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.

Familial hypercholesterolemia results from mutations in the low-density lipoprotein (LDL) receptor or apolipoprotein B genes. We have previously reported the identification of a Utah autosomal-dominant hypercholesterolemia pedigree (kindred 1173) that did not show linkage to either of these loci (Hunt et al. 2000). Expansion of the pedigree and increased marker density within the region of interest have resulted in a multipoint LOD score of 9.6 and enabled us to decrease the size of the linked region to approximately 7.5 Mbp. In addition, we were able to identify additional families sharing the same microsatellite haplotype. While all haplotype carriers in kindred 1173 (K1173) are affected, the haplotype carriers within the newly identified families are unaffected, suggesting that the causal mutation in K1173 had occurred after divergence of these pedigrees from a common ancestor. Mutation screening of genes in the region identified a single nucleotide variant (G-->T) present on the K1173 haplotype that was not present on the same haplotype in the other kindreds. This variant results in a D374Y missense change in the gene PCSK9.

A number of bacterial species secrete phosphatidylinositol-specific phospholipase C (PI-PLC). In this report, we show that the facultative intracellular bacterial pathogen, Listeria monocytogenes, contains a gene, plcA, predicting a polypeptide with 31% amino acid identity to a Bacillus thuringiensis PI-PLC. Accordingly, L. monocytogenes secretes PI-PLC activity, while a mutant with a transposon insertion in plcA lacks detectable PI-PLC activity. In addition, expression of plcA in B. subtilis resulted in secretion of PI-PLC activity. The L. monocytogenes PI-PLC-defective mutant was three logs less virulent for mice and failed to grow in host tissues. The mutant was also defective for in vitro growth in mouse peritoneal macrophages. These results strongly suggest that PI-PLC is an essential determinant of L. monocytogenes pathogenesis. Whether the PI-PLC acts on a bacterial or host substrate remains to be determined.

Listeria monocytogenes is a facultative intracellular bacterial pathogen that spreads cell to cell without exposure to the extracellular environment. Bacterial cell-to-cell spread is mediated in part by two secreted bacterial phospholipases C (PLC), a broad spectrum PLC (PC-PLC) and a phosphatidylinositolspecific PLC (PI-PLC). PI-PLC is secreted in an active state, whereas PC-PLC is secreted as an inactive proenzyme (proPC-PLC) whose activation is mediated in vitro by an L. monocytogenes metalloprotease (Mpl). Analysis of PI-PLC, PC-PLC, and Mpl single and double mutants revealed that Mpl also plays a role in the spread of an infection, but suggested that proPC-PLC has an Mpl-independent activation pathway. Using biochemical and microscopic approaches, we describe three intracellular proteolytic pathways regulating PCPLC activity. Initially, proPC-PLC secreted in the cytosol of infected cells was rapidly degraded in a proteasome-dependent manner. Later during infection, PCPLC colocalized with bacteria in lysosome-associated membrane protein 1–positive vacuoles. Activation of proPC-PLC in vacuoles was mediated by Mpl and an Mpl-independent pathway, the latter being sensitive to inhibitors of cysteine proteases. Lastly, proPC-PLC activation by either pathway was sensitive to bafilomycin A1, a specific inhibitor of vacuolar ATPase, suggesting that activation was dependent on acidification of the vacuolar compartment. These results are consistent with a model in which proPC-PLC activation is compartment specific and controlled by a combination of bacterial and host factors.

Plasmalogens, 1-O-alk-1'-enyl 2-acyl glycerol phospholipids and glycolipids, seem to have evolved first in anaerobic bacteria, but they did not persist when facultative and aerobic species appeared after the concentration of oxygen increased in the early earth's history. Later, when aerobic animal cells appeared with their mitochondria and other intracellular organelles, plasmalogen biosynthesis requiring molecular oxygen, reappeared. The possible reasons for the disappearance and reappearance of plasmalogens in the evolution of life on earth are discussed. The sensitivity of plasmalogens to reactive oxygen species may have caused their disappearance when respiration first evolved. Special features of plasmalogen structure and the resulting lipid packing may account for their reappearance.

