Papers by Howard Goldfine
Infection and immunity, 1999
Listeria monocytogenes secretes several proteins that have been shown to contribute to virulence.... more Listeria monocytogenes secretes several proteins that have been shown to contribute to virulence. Among these is listeriolysin O (LLO), a pore-forming hemolysin that is absolutely required for virulence. Two other virulence factors are phospholipases: a phosphatidylinositol-specific phospholipase C (PI-PLC [plcA]) and a broad-range PLC (plcB). Although mutations in plcA or plcB resulted in small increases in mouse 50% lethal dose (LD50), deletions in both genes resulted in a 500-fold increase in LD50. We have examined the role of these secreted proteins in host intracellular signaling in the J774 macrophage-like cell line. Measurements of cytosolic free calcium ([Ca2+]i) have revealed a rapid spike upon exposure of these cells to wild-type L. monocytogenes. This is followed by a second peak at 5 min and a third prolonged peak with a maximal [Ca2+]i of 800 to 1,000 nM. The pattern of calcium changes was greatly altered by deletion of any of the three virulence factors. An LLO mutant ...
Infection and immunity, 1999
Listeria monocytogenes, a gram-positive facultative intracellular pathogen, produces two distinct... more Listeria monocytogenes, a gram-positive facultative intracellular pathogen, produces two distinct phospholipases C. PC-PLC, encoded by plcB, is a broad-range phospholipase, whereas PI-PLC, encoded by plcA, is specific for phosphatidylinositol. It was previously shown that PI-PLC plays a role in efficient escape of L. monocytogenes from the primary phagosome. To further understand the function of PI-PLC in intracellular growth, site-directed mutagenesis of plcA was performed. Two potential active-site histidine residues were mutated independently to alanine, serine, and phenylalanine. With the exception of the activity of the enzyme containing H38F, which was unstable, the PI-PLC enzyme activities of culture supernatants containing each mutant enzyme were <1% of wild-type activity. In addition, the levels of expression of the mutant PI-PLC proteins were equivalent to wild-type expression. Derivatives of L. monocytogenes containing these specific plcA mutations were found to have p...
Infection and immunity, 1998
The secreted broad-range phosphatidylcholine (PC)-preferring phospholipase C (PC-PLC) of Listeria... more The secreted broad-range phosphatidylcholine (PC)-preferring phospholipase C (PC-PLC) of Listeria monocytogenes plays a role in the bacterium's ability to escape from phagosomes and spread from cell to cell. Based on comparisons with two orthologs, Clostridium perfringens alpha-toxin and Bacillus cereus PLC (PLCBc), we generated PC-PLC mutants with altered enzymatic activities and substrate specificities and analyzed them for biological function in tissue culture and mouse models of infection. Two of the conserved active-site zinc-coordinating histidines were confirmed by single amino acid substitutions H69G and H118G, which resulted in proteins inactive in broth culture and unstable intracellularly. Substitutions D4E and H56Y remodeled the PC-PLC active site to more closely resemble the PLCBc active site, while a gene replacement resulted in L. monocytogenes secreting PLCBc. All of these mutants yielded similar amounts of active enzyme as wild-type PC-PLC both in broth culture ...
Journal of bacteriology, 1997
The sn-glycerol-3-phosphate acyltransferase (plsB) of Escherichia coli is a key regulatory enzyme... more The sn-glycerol-3-phosphate acyltransferase (plsB) of Escherichia coli is a key regulatory enzyme that catalyzes the first committed step in phospholipid biosynthesis. We report the initial characterization of a novel gene (termed plsD) from Clostridium butyricum, cloned based on its ability to complement the sn-glycerol-3-phosphate auxotrophic phenotype of a plsB mutant strain of E. coli. Unlike the 83-kDa PlsB acyltransferase from E. coli, the predicted plsD open reading frame encoded a protein of 26.5 kDa. Two regions of strong homology to other lipid acyltransferases, including PlsB and PlsC analogs from mammals, plants, yeast, and bacteria, were identified. PlsD was most closely related to the 1-acyl-sn-glycerol-3-phosphate acyltransferase (plsC) gene family but did not complement the growth of plsC(Ts) mutants. An in vivo metabolic labeling experiment using a plsB plsX plsC(Ts) strain of E. coli confirmed that the plsD expression restored the ability of the cells to synthesize...
