Howard Goldfine

Comparative aspects of bacterial lipids.

Publisher Summary This chapter deals with the comparative aspects of bacterial lipids. The chapter attempts to outline their differences, to describe the biosynthetic systems—the presence or absence of which lead to the observed compositions, and to assess the significance of these differcnces. Bacterial lipids have been discussed extensively in monographs. Research on bacterial lipids focuses on such questions as their location in functional membrane units, the relationship of individual lipid species to vitrous enzymes, multi-enzyme complexes, and transport systems. The regulation of membrane–lipid biosynthesis at the enzyme level is receiving more attention in bacteria, and the isolation of a variety of mutants with lesions in the synthesis of lipids promises to lead to a better understanding of the genetics and control of lipid biosynthesis. It has long been recognized that cell diversity among the bacteria tends to increase toward the periphery of the cell, and the membrane lipids clearly offer a fertile ground for the study of comparitive biochemistry.

Lipids of Prokaryotes–Structure and Distribution

Publisher Summary This chapter reviews the structures of the lipids of prokaryotes and the distribution of these lipids in various groups of organisms. It discusses the arrangement of the prokaryotes in the eighth edition of Bergeys Manual of Determinative Bacteriology . The presence of acyl and alk-1-enyl chains containing double bonds, methyl branches, or small rings is critical for the maintenance of membrane fluidity that is important for the function of membrane enzymes and transport systems. Variation in polar head groups may also affect membrane fluidity and the association of certain lipid classes with these enzymes and transport systems. The chapter discusses the location of membrane lipids with respect to intracellular membranes. Lipids are often distributed asymmetrically in the two leaflets of a given membrane. Some organisms have evolved dual lipid-biosynthetic capacities that allow them to substitute glycosyl diglycerides or ornithine lipids for phosphatidylethanolamine in the absence of phosphate. Within limits of size and charge, polar lipids may replace one another in biological membranes. Exogenous fatty acyl chains and alk-1-enyl chains may also be substituted for the naturally occurring chains in bacterial auxotrophs. Much more work on membrane mutants is needed to understand the multifaceted roles of prokaryotic lipids. Work on prokaryotic organisms has not only provided an abundance of new insights into their membrane structure and functions, it is also leading the way to a more complete understanding of eukaryotic cell membranes.

Mobilization of Protein Kinase C in Macrophages Induced by Listeria monocytogenes Affects Its Internalization and Escape from the Phagosome

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 δ (PKC δ) to the periphery of J774 cells and for translocation of PKC β 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 β II but not PKC δ. 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 δ is responsible for the initial calcium signal and the subsequent PKC β II translocation. LLO-dependent translocation of PKC β 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 β 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 transfer of choline from the host to the bacterial cell surface requires glpQ in Haemophilus influenzae.

Haemophilus influenzae incorporates choline obtained from environmental sources onto its lipopolysaccharide as phosphorylcholine (ChoP). The decoration of the bacterial surface with ChoP contributes to pathogenesis by allowing for mimicry of the host. As the main reservoir for choline in the host is phosphatidylcholine, we tested whether other choline‐containing molecules associated with eukaryotic membranes could provide an alternative source of choline. H. influenzae was able to use glycerophosphorylcholine (GPC), an abundant degradation product of phospholipids, as efficiently as free choline. Utilization of GPC required glpQ, which expresses an enzyme with glycerophosphodiester phosphodiesterase activity. In the absence of free choline, this gene was required for adherent H. influenzae to obtain choline directly from epithelial cells in culture. GlpQ therefore allows choline to be transferred from the host to the bacterial cell surface.

Lipid Composition in the Classification of the Butyric Acid-producing Clostridia

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.

Macrophage intracellular signaling induced by Listeria monocytogenes.

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.

A Complex Extracellular Sphingomyelinase of Pseudomonas aeruginosa Inhibits Angiogenesis by Selective Cytotoxicity to Endothelial Cells

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 angiogenesis (i.e., formation of new blood vessels from existing vasculature). Finally, the impact of PlcHR in an in vivo model of angiogenesis in transgenic zebrafish, and ones treated with an antisense morpholino to knock down a key blood cell regulator, were evaluated because in vitro assays cannot fully represent the complex processes of angiogenesis. As little as 2 ng/embryo of PlcHR was lethal to ∼50% of EGFP-labeled EC at 6 h after injection of embryos at 48 hpf (hours post-fertilization). An active site mutant of PlcHR (Thr178Ala) exhibited 120-fold reduced inhibitory activity in the EC invasion assay, and 20 ng/embryo elicited no detectable inhibitory activity in the zebrafish model. Taken together, these observations are pertinent to the distinctive vasculitis and poor wound healing associated with P. aeruginosa sepsis and suggest that the potent antiangiogenic properties of PlcHR are worthy of further investigation for the treatment of diseases where angiogenesis contributes pathological conditions (e.g., vascularization of tumors, diabetic retinopathy).

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

ABSTRACT Infection of the J774 murine macrophage-derived cell line withListeria 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 broad-range 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 marrow-derived 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 δ in J774 cells, also inhibited the activation of PLD, but hispidin, an inhibitor of PKC βI and βII, did not. Pretreatment of J774 cells with the PLD inhibitor, 2,3-diphosphoglycerate partially inhibited escape of the bacteria from the primary phagocytic vacuole.

Ornithine lipid is required for optimal steady‐state amounts of c‐type cytochromes in Rhodobacter capsulatus

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 post‐translational 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.

Listeria monocytogenes : pathogenesis and host response

A 20-Year Perspective on Listeria monocytogenes Pathogenesis.- Listeriosis.- Listeria Genomics.- Metabolism and Physiology of Listeria monocytogenes.- The Cell Wall of Listeria monocytogenes and its Role in Pathogenicity.- Environmental Reservoir and Transmission into the Mammalian Host.- Regulation of Listeria monocytogenes Virulence Genes.- Invasion of Host Cells by Listeria monocytogenes.- Escape of Listeria monocytogenes from a Vacuole.- Actin-Based Motility and Cell-to-Cell Spread of Listeria monocytogenes.- Adaptive Immunity to Listeria monocytogenes.- Immune Evasion and Modulation by Listeria monocytogenes.- Bacteriophages of Listeria.