The composition of the cell envelope of a heptose-deficient lipopolysaccharide mutant of Escherichia coli, GR467, was studied after fractionation into its outer and cytoplasmic membrane components by means of sucrose density gradient centrifugation. The outer membrane of GR467 had a lower density than that of its parent strain, CR34. Analysis of the fractionated membranes of GR467 indicated that the phospholipid-to-protein ratio had increased 2.4-fold in the outer membrane. The ratio in the mutant cytoplasmic membrane was also increased, although to a lesser extent. By employing a third parameter, the lipid A content of the outer membrane, it was found that the observed phospholipidto-protein change in the outer membrane was due predominantly to a decrease in the relative amount of protein. This decrease in protein was particularly significant, since it was concomitant with a 68% decrease in the lipid A recovered in the outer membrane of GR467 relative to the lipid A recovered in the outer membrane of CR34. Similar findings were observed in a second heptose-deficient mutant of E. coli, RC-59. The apparent protein deficiency in GR467 was further studied by subjecting solubilized envelope proteins to sodium dodecyl sulfatepolyacrylamide gel electrophoresis. It was found that major envelope proteins which were localized in the outer membrane were greatly diminished in GR467. Two revertants of GR467 with the wild-type amounts of heptose had wild-type relative levels of protein in their outer membranes. A partial heptose revertant had a relative level of protein in its outer membrane between those of the mutant and wild type. The organization of the gram-negative bacterial cell envelope may be considered on a number of levels: (i) the chemical composition and linear structure of the major macromolecules, e.g., lipopolysaccharide, lipoprotein, protein, phospholipid, and peptidoglycan; (ii) the stereochemistry of these macromolecules; (iii) the cross-linking of repeating units of a given macromolecule; and (iv) the bonds linking different macromolecules. These factors all contribute to the formation of a multilayered structure which supports such phenomena as active transport of small molecules, enzymatic reactions, and, indeed, the growth of existing envelope structures involving translocation of preformed subunits or in situ synthesis. The cell envelope is generally thought to consist of three distinct layers-the outermost layer containing lipopolysaccharide (LPS), protein, and phospholipid, a rigid peptidoglycan layer, and the cytoplasmic membrane consisting mainly of protein and phospholipid (7, 22, 24, 28, 37, 44). Extensive literature exists describing the chemical composition and linear structure of the LPS in gram-negative bacteria. A large portion of the information available has been derived from analyses of Salmonella strains (19). These analyses indicate that the LPS structure can be divided into three regions, designated as 0-antigen polysaccharide, core polysaccharide, and lipid A. The 0-antigen sugars, wh...

No abstract

Listeriolysin O (LLO) and a phosphatidylinositol-specific phospholipase C (PI-PLC) are known virulence factors of Listeria monocytogenes in both tissue cultures and the murine model of infection. LLO is a member of a family of pore-forming cholesterol-dependent cytotoxins and is known to play an essential role in escape from the primary phagocytic vacuole of macrophages. PI-PLC plays an accessory role, in that PI-PLC mutants are partially defective in escape. We have shown that both of these molecules are essential for initiating rapid increases in the calcium level in the J774 murine macrophage cell line (S. J. Wadsworth and H. Goldfine, Infect. Immun. 67:1770-1778, 1999). Here we show that both LLO and PI-PLC are required for translocation of protein kinase C ␦ (PKC ␦) to the periphery of J774 cells and for translocation of PKC ␤ II to early endosomes beginning within the first minute after addition of bacteria to the culture medium. Treatment with the calcium channel blocker SK&F 96365 inhibited translocation of PKC ␤ II but not PKC ␦. Our findings lead us to propose a host signaling pathway requiring LLO and the formation of diacylglycerol by PI-PLC in which calcium-independent PKC ␦ is responsible for the initial calcium signal and the subsequent PKC ␤ II translocation. LLO-dependent translocation of PKC ␤ I to early endosomes also occurs between 1 and 4 min after infection, but this occurs in the absence of PI-PLC. All of these signals were observed in cells that had not internalized bacteria. Blocking PKC ␤ translocation with hispidin resulted in more rapid uptake of wild-type bacteria and greatly reduced escape from the primary phagocytic vacuoles of J774 cells.The capacity to survive and grow within macrophages is a hallmark of infections with Listeria monocytogenes. Our recent studies have focused on the earliest stages of the encounter between L. monocytogenes and a macrophage, as represented by the J774 murine macrophage cell line, a well-studied tissue culture model of infection (5,30,33). We have observed that the cytosolic calcium level is elevated at 1, 5, and 10 min after infection with wild-type L. monocytogenes but not after infection with a strain with a listeriolysin O (LLO) mutation. Strains with deletions in the genes encoding two secreted phospholipases C did not produce some or all of these signals (35).Of specific interest to workers in our laboratory are signal transduction pathways activated by the two phospholipases that contribute significantly to virulence in the mouse model of infection (5, 30). One of these, a phosphatidylinositol (PI)-specific phospholipase C (PI-PLC), encoded by plcA, hydrolyzes host PI, leading to production of the eukaryotic signaling molecule diacylglycerol (DAG) (4,12,23,30). The other, a broad-range phospholipase C (BR-PLC), encoded by plcB, acts on many host polar lipids, including sphingomyelin, leading to production of DAG and ceramide (10, 11). The elevated calcium levels produced by the combined actions of LLO and PI-PLC appear to be part of a signali...