins. The TAT secretion system is reported to be essential for many important bacterial processes that include cell wall biosynthesis. The M. tuberculosis secretion and invasion protein RipA has endopeptidase activity and interacts with one of the resuscitation antigens (RpfB) that are expressed during pathogen reactivation. MoxR1, a member of the ATPase family that is associated with various cellular activities, was predicted to interact with RipA based on in silico analyses. A bimolecular fluorescence complementation (BiFC) assay confirmed the interaction of these two proteins in HEK293T cells. The overexpression of RipA in Mycobacterium smegmatis and copurification with MoxR1 further validated their interaction in vivo. Recombinant MoxR1 protein, expressed in Escherichia coli, displays ATP-enhanced chaperone activity. Secretion of recombinant RipA (rRipA) protein into the E. coli culture filtrate was not observed in the absence of RipA-MoxR interaction. Inhibition of this export system in M. tuberculosis, including the key players, will prevent localization of peptidoglycan hydrolase and result in sensitivity to existing ?-lactam antibiotics, opening up new candidates for drug repurposing.ImportanceThe virulence mechanism of mycobacteria is very complex. Broadly, the virulence factors can be classified as secretion factors, cell surface components, enzymes involved in cellular metabolism, and transcriptional regulators. The mycobacteria have evolved several mechanisms to secrete its proteins. Here, we have identified one of the virulence proteins of Mycobacterium tuberculosis, RipA, possessing peptidoglycan hydrolase activities secreted by the TAT secretion pathway. We also identified MoxR1 as a protein-protein interaction partner of RipA and demonstrated chaperone activity of this protein. We show that MoxR1-mediated folding is critical for the secretion of RipA within the TAT system. Inhibition of this export system in M. tuberculosis will prevent localization of peptidoglycan hydrolase and result in sensitivity to existing ?-lactam antibiotics, opening up new candidates for drug repurposing.

Project description:BACKGROUND:Microbial taxonomy remains a conservative discipline, relying on phenotypic information derived from growth in pure culture and techniques that are time-consuming and difficult to standardize, particularly when compared to the ease of modern high-throughput genome sequencing. Here, drawing on the genus Acinetobacter as a test case, we examine whether bacterial taxonomy could abandon phenotypic approaches and DNA-DNA hybridization and, instead, rely exclusively on analyses of genome sequence data. RESULTS:In pursuit of this goal, we generated a set of thirteen new draft genome sequences, representing ten species, combined them with other publically available genome sequences and analyzed these 38 strains belonging to the genus. We found that analyses based on 16S rRNA gene sequences were not capable of delineating accepted species. However, a core genome phylogenetic tree proved consistent with the currently accepted taxonomy of the genus, while also identifying three misclassifications of strains in collections or databases. Among rapid distance-based methods, we found average-nucleotide identity (ANI) analyses delivered results consistent with traditional and phylogenetic classifications, whereas gene content based approaches appear to be too strongly influenced by the effects of horizontal gene transfer to agree with previously accepted species. CONCLUSION:We believe a combination of core genome phylogenetic analysis and ANI provides an appropriate method for bacterial species delineation, whereby bacterial species are defined as monophyletic groups of isolates with genomes that exhibit at least 95% pair-wise ANI. The proposed method is backwards compatible; it provides a scalable and uniform approach that works for both culturable and non-culturable species; is faster and cheaper than traditional taxonomic methods; is easily replicable and transferable among research institutions; and lastly, falls in line with Darwin's vision of classification becoming, as far as is possible, genealogical.

Dataset Information

Defining the interaction of the protease CpaA with its type II secretion chaperone CpaB and its contribution to virulence in Acinetobacter species.

Publications

Defining the interaction of the protease CpaA with its type II secretion chaperone CpaB and its contribution to virulence in <i>Acinetobacter</i> species.

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