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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). The roles of the remaining plsC316 and plsC3498 genes remained unknown. In this work, these genes were cloned, and chromosomal insertion-deletion mutations inactivating them were obtained to define their function. Characterization of these mutants indicated that, unlike the Escherichia coli plsC, neither plsC316 nor plsC3498 was essential in R. capsulatus. In contrast, no plsC316 olsA double mutant could be isolated, indicating that an intact copy of either olsA or plsC316 was required for R. capsulatus growth under the conditions tested. Compared to OlsA null mutants, PlsC316 null mutants contained ornithine lipid and had no c-type cytochrome-related phenotype. However, they exhibited slight growth impairment and highly altered total fatty acid and phospholipid profiles. Heterologous expression in an E. coli plsC(Ts) mutant of either R. capsulatus plsC316 or olsA gene products supported growth at a nonpermissive temperature, exhibited AGPAT activity in vitro, and restored phosphatidic acid biosynthesis. The more vigorous AGPAT activity displayed by PlsC316 suggested that plsC316 encodes the main AGPAT required for glycerophospholipid synthesis in R. capsulatus, while olsA acts as an alternative AGPAT that is specific for ornithine lipid synthesis. This study therefore revealed for the first time that some OlsA enzymes, like the enzyme of R. capsulatus, are bifunctional and involved in both membrane ornithine lipid and glycerophospholipid biosynthesis.

The clostridial clone pHH37 was determined by complementation of the plsB defect in mutant strains of Escherichia coli to investigate whether the plasmid expressed a gene that encodes a G3P acyltransferase. The PlsD protein sequences were characterized by a high degree of homology to the PlsC 1-acyl-G3P acyltransferase. However, the plasmid pSJ342 did not complement the 1-acyl-G3P acyltransferase mutation in strain SM2-1.

The study indicate a postulated accessory role for phoshatidylinositol-specific phospholipase C (PI-PLC) with listeriolysin O (LLO) in lysing the primary phagosome of a macrophage. Potential cooperative interactions of Listeria monocytogenes PI-PLC and LLO, a pore-forming hemolysin in a liposome lysis assay was studied. The effect of PI-PLC was not dependent on lipid hydrolysis and both proteins released insulin from liposomes. Membrane fusion did not follow membrane permeabilization.

The taxonomic status of Clostridium innocuum was based on its novel polar lipid composition, as the lipid amphiphiles exhibited differences from those of Acholeplasma laidlawii. The polar lipids of C. innocuum consisted of glycosyldiradylglycerols, phospholipids and phosphoglycolipids. A PG acetyl of cardiolipin plasmalogen was one of the newly found lipids.

The phosphatidylinositol-specific phospholipase C from Listeria monocytogenes has lower activity on glycosyl phosphatidylinositol-anchored proteins than phospholipase C from Bacillus thuringiensis. The L. monocytogenes enzyme have no activity on phosphatidylinositol 4,5-biphosphate and phosphatidylinositol 4-phosphate and acts specifically on phosphatidylinositol.

Phospholipase C (PLC), an enzyme secreted by Listeria monocytogenes, is responsible for the hydrolylsis of phosphatidylethanolamine and phosphatidylglycerol that takes place after the dispersion of Escherichia coli phospholipids in Triton X-100. PCL promotes the hydrolysis of PE, the plasmalogen form of PE, the glycerol acetal of PE and cardiolipin. The hydrolytic activity of cardiolipin is stimulated by I-, Cl- and F- on Pc-Triton X-100 mixed micelles.

The effect of modifying the fatty acid substrates in a biotin free medium on the membrane lipid composition in Clostridium acetobutylicum was examined in order to develop strategies for enhanced solvent resistance. The results showed that C. acetobutylicum grown in the absence of biotin can alter its lipid composition to promote bilayer stability. These changes involve phospholipids, glycolipids of the diglyceride type and phosphoglycolipids of unknown structures. These lipids are regulated in order to provide a relatively constant phase state of the membrane lipids.

