Listeria monocytogenes secretes two distinct phospholipases C, a phosphatidylinositol-specific phospholipase C (PI-PLC) and a broad-range phospholipase C (PC-PLC). In this study, single in-frame deletion mutants with mutations in each PLC and a double mutant lacking both PLCs were characterized with regard to virulence in mice, escape from a primary vacuole, and cell-to-cell spread in cell culture. The mutant lacking PI-PLC, previously shown to be twofold less virulent than the wild type in mice, had a minor defect in escape from a primary vacuole but was not notably affected in cell-to-cell spread. The mutant lacking PC-PLC was 20-fold less virulent in mice and was defective in cell-to-cell spread but had no measurable defect in escape from a primary vacuole. The mutant lacking both PLCs was 500-fold less virulent in mice and was severely diminished in its ability to escape from the primary vacuole and to spread cell to cell. Cellular levels of diacylglycerol and ceramide, products of PLC activity, accumulated beginning 3 to 4 h after infection of cells with wild-type bacteria. The bacterial PLCs were partially responsible for this activity, since cells infected with the mutant lacking both PLCs had a reduced increase in diacylglycerol and no increase in ceramide. Elevation of diacylglycerol in the absence of bacterial PLCs indicated that host cell phospholipase(s) was activated during infection. The results of this study were consistent with the two bacterial PLCs having overlapping functions throughout the course of intracellular infection. Furthermore, the PC-PLC, and possibly PI-PLC, appeared to be enzymatically active intracellularly.
Listeria monocytogenes secretes a phospholipase C (PLC) which has 39% amino acid sequence identity with the broad-specificity PLC from Bacillus cereus. Recent work indicates that the L. monocytogenes enzyme plays a role during infections of mammalian cells (J.-A. Vazquez-Boland, C. Kocks, S. Dramsi, H. Ohayon, C. Geoffroy, J. Mengaud, and P. Cossart, Infect. Immun. 60:219-230, 1992). The homogeneous enzyme has a specific activity of 230 mumol/min/mg when phosphatidylcholine (PC) is dispersed in sodium deoxycholate. With phospholipid-Triton X-100 mixed micelles, the enzyme had a broad pH optimum between 5.5 and 8.0, and the rates of lipid hydrolysis were in the following order: PC > phosphatidylethanolamine (PE) > phosphatidylserine > sphingomyelin >> phosphatidylinositol (PI). Activity on PC was stimulated 35% by 0.5 M NaCl and 60% by 0.05 mM ZnSO4. When Escherichia coli phospholipids were dispersed in Triton X-100, PE and phosphatidylglycerol, but not cardiolipin, were hydrolyzed. The enzyme was active on all phospholipids of vesiculated human erythrocytes including PI, which was rapidly hydrolyzed at pH 7.0. PI was also hydrolyzed in PI-PC-cholesterol liposomes by the nonspecific PLC from L. monocytogenes and by the homologous enzyme from B. cereus. The water-soluble hydrolysis product was identified as inositol-1-phosphate. For the hydrolysis of human erythrocyte ghost phospholipids, a broad pH optimum was also observed. 32P-labelled Clostridium butyricum protoplasts, which are rich in ether lipids, were treated with PLC. The enzyme hydrolyzed the plasmalogen form of PE, its glycerol acetal, and cardiolipin, in addition to PE. I-, Cl- and F- stimulated activity on either PC- Triton X-100 mixed micelles or human erythrocyte ghosts, unlike the enzyme from B. cereus which is strongly inhibited by halides. Tris-HCl, phosphate, and calcium nitrate had similar inhibitory effects on the enzyme on the enzymes from L. monocytogenes and B. cereus.
An examination of 20 strains of butyric acid-producing Clostridium species for phospholipid class compositions, plasmalogen content, and acyl and alk-l-enyl chains showed that the deoxyribonucleic acid homology groups I (Clostridium butyricum) and II (Clostridium beijerinckii) could be distinguished by their lipid compositions. The phospholipids of C. butyricum strains had ethanolamine as the major nitrogenous lipid polar head-group moiety, more octadecenoate plus C19-cyclopropane than hexadecenoate plus C17-cyclopropane acyl chains, and the predominant alk-l-enyl chain was C18-monounsaturated. Clostridium beijerinckii strains had N-methylethanolamine plus ethanolamine in phospholipid head-groups, more hexadecenoate plus C17-cyclopropane than octadecenoate plus C19-cyclopropane acyl chains, and the major alk-l-enyl chain was C16-saturated. Three species falling outside the two homology groups Clostridium fallax, Clostridium pseudofallax and Clostridium acetobutylicum had ethanolamine as the major phospholipid base, but these species could be distinguished from C. butyricum by their acyl and alk-l-enyl chain compositions. The lipid composition of Clostridium pasteurianum is even more distinct.
Three of the major phospholipids of the cell membrane of Clostridium butyricum are phosphatidylethanolamine (PE), plasmenylethanolamine (PlaE), and the glycerol acetal of plasmenylethanolamine. When cultured in the absence of biotin in media supplemented with a cis-unsaturated fatty acid, the cellular lipids become highly enriched with the fed fatty acid. Under these conditions, the ratio of the glycerol acetal of PlaE to the sum of PE plus PlaE increases markedly over that seen in cells containing mixtures of saturated and unsaturated fatty acids [Johnston, N.C., & Goldfine, H. (1985) Biochim. Biophys. Acta 813, 10-18]. We have studied the polymorphic phase behavior of the phospholipids from C. butyricum grown on oleic acid using differential scanning calorimetry, 31P nuclear magnetic resonance, and X-ray diffraction. The mixed PE plus PlaE fraction undergoes a transition from the gel to liquid-crystalline state at -1.9 degrees C and a lamellar to reversed hexagonal (L----H) transition at or near 0 degrees C. The glycerol acetal of PlaE melts at 16.1 degrees C, and as predicted from lipid packing theory, the lamellar phase is stabilized, up to 50 degrees C. Addition of the oleate-enriched glycerol acetal of PlaE to dioleoylphosphatidylethanolamine, or the PE plus PlaE fraction from oleate-grown cells, stabilized the lamellar arrangement of the mixtures. A ratio of glycerol acetal of PlaE to total PE (PE plus PlaE) of 0.5, which is close to that found in cells grown on palmitic plus oleic acid, 0.6-0.7, did not produce a lamellar phase at 37 degrees C when the lipids enriched with oleic acid were tested,(ABSTRACT TRUNCATED AT 250 WORDS)
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