Science (New York, N.Y.), Jan 25, 1966
Examination of the lipids of three species of nonphotosynthetic bacteria with extensive internal ... more Examination of the lipids of three species of nonphotosynthetic bacteria with extensive internal membranes revealed phosphatidyl choline (lecithin) in two species. In one of these there was an unusual accumulation of phosphatidyl N-dimethylethanolamine. (The relation between lecithin and membrane elaboration in microorganisms is discussed.)
Journal of lipid research, 1985
Phosphatidylserine decarboxylase activity has been characterized in membrane preparations from Cl... more Phosphatidylserine decarboxylase activity has been characterized in membrane preparations from Clostridium butyricum ATCC 19398. A particulate fraction was shown to catalyze the formation of phosphatidylethanolamine and plasmenylethanolamine when vesicles containing phosphatidylserine and plasmenylserine were used as substrate. No plasmenylethanolamine was formed when phosphatidylserine alone was used as substrate. The activity with phosphatidylserine was activated by divalent cations and was optimal under anaerobic conditions. Ionic detergents inhibited phosphatidylethanolamine formation strongly and nonionic detergents inhibited partially. In the presence of Triton X-100, phosphate from [32P]phosphatidylserine appeared in three unidentified lipid products, in addition to phosphatidylethanolamine. The formation of these products was time- and Triton X-100 concentration-dependent. Hydroxylamine inhibited phosphatidylserine decarboxylase, but did not prevent the reactions stimulated ...
Listeria monocytogenes: Pathogenesis and Host Response, 2007
The ability of Listeria monocytogenes to escape from vacuoles of infected cells and subsequently ... more The ability of Listeria monocytogenes to escape from vacuoles of infected cells and subsequently to replicate in the cytosol and spread from cell to cell is one of the distinctive features of this facultative intracellular pathogen. The process of escape is mediated by several proteins that are encoded by genes in the PrfA regulon cluster. These include listeriolysin O, a
Summary A number of bacterial species secrete phosphatidylinositol-specific phospholipase C (PI-P... more Summary 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, pkA, predicting a polypeptide with 31% amino acid identity to a Bacillus thuringiewis PI-PLC . Accordingly, L. monocytogenes secretes PI-PLC activity, while a mutant with a transposon insertion in p1cA lacks detectable
Lipids, 1983
We have studied the phospholipid composition ofRhizobium meliloti strains which do or do not cont... more We have studied the phospholipid composition ofRhizobium meliloti strains which do or do not contain the large, tumor-inducing (Ti) plasmid ofAgrobacterium tumefaciens. The major phospholipids of stationary phase cells were phosphatidylethanolamine (PE) (22%), phosphatidyl-N-methylethanolamine (22%), phosphatidylcholine (PC) (27%), phosphatidylglycerol (11.4%), and cardiolipin (11%); as average percent of lipid phosphorus. Phosphatidyl-N,N-dimethylethanolamine (3.7%) was also present. The proportions of PE were higher,
PLoS pathogens, 2009
The hemolytic phospholipase C (PlcHR) expressed by Pseudomonas aeruginosa is the original member ... more The hemolytic phospholipase C (PlcHR) expressed by Pseudomonas aeruginosa is the original member of a Phosphoesterase Superfamily, which includes phosphorylcholine-specific phospholipases C (PC-PLC) produced by frank and opportunistic pathogens. PlcHR, but not all its family members, is also a potent sphingomyelinase (SMase). Data presented herein indicate that picomolar (pM) concentrations of PlcHR are selectively lethal to endothelial cells (EC). An RGD motif of PlcHR contributes to this selectivity. Peptides containing an RGD motif (i.e., GRGDS), but not control peptides (i.e., GDGRS), block the effects of PlcHR on calcium signaling and cytotoxicity to EC. Moreover, RGD variants of PlcHR (e.g., RGE, KGD) are significantly reduced in their binding and toxicity, but retain the enzymatic activity of the wild type PlcHR. PlcHR also inhibits several EC-dependent in vitro assays (i.e., EC migration, EC invasion, and EC tubule formation), which represent key processes involved in angiog...
