Listeria monocytogenes phosphatidylinositol (PI)-specific phospholipase C has low activity on glycosyl-PI-anchored proteins

Gandhi, Avani; Perussia, Bice; Goldfine, Howard

doi:10.1128/jb.175.24.8014-8017.1993

Cited by 32 publications

(25 citation statements)

References 27 publications

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“…Our data suggest that LmPI-PLC has evolved as an essential determinant of L. monocytogenes pathogenesis by loss or absence of the Vb ␤-strand needed for efficient GPI-anchor hydrolysis. As predicted from a structural comparison, the absence of the Vb ␤-strand leads to greatly reduced activity on GPI anchors (8,10). There are hundreds of diverse GPI-anchored proteins on eukaryotic cell surfaces, some of which are important for innate immunity and intracellular signaling (26).…”

Section: Discussionmentioning

confidence: 99%

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Listeria monocytogenes phosphatidylinositol-specific phospholipase C has evolved for virulence by greatly reduced activity on GPI anchors

Wei

Zenewicz

Goldfine

2005

Proc. Natl. Acad. Sci. U.S.A.

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Listeria monocytogenes phosphatidylinositol-specific phospholipase C (PI-PLC) plays a critical role in escape of this human pathogen from host cell vacuoles. Unlike classical bacterial PI-PLCs, the L. monocytogenes enzyme has very weak activity on glycosylphosphatidylinositol (GPI)-anchored proteins. Previous crystal structure analysis has revealed that a small ␤-strand (Vb) is present in Bacillus cereus PI-PLC and is absent in the enzyme from L. monocytogenes. This Vb ␤-strand in B. cereus PI-PLC forms contacts with the glycan linker of GPI anchors, which presumably increases its activity on GPI-anchored proteins. In this study, we show that, of all known bacterial PI-PLCs, those from listeriae are the only ones that lack the ␤-strand. Expression by L. monocytogenes of B. cereus PI-PLC, which has strong activity on GPI-anchored proteins, inhibited bacterial escape from a vacuole and cell-to-cell spread, resulting in greatly reduced virulence in mice. Deletion of the Vb ␤-strand from B. cereus PI-PLC abolished its ability to cleave GPI-anchored proteins, decreased its inhibitory effects, and increased its virulence in mice. These results strongly suggest that L. monocytogenes PI-PLC has evolved as an important determinant of L. monocytogenes pathogenesis by absence of the Vb ␤-strand, thus leading to greatly reduced activity on GPI-anchored proteins. glycosylphosphatidylinositol-anchored proteins

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Section: Discussionmentioning

confidence: 99%

“…LmPI-PLC (encoded by plcA) is comparable with Bacillus thuringiensis PI-PLC (BtPI-PLC) and Bacillus cereus PI-PLC (BcPI-PLC) in its ability to cleave PI; however, its activity on GPI-anchored proteins is much lower (8,9). LmPI-PLC is an ortholog of BcPI-PLC, but their primary sequences are quite different, with 29% amino acid identity and 42% similarity.…”

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confidence: 99%

Listeria monocytogenes phosphatidylinositol-specific phospholipase C has evolved for virulence by greatly reduced activity on GPI anchors

Wei

Zenewicz

Goldfine

2005

Proc. Natl. Acad. Sci. U.S.A.

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“…Like other bacterial PI-PLCs, the enzyme from L. monocytogenes has high specificity for PI with no detectable activity on PI-4-P or PI-4,5-P 2 , eukaryotic lipids involved in intracellular signaling. It has relatively low activity on glycosyl-PI-anchored eukaryotic membrane proteins, which are actively cleaved by other bacterial PI-PLCs (14,16).…”

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confidence: 99%

Activation of Host Phospholipases C and D in Macrophages after Infection withListeria monocytogenes

2000

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The earliest events in the interaction of Listeria monocytogenes with mammalian cells appear to involve the activities of bacterial secreted proteins before internalization of these bacteria. On infection of the J774 murine macrophage cell line, these activities delay uptake of wild-type bacteria into the phagosome (36). Subsequent growth in the cytoplasm and cellto-cell spread are completely dependent on the ability of the bacterium to mediate escape from a vacuole (12,26,35). Two genes, hly and plcA, in a cluster of six genes on the bacterial chromosome have been implicated in escape from the primary vacuole of a macrophage. They encode listeriolysin O (LLO) and a phosphatidylinositol-specific phospholipase C (PI-PLC), respectively. A third gene, prfA, which is adjacent to plcA, encodes a positive regulatory protein, PrfA, which is required for the induction of all genes in this virulence cluster (25,29). LLO has been shown to be absolutely required for escape from the primary vacuole of a macrophage (12, 26) and for mouse virulence (9,13,18,26,29). Assays for escape from the primary vacuole show that mutants in PI-PLC are between 30 and 65% less likely to be found in the cytoplasm of a bone marrowderived macrophage than a wild-type strain at 1.5 h postinfection (7, 33). These mutants show reduced growth compared to the wild type in mouse liver; however, the mouse 50% lethal dose is only slightly increased upon intravenous infection (7).PI-PLC of L. monocytogenes is a member of a family of homologous enzymes secreted by gram-positive bacteria. Like other bacterial PI-PLCs, the enzyme from L. monocytogenes has high specificity for PI with no detectable activity on PI-4-P or PI-4,5-P 2 , eukaryotic lipids involved in intracellular signaling. It has relatively low activity on glycosyl-PI-anchored eukaryotic membrane proteins, which are actively cleaved by other bacterial PI-PLCs (14, 16).The ability of L. monocytogenes to escape from a phagosome, grow in the cytoplasm, and spread from cell to cell is essential for the pathogenesis of this food-borne, human and animal pathogen. In humans, infections with L. monocytogenes tend to occur in immunocompromised adults, pregnant women, and the elderly. They can produce septic abortions of the fetus and meningoencephalitis and are often fatal (11,28).Since bacterial LLO and PI-PLC activities appear to be important for elevation of intracellular Ca 2ϩ in host cells (36), it seemed possible that there is a connection between escape from the vacuole and activation of certain host cell functions that are dependent on elevated intracellular Ca 2ϩ . Among these is the activation of host PLC isoforms, which hydrolyze PI-4-P and PI-4,5-P 2 (27, 31). The hydrolysis of host phosphoinositides by bacterial and host PLCs also results in the formation of diacylglycerol (DAG), which is an activator of eukaryotic protein kinase C (PKC) isoforms (24). Activation of the classical isoforms of PKC also requires elevated intracellular Ca 2ϩ . Since PKC has been implicated in activation of pho...

