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Bacteria induce prolonged PMN survival via a phosphatidylcholine-specific phospholipase C- and protein kinase C-dependent mechanism.

ABSTRACT: Polymorphonuclear leukocytes (PMNs) are essential for the human innate immune defense, limiting expansion of invading microorganisms. PMN turnover is controlled by apoptosis, but the regulating signaling pathways remain elusive, largely due to inherent differences between mice and humans that undermine use of mouse models for understanding human PMN biology. Here, we aim to elucidate signal transduction mediating survival of human peripheral blood PMNs in response to bacteria, such as Yersinia pseudotuberculosis, an enteropathogen that causes the gastro-intestinal disease yersiniosis, as well as Escherichia coli and Staphylococcus aureus. Determinations of cell death reveal that uninfected control cells undergo apoptosis, while PMNs infected with either Gram-positive or -negative bacteria show profoundly increased survival. Infected cells exhibit decreased caspase 3 and 8 activities, increased mitochondrial integrity and are resistant to apoptosis induced by a death receptor ligand. This bacteria-induced response is accompanied by pro-inflammatory cytokine production including interleukin-8 and tumor necrosis factor-? competent to attract additional PMNs. Using agonists and pharmacological inhibitors, we show participation of Toll-like receptor 2 and 4, and interestingly, that protein kinase C (PKC) and phosphatidylcholine-specific phospholipase C (PC-PLC), but not tyrosine kinases or phosphatidylinositol-specific phospholipase C (PI-PLC) are key players in this dual PMN response. Our findings indicate the importance of prolonged PMN survival in response to bacteria, where general signaling pathways ensure complete exploitation of PMN anti-microbial capacity.

SUBMITTER: Erttmann SF

PROVIDER: S-EPMC3909253 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Bacteria induce prolonged PMN survival via a phosphatidylcholine-specific phospholipase C- and protein kinase C-dependent mechanism.

Erttmann Saskia F SF Gekara Nelson O NO Fällman Maria M

PloS one 20140131 1

Polymorphonuclear leukocytes (PMNs) are essential for the human innate immune defense, limiting expansion of invading microorganisms. PMN turnover is controlled by apoptosis, but the regulating signaling pathways remain elusive, largely due to inherent differences between mice and humans that undermine use of mouse models for understanding human PMN biology. Here, we aim to elucidate signal transduction mediating survival of human peripheral blood PMNs in response to bacteria, such as Yersinia p ...[more]

PMID: 24498214

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Project description:BackgroundVibrio anguillarum is the causative agent of vibriosis in fish. Several extracellular proteins secreted by V. anguillarum have been shown to contribute to virulence. While two hemolysin gene clusters, vah1-plp and rtxACHBDE, have been previously identified and described, the activities of the protein encoded by the plp gene were not known. Here we describe the biochemical activities of the plp-encoded protein and its role in pathogenesis.ResultsThe plp gene, one of the components in vah1 cluster, encodes a 416-amino-acid protein (Plp), which has homology to lipolytic enzymes containing the catalytic site amino acid signature SGNH. Hemolytic activity of the plp mutant increased 2-3-fold on sheep blood agar indicating that plp represses vah1; however, hemolytic activity of the plp mutant decreased by 2-3-fold on fish blood agar suggesting that Plp has different effects against erythrocytes from different species. His6-tagged recombinant Plp protein (rPlp) was over-expressed in E. coli. Purified and re-folded active rPlp exhibited phospholipase A2 activity against phosphatidylcholine and no activity against phosphatidylserine, phosphatidylethanolamine, or sphingomyelin. Characterization of rPlp revealed broad optimal activities at pH 5-9 and at temperatures of 30-64°C. Divalent cations and metal chelators did not affect activity of rPlp. We also demonstrated that Plp was secreted using thin layer chromatography and immunoblot analysis. Additionally, rPlp had strong hemolytic activity towards rainbow trout erythrocytes, but not to sheep erythrocytes suggesting that rPlp is optimized for lysis of phosphatidylcholine-rich fish erythrocytes. Further, only the loss of the plp gene had a significant effect on hemolytic activity of culture supernatant on fish erythrocytes, while the loss of rtxA and/or vah1 had little effect. However, V. anguillarum strains with mutations in plp or in plp and vah1 exhibited no significant reduction in virulence compared to the wild type strain when used to infect rainbow trout.ConclusionThe plp gene of V. anguillarum encoding a phospholipase with A2 activity is specific for phosphatidylcholine and, therefore, able to lyse fish erythrocytes, but not sheep erythrocytes. Mutation of plp does not affect the virulence of V. anguillarum in rainbow trout.

Dataset Information

Bacteria induce prolonged PMN survival via a phosphatidylcholine-specific phospholipase C- and protein kinase C-dependent mechanism.

Publications

Bacteria induce prolonged PMN survival via a phosphatidylcholine-specific phospholipase C- and protein kinase C-dependent mechanism.

Similar Datasets

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