Proceedings of the National Academy of Sciences, 1995
We have examined potential cooperative interactions of Listeria monocytogenes phosphatidylinosito... more We have examined potential cooperative interactions of Listeria monocytogenes phosphatidylinositol-specific phospholipase C (PI-PLC) and listeriolysin O (LLO), a pore-forming hemolysin, in a liposome lysis assay. Large unilamellar vesicles, approximately 0.1 micron in diameter, encapsulating the fluorescent probe calcein, were treated with PI-PLC or LLO at pH 6.0, and each was capable of causing dye release. With phosphatidylcholine/phosphatidylinositol/cholesterol liposomes at 0.1 microM lipid, minimal release of dye was observed on addition of 80 pM LLO or 7 nM PI-PLC. Addition of the two proteins together produced rapid dye release. Unexpectedly, essentially identical results were obtained with phosphatidylcholine/cholesterol liposomes. Thus, the effect of PI-PLC did not depend on lipid hydrolysis. Both proteins also released inulin (M(r) 5200) from liposomes. Membrane permeabilization was not accompanied by membrane fusion. Very little dye release from phosphatidylcholine/phosphatidylinositol/cholesterol liposomes was seen with PI-PLC from Bacillus thuringiensis, and addition of this enzyme to LLO produced no additional dye release; however PI-PLC from L. monocytogenes cooperated with perfringolysin O from Clostridium perfringens. PI-PLC from L. monocytogenes and LLO bind to phosphatidylcholine/cholesterol liposomes, and the rate of binding of each protein was not influenced by the presence of the other. These data support a postulated accessory role for PI-PLC with LLO in lysing the primary phagosome of a macrophage.
Molecular Microbiology, 2006
The c-type cytochromes are haemoproteins that are subunits or physiological partners of electron ... more The c-type cytochromes are haemoproteins that are subunits or physiological partners of electron transport chain components, like the cytochrome bc1 complex or the cbb3-type cytochrome c oxidase. Their haem moieties are covalently attached to the corresponding apocytochromes via a complex posttranslational maturation process. During our studies of cytochrome biogenesis, we uncovered a novel class of mutants that are unable to produce ornithine lipid and that lack several c-type cytochromes. Molecular analyses of these mutants led us to the ornithine lipid biosynthesis genes of Rhodobacter capsulatus. Herein, we have characterized these mutants, and established the chemical structure of this non-phosphorus membrane lipid from R. capsulatus. Ornithine lipids are known to induce potent host immune responses, including B-lymphocyte mitogenicity, adjuvanticity and macrophage activation. Yet, despite their widespread occurrence in Eubacteria, and the diverse biological effects they elicit in mammals, their physiological role in bacterial cells remained hitherto poorly defined. Our findings now indicate that under certain bacterial growth conditions ornithine lipids are crucial for optimal steady-state amounts of some extracytoplasmic proteins, including several c-type cytochromes, and attribute them a novel and important biological function.