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“…L. monocytogenes expressing B. anthracis PI-PLC was able to form plaques in L2 monolayers, but the plaque size was significantly reduced compared to both strain Lmdd and a strain expressing inactive L. monocytogenes PI-PLC (Table 2). A comparison of their crystal structures has shown that L. monocytogenes PI-PLC and B. cereus PI-PLC have similar molecular structures; however, B. cereus PI-PLC has an extra ␤-strand (Vb) which is thought to be needed for recognition of GPI anchors (9,23). B. anthracis PI-PLC has 97% similarity to B. cereus PI-PLC, and both have exactly the same Vb ␤-strand amino acid sequence.…”

Section: Discussionmentioning

confidence: 99%

Characterization ofListeria monocytogenesExpressing Anthrolysin O and Phosphatidylinositol-Specific Phospholipase C fromBacillus anthracis

et al. 2005

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Two virulence factors of Listeria monocytogenes, listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC), mediate escape of this pathogen from the phagocytic vacuole of macrophages, thereby allowing the bacterium access to the host cell cytosol for growth and spread to neighboring cells. We characterized their orthologs from Bacillus anthracis by expressing them in L. monocytogenes and characterizing their contribution to bacterial intracellular growth and cell-to-cell spread. We generated a series of L. monocytogenes strains expressing B. anthracis anthrolysin O (ALO) and PI-PLC in place of LLO and L. monocytogenes PI-PLC, respectively. We found that ALO was active at both acidic and neutral pH and could functionally replace LLO in mediating escape from a primary vacuole; however, ALO exerted a toxic effect on the host cell by damaging the plasma membrane. Listeria monocytogenes and Bacillus anthracis are gram-positive bacteria in the low-GϩC lineage that cause rare but fatal infections of humans and animals. Although L. monocytogenes has been studied as a model facultative intracellular pathogen for decades, B. anthracis had, until recently, received less attention. Interestingly, the B. anthracis genome sequence has revealed the presence of a number of potential determinants of pathogenesis shared with those of L. monocytogenes, including a phosphatidylinositol-specific phospholipase C (PI-PLC), a phosphatidylcholine-preferring phospholipase C (PC-PLC), and listeriolysin O (LLO) (27). In L. monocytogenes, these determinants are integral to its intracellular growth cycle. LLO is essential for escape from a phagosome, the phospholipases contribute to that process, and all three are important for cell-to-cell spread (3, 10, 27, 34). Although B. anthracis is traditionally considered an extracellular pathogen, there is considerable evidence that spores germinate and grow in macrophages during the early phase of infection (8, 30). We expressed here the B. anthracis orthologs of L. monocytogenes LLO and PI-PLC in L. monocytogenes in order to characterize them in a well-studied model of intracellular pathogenesis. MATERIALS AND METHODSBacterial strains and growth conditions. The L. monocytogenes strains used in the present study are listed in Table 1. Unless otherwise specified, all bacterial strains were grown in brain heart infusion broth, and strains derived from L. monocytogenes ⌬dal ⌬dat (Lmdd) were supplemented with 100 g of D-alanine/ml to stationary phase at 30°C. Strains containing pKSV7 were selected and maintained with ampicillin (50 g/ml) in Escherichia coli and chloramphenicol (10 g/ml) in L. monocytogenes.Tissue-culture cells and growth media. The mouse macrophage-like cell line J774 and murine L2 fibroblasts were grown in Dulbecco modified Eagle medium plus 7.5% fetal bovine serum (FBS) and 1 mM L-glutamine. Bone marrowderived macrophages obtained from C57BL/6 mice were cultured in RPMI 1640-10% FBS-1 mM L-glutamine. All were incubated at 37°C with 5% CO 2 . For bacterial infec...

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Listeria monocytogenes phosphatidylinositol (PI)-specific phospholipase C has low activity on glycosyl-PI-anchored proteins

Cited by 32 publications

References 27 publications

Listeria monocytogenes phosphatidylinositol-specific phospholipase C has evolved for virulence by greatly reduced activity on GPI anchors

Listeria monocytogenes phosphatidylinositol-specific phospholipase C has evolved for virulence by greatly reduced activity on GPI anchors

Activation of Host Phospholipases C and D in Macrophages after Infection withListeria monocytogenes

Characterization ofListeria monocytogenesExpressing Anthrolysin O and Phosphatidylinositol-Specific Phospholipase C fromBacillus anthracis

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