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 ∼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).

Homeostasis and Catabolism of Choline and Glycine Betaine: Lessons from Pseudomonas aeruginosa

“…Bacteria commonly produce secreted phospholipases C and/or D, which liberate phosphorylcholine or choline from choline phosphate-containing phospholipids and sphingolipids, respectively (32,33). These phospholipases are often considered virulence factors, when studied in opportunistic pathogens (reviewed in reference 33), that can alter bacterial survival, cause cell damage, induce inflammatory cytokine production, and suppress antibacterial responses of innate immune cells (34)(35)(36)(37). However, they could also be categorized as choline/nutrient acquisition systems beneficial for niche survival (38), with the side effects of host cell damage and potential hemolysis.…”

Bacterial Sphingomyelinases and Phospholipases as Virulence Factors

“…PlcN hydrolyzes PC and PS, but not SM, and is nonhemolytic (247), whereas PlcH hydrolyzes PC, SM, and lyso-PC at equal rates (248) and also acts on cardiolipin, PE, and PG (249). P. aeruginosa PlcH, which is expressed during lung infections in humans (250), is hemolytic to human erythrocytes (26) and selectively cytotoxic to endothelial cells (248). Furthermore, it suppresses the neutrophil respiratory burst and induces a strong chemokine response in mice and human granulocytes (248,251).…”

“…aeruginosa secretes two PLCs from the acid phosphatase superfamily, PlcN and PlcH/PlcS (Table 5), via the twin-arginine translocation (Tat) and type II Xcp-dependent systems (246). PlcN hydrolyzes PC and PS, but not SM, and is nonhemolytic (247), whereas PlcH hydrolyzes PC, SM, and lyso-PC at equal rates (248) and also acts on cardiolipin, PE, and PG (249). P. aeruginosa PlcH, which is expressed during lung infections in humans (250), is hemolytic to human erythrocytes (26) and selectively cytotoxic to endothelial cells (248).…”

Imaging the early stages of phospholipase C/sphingomyelinase activity on vesicles containing coexisting ordered-disordered and gel-fluid domains

“…A 1,000 bp region of the plcH gene was mutagenized, and the pooled mutated DNA was used to replace the corresponding wild-type plcH gene in an Escherichia coli plasmid ( 51 ). Individual clones carrying the mutated plcH gene were then screened for PLC activity using the synthetic PLC substrate p -nitrophenyl phosphatidylcholine (NPPC) ( 9 ).…”

“…However, unlike PlcH, AcpA has phosphatase activity but not authentic PLC activity ( 51 ). The Ser175 of AcpA was identifi ed in the active site of AcpA, which is only defi ned based on the crystal structure of AcpA in context of the interactions of 10 amino acids, including Ser175, with vanadate (a phosphate analog), which provides a model for the phosphate moiety of a phosphomonoester or phosphodiester bond of an AcpA or PLC substrate ( 52 ).…”