Microbiology, 1983
An examination of 20 strains of butyric acid-producing Clostridium species for phospholipid class... more An examination of 20 strains of butyric acid-producing Clostridium species for phospholipid class compositions, plasmalogen content, and acyl and alk-1-enyl chains showed that the deoxyri bonucleic acid homology groups I (Clostridium butyricum) and I1 (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 C ,-cyclopropane than hexadecenoate plus C ,-cyclopropane acyl chains, and the predominant alk-1 -enyl chain was C1 ,-monounsaturated. Clostridium beijerinckii strains had N-methylethanolamine plus ethanolamine in phospholipid head-groups, more hexadecenoate plus C ,-cyclopropane than octadecenoate plus C ,-cyclopropane acyl chains, and the major alk-1-enyl chain was C,,-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-1 -enyl chain compositions. The lipid composition of Clostridium pasteurianum is even more distinct.
Microbiology, 1994
The extractable polar lipids of Clostridium innocuum have been shown to consist of glycosyldirady... more The extractable polar lipids of Clostridium innocuum have been shown to consist of glycosyldiradylglycerols, phospholipids and phosphoglycolipids. The major glycosyldiradylglycerols are ~-Glcp(al-3)radyl,Gro and ~-G a l p ( a l -2 )~-Glcp(al-3)rady12Gro. Both glycolipids have some l-O-(alk-l-enyl)-2-O-acyl species, in addition to diacyl species. The phospholipids include bisphosphatidylglycerol (cardiolipin), lysocardiolipin and phosphatidylglycerol (PG). In addition, several novel lipids have been found, including a PG acetal of cardiolipin plasmalogen, smaller amounts of a lyso form of this lipid, a PG acetal of PG plasmalogen, and two phosphoglycolipids, which represent 65 O/ O of t o t a l polar lipids. The latter have been identified as 2'-amino-1',3'dihydroxypropane-3'-P-6-~-Galp(al-2)~-Glcp(al-3)radyl,Gro and a derivative of this lipid containing a n acyl chain esterified to 0-6 of t h e glucopyranosyl ring. Based on rRNA sequence data, C. innocuum is considered to b e a relative of t h e mycoplasmas. Its unique lipid composition permits a n assessment of t h e taxonomic status of C. innocuum, since t h e lipid amphiphiles display marked differences from those of Acholeplasma laidlawii.
Microbes and Infection, 2002
Macrophages are critical for control of Listeria monocytogenes infections; accordingly, the inter... more Macrophages are critical for control of Listeria monocytogenes infections; accordingly, the interactions of L. monocytogenes with these cells have been intensively studied. It has become apparent that this facultative intracellular pathogen interacts with macrophages both prior to entry and during the intracellular phase. This review covers recent work on signaling induced in macrophages by L. monocytogenes, especially intracellular signals induced by secreted proteins including listeriolysin O and two distinct phospholipases C.
Journal of Biological Chemistry, 2008
Eicosanoid production by macrophages is an early response to microbial infection that promotes ac... more Eicosanoid production by macrophages is an early response to microbial infection that promotes acute inflammation. The intracellular pathogen Listeria monocytogenes stimulates arachidonic acid release and eicosanoid production from resident mouse peritoneal macrophages through activation of group IVA cytosolic phospholipase A2 (cPLA2alpha). The ability of wild type L. monocytogenes (WTLM) to stimulate arachidonic acid release is partially dependent on the virulence factor listeriolysin O; however, WTLM and L. monocytogenes lacking listeriolysin O (DeltahlyLM) induce similar levels of cyclooxygenase 2. Arachidonic acid release requires activation of MAPKs by WTLM and DeltahlyLM. The attenuated release of arachidonic acid that is observed in TLR2-/- and MyD88-/- macrophages infected with WTLM and DeltahlyLM correlates with diminished MAPK activation. WTLM but not DeltahlyLM increases intracellular calcium, which is implicated in regulation of cPLA2alpha. Prostaglandin E2, prostaglandin I2, and leukotriene C4 are produced by cPLA2alpha+/+ but not cPLA2alpha-/- macrophages in response to WTLM and DeltahlyLM. Tumor necrosis factor (TNF)-alpha production is significantly lower in cPLA2alpha+/+ than in cPLA2alpha-/- macrophages infected with WTLM and DeltahlyLM. Treatment of infected cPLA2alpha+/+ macrophages with the cyclooxygenase inhibitor indomethacin increases TNFalpha production to the level produced by cPLA2alpha-/- macrophages implicating prostaglandins in TNFalpha down-regulation. Therefore activation of cPLA2alpha in macrophages may impact immune responses to L. monocytogenes.
