Bacterial phospholipid hydrolysis enhances the destruction of Escherichia coli ingested by rabbit neutrophils. Role of cellular and extracellular phospholipases.

Abstract

IntroductionEscherichia coli ingested by PMN are promptly growth arrested but undergo limited destruction. We have studied bacterial phospholipid hydrolysis as a possible limiting factor in the disassembly of ingested E. coli, comparing the fates, during phagocytosis by rabbit peritoneal exudate PMN, of three isogenic strains, differing in their content of the pldA gene encoding the principal E. coli phospholipase A (PLA), i.e., pldA-, pldA+, pldA+++ (the latter strain bearing the pldA gene in a multicopy plasmid resulting in a 20-fold increase in PLA content). Ingestion and growth inhibition (> 99% within 15 min) were the same for the three strains, but phospholipid degradation differed according to bacterial PLA content: pldA+++ up to 60%, pldA+ up to 30%, and pidA-up to 20%. Since the pidA-strain has no activatable PLA, phospholipid degradation in this strain demonstrates the action of a PMN PLA. Added PLA2-rich ascitic fluid (AF) or purified AF PLA2 increased the rate and extent of degradation of the pldA-strain, provided the enzyme was added before ingestion was complete.125I-AF-PLA2 binds to both E. coli and PMN and thus can enter the vacuole during phagocytosis. Although up to 50-fold more AF-PLA2 than the PLA2 content of the PMN could be loaded into the PMN in this way, degradation of pldA-E. coli did not exceed 30%. Increased phospholipid degradation had no effect on the degradation of bacterial macromolecules. In contrast, bacterial disassembly manifest as structural disorganization, release of bacterial protein derived material, and inhibition of protein synthesis were markedly enhanced when > 50% of prelabelled bacterial phospholipids were degraded. These findings reveal a link between envelope phospholipid degradation and overall bacterial destruction, suggesting therefore that factors limiting PLA action limit the destruction of E. coli ingested by PMN. (J.

Groups IV, V, and X Phospholipases A2s in Human Neutrophils

“…Recombinant GIIA and GV sPLA 2 , but Not Recombinant GX sPLA 2 , Catalyze the Hydrolysis of Phospholipids in Live E. coli-A clear link between E. coli envelope phospholipid degradation and overall bacterial destruction by rabbit neutrophils has been established (69). We have demonstrated that human neutrophils contain GV sPLA 2 and release this enzyme upon exposure to fMLP or OZ, so we evaluated the ability of recom- binant GV sPLA 2 to hydrolyze bacterial phospholipids.…”

“…The highly dense and anionic cell wall and lipopolysaccharide layers limit the ability of extracellular sPLA 2 enzymes to access and hydrolyze the plasma membrane. However, following disruption of the cell wall by polymyxin antibiotics or BPI (48,72), cationic enzymes such as GIIA sPLA 2 may gain access to the plasma membrane and kill the bacteria (59, 102), and a clear link has been established between envelope phospholipid deg-radation and overall Gram-negative bacterial destruction (69). We found that recombinant GIIA sPLA 2 , in the presence of serum, hydrolyzed phospholipids from E. coli that lack PLA 2 activity more efficiently than recombinant GV sPLA 2 , whereas recombinant GX sPLA 2 did not catalyze detectable bacterial phospholipid hydrolysis.…”

“…As GV sPLA 2 was the only sPLA 2 enzyme released by neutrophils, these results point to a potential role for extracellular GV sPLA 2 in neutrophil-mediated E. coli phospholipid hydrolysis in vivo. It is also possible that GV sPLA 2 bound to E. coli may augment bacterial phospholipid hydrolysis after phagocytosis of the bacteria by neutrophils, as described for GIIA sPLA 2 (3,69). Extracellular GV sPLA 2 released by neutrophils may also participate in the eradication of Gram-positive bacteria, including Listeria monocytogenes, Enterobacter faecium, Enterococcus faecalis, and methicillin-sensitive and -resistant Staphylococcus aureus, because co-incubation of these bacteria with rat, human, and mouse GV sPLA 2 s resulted in significant bactericidal activity in vitro (69,89).…”

Substrate Selectivity of Lysophospholipid Transporter LplT Involved in Membrane Phospholipid Remodeling in Escherichia coli

“…Disrupting bacterial membranes by phospholipases is a potent mechanism in host cell defense (4). In polymorphonuclear neutrophils, phospholipase A 2 (PLA 2 ) is delivered into the ingested bacterial cells by bacterial permeability protein, causing degradation of Ͼ50% of target cell membranes and consequent membrane disorganization and cell disassembly (4,5).…”

Group V Phospholipase A2 in Bone Marrow-derived Myeloid Cells and Bronchial Epithelial Cells Promotes Bacterial Clearance after Escherichia coli Pneumonia

“…Fig. 4D) may also explain why bacterial clearance was impaired in GV Ϫ/Ϫ mice, because PMN efficiently ingest and kill E. coli in vitro (35). Attenuated accumulation of inflammatory cells in the alveolar space of GV Ϫ/Ϫ in comparison with GV ϩ/ϩ mice may be due to decreased expression of ICAM-1 and/or PECAM-1 on pulmonary endothelial cells in GV Ϫ/Ϫ mice (cf.…”