Characterization of phospholipase C from Pseudomonas aeruginosa as a potent inflammatory agent

Abstract

Pseudomonas aeruginosa induced a marked inflammatory response when injected intraperitoneally in C3H/HeJ mice. This inflammation was characterized by the accumulation of inflammatory cells and plasma protein and the release of arachidonic acid metabolites (6-trans-12-epileukotriene B4 [LTB4], 6-trans-LTB4, LTB4, 5-HETE (5-hydroxyeicosatetraenoic acid), LTC4, LTD4, LTE4, prostaglandin E2 [PGE2], PGF2-et, and thromboxane B2 [T.B2]) in the peritoneal cavity of the mice. Heat-inactivated PLC did not evoke any of these effects, suggesting that enzyme activity is necessary for PLC-induced inflammation. When human granulocytes were incubated with PLC in vitro, 6-trans-12epi-LTB4, 6-trans-LTB4, LTB4, 5-HETE, and PGE2 were generated. Mouse peritoneal cells stimulated with PLC released 6-trans-LTB4, LTB4, PGE2, PGF2-a, and TxB2. Both human granulocytes and mouse peritoneal cells stimulated with PLC generated significantly increased levels of arachidonic acid metabolites as compared with cells incubated with heat-inactivated PLC. Leukotriene production by both populations of cells was inhibited when the cells were preincubated with nordihydroguaiaretic acid and subsequently stimulated with PLC. Similarly, both cell types released significantly lower amounts of cyclooxygenase pathway products when they were preincubated with indomethacin and subsequently stimulated with PLC.

Bacterial Phospholipases

“…pefringens a-toxin and Ps. aeruginosa Plc-H have been shown to activate the arachidonic acid cascade in eukaryotic cells (Fujii and Sakurai 1989;Gustafson et al 1990;Meyers and Berk 1990) leading to the production of leukotrienes, thromboxanes and pro-staglandins. The production of these mediators might be especially important in pulmonary infections caused by Ps.…”

A simple and reliable method for rapid production and purification of Pseudomonas aeruginosa haemolytic phospholipase C

“…Indeed, PLC synthesis has also been observed under high osmolarity conditions and in cases of CF, the choline produced by degradation of PC and sphingomyelin by PLC could act as an osmoprotectant in the high osmolarity of the lung surfactant. In terms of signalling, haemolytic PLC activates lipoxygenase and cyclooxygenase pathways leading to the production of thromboxanes, leukotrienes and prostaglandins with dramatic consequences in terms of inflammatory responses that can be deleterious to the patient (Meyers and Berk 1990). In any case, so far, studies on phospholipases had to be interpreted with caution because of the possibility of contamination by trace amount of another active molecule that would co-purify during the long processes involved.…”

“…The most important list is as follows: toxin A and exoenzyme S (ADP-ribosyl transferases that inhibit protein synthesis and therefore lead to cell death); elastastase (metalloprotease that causes degradation of extracellular matrix components, and inactivates human immunoglobulin G and some complement components) and finally the haemolytic and nonhaemolytic phospholipases C (PLC). The haemolytic PLC is one of the major virulence factors of the bacteria and has been associated with inflammatory response (Meyers and Berk 1990;Vasil et al 1991), adhesion to epithelial cells (Saiman et al 1992), cytotoxicity and degradation of the pulmonary surfactant leading to atelectasis (Berk et al 1987). The two PLCs secreted by P. aeruginosa, e.g.…”

Pseudomonas aeruginosa and cystic fibrosis: Correlation between exoenzyme production and patient's clinical state

“…This enzyme activates the arachidonic acid cascade, leading to prostaglandin formation and inflammation. 14,34 Both PLCs are able to induce IL-8 production by monocytes, thus increasing neutrophil accumulation during infection, and PLC-H abolishes the oxidative burst from polymorphonuclear cells. 13,14 In conclusion, our results suggest that the production of elastase and neuraminidase by P. aeruginosa is particularly important in CF patients with good clinical status, and that PLC production by isolates may be involved in the decrease in lung function observed in later stages.…”

“…11,12 This exoenzyme is also a potent agent of the inflammatory reaction. [13][14][15] In addition, the adhesion of a strain of P. aeruginosa serotype O1 to bovine trachea epithelial cell monolayers may be significantly increased by PLC activity. 2 The role of the second PLC, which is nonhemolytic (PLC-N), is unclear.…”