Journal of Bacteriology, 2007
The Rhodobacter capsulatus genome contains three genes (olsA [plsC138], plsC316, and plsC3498) th... more The Rhodobacter capsulatus genome contains three genes (olsA [plsC138], plsC316, and plsC3498) that are annotated as lysophosphatidic acid (1-acyl-sn-glycerol-3-phosphate) acyltransferase (AGPAT). Of these genes, olsA was previously shown to be an O-acyltransferase in the second step of ornithine lipid biosynthesis, which is important for optimal steady-state levels of c-type cytochromes (S. Aygun-Sunar, S. Mandaci, H.-G. Koch, I. V. J. Murray, H. Goldfine, and F. Daldal. Mol. Microbiol. 61:418-435, 2006)
Infection and Immunity, 2002
Listeriolysin O (LLO) and a phosphatidylinositol-specific phospholipase C (PI-PLC) are known viru... more 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.
Infection and Immunity, 2000
Infection of the J774 murine macrophage-derived cell line with Listeria monocytogenes results in ... more Infection of the J774 murine macrophage-derived cell line with Listeria monocytogenes results in several elevations of intracellular calcium during the first 15 min of infection. These appear to result from the actions of secreted bacterial proteins, including phosphatidylinositol-specific phospholipase C (PI-PLC), a broadrange phospholipase C, and listeriolysin O (LLO) (S. J. Wadsworth and H. Goldfine, Infect. Immun. 67:1770-1778, 1999). We have measured hydrolysis of host PI and the activation of host polyphosphoinositide-specific PLC and host phospholipase D (PLD) during infection with wild-type and mutant L. monocytogenes. Elevated hydrolysis of host PI occurred within the first 10 min of infection and was dependent on both bacterial PI-PLC and LLO, both of which were required for the earliest elevations of intracellular calcium in the host cell. A more rapid hydrolysis of host PI was observed at 30 min after infection, at the time when wild-type bacteria have been internalized. Activation of host PLC, also occurred in the first 10 min of infection but was not dependent on the presence of bacterial PI-PLC. Similar observations were made in murine bone marrowderived macrophages. In J774 cells, activation of host PLD was observed after 20 min of infection and was dependent on bacterial LLO. Mutants in the bacterial phospholipases produced levels of PLD activation similar to those produced by the wild type. Phorbol myristate acetate (PMA) also activated host PLD, while long-term treatment with PMA resulted in loss of the ability of L. monocytogenes to activate host PLD, suggesting an involvement of protein kinase C (PKC) in the activation of PLD. Rottlerin, an inhibitor of PKC ␦ in J774 cells, also inhibited the activation of PLD, but hispidin, an inhibitor of PKC I and II, did not. Pretreatment of J774 cells with the PLD inhibitor, 2,3-diphosphoglycerate partially inhibited escape of the bacteria from the primary phagocytic vacuole.
Infection and Immunity, 2005
Two virulence factors of Listeria monocytogenes, listeriolysin O (LLO) and phosphatidylinositol-s... more 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. B. anthracis PI-PLC, unlike the L. monocytogenes ortholog, had high activity on glycosylphosphatidylinositol-anchored proteins. L. monocytogenes expressing B. anthracis PI-PLC showed significantly decreased efficiencies of escape from a phagosome and in cell-to-cell spread. We further compared the level of cytotoxicity to host cells by using mutant strains expressing ALO in combination either with L.
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Papers by Howard Goldfine