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A complex extracellular sphingomyelinase of Pseudomonas aeruginosa inhibits angiogenesis by selective cytotoxicity to endothelial cells.

ABSTRACT: The hemolytic phospholipase C (PlcHR) expressed by Pseudomonas aeruginosa is the original member of a Phosphoesterase Superfamily, which includes phosphorylcholine-specific phospholipases C (PC-PLC) produced by frank and opportunistic pathogens. PlcHR, but not all its family members, is also a potent sphingomyelinase (SMase). Data presented herein indicate that picomolar (pM) concentrations of PlcHR are selectively lethal to endothelial cells (EC). An RGD motif of PlcHR contributes to this selectivity. Peptides containing an RGD motif (i.e., GRGDS), but not control peptides (i.e., GDGRS), block the effects of PlcHR on calcium signaling and cytotoxicity to EC. Moreover, RGD variants of PlcHR (e.g., RGE, KGD) are significantly reduced in their binding and toxicity, but retain the enzymatic activity of the wild type PlcHR. PlcHR also inhibits several EC-dependent in vitro assays (i.e., EC migration, EC invasion, and EC tubule formation), which represent key processes involved in angiogenesis (i.e., formation of new blood vessels from existing vasculature). Finally, the impact of PlcHR in an in vivo model of angiogenesis in transgenic zebrafish, and ones treated with an antisense morpholino to knock down a key blood cell regulator, were evaluated because in vitro assays cannot fully represent the complex processes of angiogenesis. As little as 2 ng/embryo of PlcHR was lethal to approximately 50% of EGFP-labeled EC at 6 h after injection of embryos at 48 hpf (hours post-fertilization). An active site mutant of PlcHR (Thr178Ala) exhibited 120-fold reduced inhibitory activity in the EC invasion assay, and 20 ng/embryo elicited no detectable inhibitory activity in the zebrafish model. Taken together, these observations are pertinent to the distinctive vasculitis and poor wound healing associated with P. aeruginosa sepsis and suggest that the potent antiangiogenic properties of PlcHR are worthy of further investigation for the treatment of diseases where angiogenesis contributes pathological conditions (e.g., vascularization of tumors, diabetic retinopathy).

SUBMITTER: Vasil ML

PROVIDER: S-EPMC2673038 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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A complex extracellular sphingomyelinase of Pseudomonas aeruginosa inhibits angiogenesis by selective cytotoxicity to endothelial cells.

Vasil Michael L ML Stonehouse Martin J MJ Vasil Adriana I AI Wadsworth Sandra J SJ Goldfine Howard H Bolcome Robert E RE Chan Joanne J

PLoS pathogens 20090508 5

The hemolytic phospholipase C (PlcHR) expressed by Pseudomonas aeruginosa is the original member of a Phosphoesterase Superfamily, which includes phosphorylcholine-specific phospholipases C (PC-PLC) produced by frank and opportunistic pathogens. PlcHR, but not all its family members, is also a potent sphingomyelinase (SMase). Data presented herein indicate that picomolar (pM) concentrations of PlcHR are selectively lethal to endothelial cells (EC). An RGD motif of PlcHR contributes to this selec ...[more]

PMID: 19424430

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Project description:Hemolytic phospholipase C, PlcH, is an important virulence factor for Pseudomonas aeruginosa. PlcH preferentially hydrolyzes sphingomyelin and phosphatidylcholine, and this hydrolysis activity drives tissue damage and inflammation and interferes with the oxidative burst of immune cells. Among other contributors, transcription of plcH was previously shown to be induced by phosphate starvation via PhoB and the choline metabolite, glycine betaine, via GbdR. Here, we show that sphingosine can induce plcH transcription and result in secreted PlcH enzyme activity. This induction is dependent on the sphingosine-sensing transcriptional regulator SphR. The SphR induction of plcH occurs from the promoter for the gene upstream of plcH that encodes the neutral ceramidase, CerN, and transcriptional readthrough of the cerN transcription terminator. Evidence for these conclusions came from mutation of the SphR binding site in the cerN promoter, mutation of the cerN terminator, enhancement of cerN termination by adding the rrnB terminator, and reverse transcriptase PCR (RT-PCR) showing that the intergenic region between cerN and plcH is made as RNA during sphingosine, but not choline, induction. We also observed that, like glycine betaine induction, sphingosine induction of plcH is under catabolite repression control, which likely explains why such induction was not seen in other studies using sphingosine in rich media. The addition of sphingosine as a novel inducer for PlcH points to the regulation of plcH transcription as a site for the integration of multiple host-derived signals.ImportancePlcH is a secreted phospholipase C/sphingomyelinase that is important for the virulence of Pseudomonas aeruginosa. Here, we show that sphingosine, which presents itself or as a product of P. aeruginosa sphingomyelinase and ceramidase activity, leads to the induction of plcH transcription. This transcriptional induction occurs from the promoter of the upstream ceramidase gene generating a conditional operon. The transcript on which plcH resides, therefore, is different depending on which host molecule or condition leads to induction, and this may have implications for PlcH post-transcriptional regulation. This work also adds to our understanding of P. aeruginosa with host-derived sphingolipids.

Dataset Information

A complex extracellular sphingomyelinase of Pseudomonas aeruginosa inhibits angiogenesis by selective cytotoxicity to endothelial cells.

Publications

A complex extracellular sphingomyelinase of Pseudomonas aeruginosa inhibits angiogenesis by selective cytotoxicity to endothelial cells.

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