Dataset Information

Sphingosine induction of the Pseudomonas aeruginosa hemolytic phospholipase C/sphingomyelinase (PlcH).

ABSTRACT: 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.

Importance

PlcH 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.

SUBMITTER: Mackinder JR

PROVIDER: S-EPMC10955842 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

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Sphingosine induction of the Pseudomonas aeruginosa hemolytic phospholipase C/sphingomyelinase (PlcH).

Mackinder Jacob R JR Hinkel Lauren A LA Schutz Kristin K Eckstrom Korin K Fisher Kira K Wargo Matthew J MJ

Journal of bacteriology 20240227 3

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 sphingos ...[more]

PMID: 38411048

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Project description:The plcHR operon of Pseudomonas aeruginosa includes the structural gene for the hemolytic phospholipase C,plcH (previously known as plcS), and two overlapping, in-phase, genes designated plcR1 and plcR2. Hemolytic and phospholipase C (PLC) activities produced by Escherichia coli and P. aeruginosa T7 expression systems were measured in strains carrying both plcH and plcR genes and in strains carrying each gene separately. When plcH was expressed by itself in the E. coli T7 system, the area of the hemolytic zone on blood agar was less than twice the area of growth. By contrast, when plcR was coexpressed with plcH in this system, the area of the hemolytic zone was approximately 10 times that of the area of the growth on blood agar. Native polyacrylamide gel electrophoretic analyses of PlcH activity expressed in either the E. coli or the P. aeruginosa T7 system carrying plcH alone, or along with the plcR genes, suggest that PlcR either posttranslationally alters the physical or biochemical nature of PlcH or releases PlcH from a complex in the cell so that it can be secreted. The hypothesis that PlcR is involved in the secretion of PlcH is supported by the observation that the ratio of extracellular to cell-associated PlcH activity produced by P. aeruginosa strains containing an in-frame deletion in the chromosomal plcR genes is significantly reduced in comparison with this ratio seen with the wild-type parental strain. This defect in the secretion of PlcH can be complemented by the plcR genes in trans. Additional data suggest that PlcR does not directly affect the secretion of the nonhemolytic phospholipase C (PlcN). PlcR is highly similar to a calcium-binding protein (CAB) from Streptomyces erythraeus. PlcR and CAB contain typical motifs (EF hands) characteristic of eucaryotic calcium-binding proteins, including calmodulin. P. aeruginosa naturally produces membrane vesicles (MVs) containing extracellular proteins including PLC. MVs from the PAO1WT strain contained at least 10-fold more PLC specific activity than those isolated from a strain carrying a deletion of plcR (PAO1 deltaR). Immunogold electron microscopy of PAO1WT and PAO1 deltaR whole cells revealed a distribution of PlcH in these strains consistent with the hypothesis that PlcR is required for the secretion of PlcH.

Project description: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).

Dataset Information

Sphingosine induction of the Pseudomonas aeruginosa hemolytic phospholipase C/sphingomyelinase (PlcH).

Importance

Publications

Sphingosine induction of the <i>Pseudomonas aeruginosa</i> hemolytic phospholipase C/sphingomyelinase (PlcH).

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Dataset Information

Sphingosine induction of the Pseudomonas aeruginosa hemolytic phospholipase C/sphingomyelinase (PlcH).

Importance

Publications

Sphingosine induction of the &lt;i&gt;Pseudomonas aeruginosa&lt;/i&gt; hemolytic phospholipase C/sphingomyelinase (PlcH).

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Sphingosine induction of the <i>Pseudomonas aeruginosa</i> hemolytic phospholipase C/sphingomyelinase (PlcH).