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Legionella pneumophila Effector LpdA Is a Palmitoylated Phospholipase D Virulence Factor.

ABSTRACT: Legionella pneumophila is a bacterial pathogen that thrives in alveolar macrophages, causing a severe pneumonia. The virulence of L. pneumophila depends on its Dot/Icm type IV secretion system (T4SS), which delivers more than 300 effector proteins into the host, where they rewire cellular signaling to establish a replication-permissive niche, the Legionella-containing vacuole (LCV). Biogenesis of the LCV requires substantial redirection of vesicle trafficking and remodeling of intracellular membranes. In order to achieve this, several T4SS effectors target regulators of membrane trafficking, while others resemble lipases. Here, we characterized LpdA, a phospholipase D effector, which was previously proposed to modulate the lipid composition of the LCV. We found that ectopically expressed LpdA was targeted to the plasma membrane and Rab4- and Rab14-containing vesicles. Subcellular targeting of LpdA required a C-terminal motif, which is posttranslationally modified by S-palmitoylation. Substrate specificity assays showed that LpdA hydrolyzed phosphatidylinositol, -inositol-3- and -4-phosphate, and phosphatidylglycerol to phosphatidic acid (PA) in vitro. In HeLa cells, LpdA generated PA at vesicles and the plasma membrane. Imaging of different phosphatidylinositol phosphate (PIP) and organelle markers revealed that while LpdA did not impact on membrane association of various PIP probes, it triggered fragmentation of the Golgi apparatus. Importantly, although LpdA is translocated inefficiently into cultured cells, an L. pneumophila ?lpdA mutant displayed reduced replication in murine lungs, suggesting that it is a virulence factor contributing to L. pneumophila infection in vivo.

SUBMITTER: Schroeder GN

PROVIDER: S-EPMC4567653 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Legionella pneumophila Effector LpdA Is a Palmitoylated Phospholipase D Virulence Factor.

Schroeder Gunnar N GN Aurass Philipp P Oates Clare V CV Tate Edward W EW Hartland Elizabeth L EL Flieger Antje A Frankel Gad G

Infection and immunity 20150727 10

Legionella pneumophila is a bacterial pathogen that thrives in alveolar macrophages, causing a severe pneumonia. The virulence of L. pneumophila depends on its Dot/Icm type IV secretion system (T4SS), which delivers more than 300 effector proteins into the host, where they rewire cellular signaling to establish a replication-permissive niche, the Legionella-containing vacuole (LCV). Biogenesis of the LCV requires substantial redirection of vesicle trafficking and remodeling of intracellular memb ...[more]

PMID: 26216420

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Project description:Several novel Legionella pneumophila virulence genes were previously discovered by use of signature-tagged mutagenesis (P. H. Edelstein, M. A. Edelstein, F. Higa, and S. Falkow, Proc. Natl. Acad. Sci. 96:8190-8195, 1999). One of these mutants appeared to be defective in multiplication in guinea pig lungs and spleens, yet it multiplies normally in guinea pig alveolar macrophages. Here we report further characterization of the mutated gene and its protein and the virulence role of the gene. The complete sequence of the gene, now called lvgA, is 627 bp long, and its protein product is approximately 27 kDa in size. lvgA was present in all 50 strains of L. pneumophila tested. No significant nucleic acid or protein homology was found in the GenBank database for the gene, nor were any distinctive motifs discovered in a search of other databases. The expression of both DotA and IcmX in the lvgA mutant was normal. Subcellular fractionation studies localized LvgA to the outer membrane fraction, and protease digestion studies suggested that at least some of the protein is surface expressed. No change in bacterial lipopolysaccharide composition or reactivity to serogroup-specific antisera was detected in the mutant. Growth competition studies with alveolar macrophages showed that the mutant was outcompeted by its parent 3-fold in 24 h and 24-fold in 48 h, in contrast to what was observed with the null phenotype in parallel testing with alveolar macrophages or with the A549 alveolar epithelial cell line. This macrophage defect of the mutant bacterium was due to slower growth, as the mutant invaded alveolar macrophages normally. Electron microscopy showed that the mutant bacterium resided in a ribosome-studded phagosome in alveolar macrophages, with no distinction from its parent. The lvgA mutant was outcompeted by its parent about sixfold in guinea pig lungs and spleens; prolonged observation of infected animals showed no late-onset virulence of the mutant. Transcomplementation of the mutant restored the parental phenotype in guinea pigs. The lvgA mutant was twofold more susceptible to killing by human beta-defensin 2 but not to killing by other cationic peptides, serum complement, or polymorphonuclear neutrophils. lvgA is a novel virulence gene that is responsible for pleiotropic functions involving both extracellular and intracellular bacterial resistance mechanisms.

Project description:Effector proteins translocated by the Dot/Icm type IV secretion system determine the virulence of Legionella pneumophila (L. pneumophila). Among these effectors, members of the SidE family (SidEs) regulate several cellular processes through a unique phosphoribosyl ubiquitination mechanism mediated by another effector, SidJ. Host-cell calmodulin (CaM) activates SidJ to glutamylate the SidEs of ubiquitin (Ub) ligases and to make a balanced Ub ligase activity. Given the central role of SidJ in this regulatory process, studying the nature of evolution of sidJ is important to understand the virulence of L. pneumophila and the interaction between the bacteria and its hosts. By studying sidJ from a large number of L. pneumophila strains and using various molecular evolution algorithms, we demonstrated that intragenic recombination drove the evolution of sidJ and contributed to sidJ diversification. Additionally, we showed that four codons of sidJ which are located in the N-terminal (NTD) (codons 58 and 200) and C-terminal (CTD) (codons 868 and 869) domains, but not in the kinase domain (KD) had been subjected to strong positive selection pressure, and variable mutation profiles of these codons were identified. Protein structural modeling of SidJ provided possible explanations for these mutations. Codons 868 and 869 mutations might engage in regulating the interactions of SidJ with CaM through hydrogen bonds and affect the CaM docking to SidJ. Mutation in codon 58 of SidJ might affect the distribution of main-chain atoms that are associated with the interaction with CaM. In contrast, mutations in codon 200 might influence the α-helix stability in the NTD. These mutations might be important to balance Ub ligase activity for different L. pneumophila hosts. This study first reported that intragenic recombination and positive Darwinian selection both shaped the genetic plasticity of sidJ, contributing to a deeper understanding of the adaptive mechanisms of this intracellular bacterium to different hosts.

Dataset Information

Legionella pneumophila Effector LpdA Is a Palmitoylated Phospholipase D Virulence Factor.

Publications

Legionella pneumophila Effector LpdA Is a Palmitoylated Phospholipase D Virulence Factor.

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