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Phospholipase D promotes Arcanobacterium haemolyticum adhesion via lipid raft remodeling and host cell death following bacterial invasion.

ABSTRACT: BACKGROUND: Arcanobacterium haemolyticum is an emerging bacterial pathogen, causing pharyngitis and more invasive infections. This organism expresses an unusual phospholipase D (PLD), which we propose promotes bacterial pathogenesis through its action on host cell membranes. The pld gene is found on a genomic region of reduced %G + C, suggesting recent horizontal acquisition. RESULTS: Recombinant PLD rearranged HeLa cell lipid rafts in a dose-dependent manner and this was inhibited by cholesterol sequestration. PLD also promoted host cell adhesion, as a pld mutant had a 60.3% reduction in its ability to adhere to HeLa cells as compared to the wild type. Conversely, the pld mutant appeared to invade HeLa cells approximately two-fold more efficiently as the wild type. This finding was attributable to a significant loss of host cell viability following secretion of PLD from intracellular bacteria. As determined by viability assay, only 15.6% and 82.3% of HeLa cells remained viable following invasion by the wild type or pld mutant, respectively, as compared to untreated HeLa cells. Transmission electron microscopy of HeLa cells inoculated with A. haemolyticum strains revealed that the pld mutant was contained within intracellular vacuoles, as compared to the wild type, which escaped the vacuole. Wild type-infected HeLa cells also displayed the hallmarks of necrosis. Similarly inoculated HeLa cells displayed no signs of apoptosis, as measured by induction of caspase 3/7, 8 or 9 activities. CONCLUSIONS: These data indicate that PLD enhances bacterial adhesion and promotes host cell necrosis following invasion, and therefore, may be important in the disease pathogenesis of A. haemolyticum infections.

SUBMITTER: Lucas EA

PROVIDER: S-EPMC2978216 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Phospholipase D promotes Arcanobacterium haemolyticum adhesion via lipid raft remodeling and host cell death following bacterial invasion.

Lucas Erynn A EA Billington Stephen J SJ Carlson Petteri P McGee David J DJ Jost B Helen BH

BMC microbiology 20101025

<h4>Background</h4>Arcanobacterium haemolyticum is an emerging bacterial pathogen, causing pharyngitis and more invasive infections. This organism expresses an unusual phospholipase D (PLD), which we propose promotes bacterial pathogenesis through its action on host cell membranes. The pld gene is found on a genomic region of reduced %G + C, suggesting recent horizontal acquisition.<h4>Results</h4>Recombinant PLD rearranged HeLa cell lipid rafts in a dose-dependent manner and this was inhibited ...[more]

PMID: 20973961

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Project description:Arcanobacterium haemolyticum, a pathogen of the human upper respiratory tract and other systems, has been reported to produce soluble toxins, including a phospholipase D (PLD). We confirmed production of PLD by this organism and cloned and sequenced pld. Arcanobacterial PLD (PLD-A) was found to be a protein of approximately 31.5 kDa with a pI of approximately 9.4. Cosmid cloning, followed by subcloning into phagemid pBluescriptIISK+, yielded Escherichia coli(pAh140), a recombinant with a gene product corresponding to PLD-A. Evidence of PLD activity was found by three assays in supernatant fluid of cultures of E. coli(pAh140) and A. haemolyticum, but not in E. coli(pBluescriptIISK+). Experiments to determine if this protein was secreted were not conducted, but previous work with PLD from Corynebacterium pseudotuberculosis suggested that the presence of the enzyme in culture supernatant fluids was due to lysis of E. coli rather than to active transport. Antibodies in polyclonal sera from goats immunized with native or recombinant PLD-A neutralized native and recombinant PLD-A activity, and antibodies against native or recombinant PLD from C. pseudotuberculosis (PLD-P) partially neutralized native and recombinant PLD-A. Antibodies prepared against recombinant PLD-A labelled both recombinant and native PLD-A in Western blots (immunoblots) and dot blots, but antibodies against PLD-P did not. Sequencing of the insert in pAh140 revealed an open reading frame of 930 bp coding for 309 amino acids, including a putative signal sequence of 26 amino acids (3.2 kDa, determined on the basis of homology with the 24-amino-acid signal sequence of pld from C. pseudotuberculosis bv. ovis) and the mature PLD protein (31.5 kDa). Sequence comparisons of coding regions revealed 65% DNA homology with pld genes from C. pseudotuberculosis and Corynebacterium ulcerans. Comparison of amino acid sequences revealed 64% homology of PLD-A both with PLD-P and with PLD produced by C. ulcerans.

Dataset Information

Phospholipase D promotes Arcanobacterium haemolyticum adhesion via lipid raft remodeling and host cell death following bacterial invasion.

Publications

Phospholipase D promotes Arcanobacterium haemolyticum adhesion via lipid raft remodeling and host cell death following bacterial invasion.

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