Endogenous glutathione (GSH) and glutathione disulfide (GSSG) status is highly sensitive to oxidative conditions and have broad application as a surrogate indicator of redox status in vivo. Established methods for GSH and GSSG quantification in whole blood display limited utility in human plasma, where GSH and GSSG levels are ~3–4 orders of magnitude below those observed in whole blood. This study presents simplified sample processing and analytical LC–MS/MS approaches exhibiting the sensitivity and accuracy required to measure GSH and GSSG concentrations in human plasma samples, which after 5‐fold dilution to suppress matrix interferences range from 200 to 500 nm (GSH) and 5–30 nm (GSSG). The utility of the methods reported herein is demonstrated by assay performance and validation parameters which indicate good sensitivity [lower limits of quantitation of 4.99 nm (GSH) and 3.65 nm (GSSG), and high assay precision (intra‐assay CVs 3.6 and 1.9%, and inter‐assay CVs of 7.0 and 2.8% for GSH and GSSG, respectively). These methods also exhibited exceptional recovery of analyte‐spiked plasma samples (98.0 ± 7.64% for GSH and 98.5 ± 12.7% for GSSG). Good sample stability at −80°C was evident for GSH for up to 55 weeks and GSSG for up to 46 weeks, with average CVs <15 and <10%, respectively. [ABSTRACT FROM AUTHOR]

An early exposure to lipid biochemistry in the laboratory of Konrad Bloch resulted in a fascination with the biosynthesis, structures, and functions of bacterial lipids. The discovery of plasmalogens (1-alk-1-enyl, 2-acyl phospholipids) in anaerobic Gram-positive bacteria led to studies on the physical chemistry of these lipids and the cellular regulation of membrane lipid polymorphism in bacteria. Later studies in several laboratories showed that the formation of the alk-1-enyl ether bond involves an aerobic process in animal cells and thus is fundamentally different from that in anaerobic organisms. Our work provides evidence for an anaerobic process in which plasmalogens are formed from their corresponding diacyl lipids. Studies on the roles of phospholipases in Listeria monocytogenes revealed distinctions between its phospholipases and those previously discovered in other bacteria and showed how the Listeria enzymes are uniquely fitted to the intracellular lifestyle of this significant human pathogen. [ABSTRACT FROM AUTHOR]

The kinetic activation and specific activities of the phosphatidylinositol-specific phospholipase C (PI-PLC) obtained from Listeria monocytogenes are discussed. PI-PLC is shown to exhibit an extremely high affinity for the anionic phospholipids and hence can be employed for L. monocytogenes infectivity.

An experimental procedure to prepare purified phospholipase C (PLC(sub LM)) free from a frequent contaminant, listeriolysin O, is reported together with its optimized activity on lipids in the form of large unilamellar vesicles (LUVs), in the absence of detergents. Results depicted that the catalytic activity of PLC(sub LM) induce aggregation and fusion of vesicles under conditions, which are compatible with the proposed pathogenic role of phospholipase C/sphingomyelinase.

The lipidomes of Clostridium fallax and Clostridium cadaveris were studied using thin‐layer chromatography (TLC) and normal phase liquid chromatography/mass spectrometry (NPLC/MS). Both species contain diradylglycerol (DRG), monohexosyldiradylglycerol (MHDRG), monohexosyl monoacylglycerol (MHMAG), phosphatidylglycerol (PtdGro), and phosphatidylethanolamine (PtdEtn). DRG, MHDRG, PtdEtn, and PtdGro are present in both diacyl and alk‐1‐enyl acyl (plasmalogen) forms. Both species contain cardiolipin (Ptd2Gro), which is present in tetraacyl, monoalkenyl‐triacyl, and dialkenyl‐diacyl forms. Both species contain small amounts of phosphatidylcholine (PtdCho). The presence of octadecadienoic (18:2) acyl chains in some PtdCho species indicates that they arise from the medium because no 18:2 is seen in the other lipids and clostridia generally lack the capacity to synthesize polyunsaturated fatty acids. The major lipidomic differences between these two species are that C. fallax contains a glycerolacetal of plasmenylethanolamine while C. cadaveris contains an ethanolamine‐phosphate‐modified diacylglycerol. The significance of these lipid compositions is discussed. [ABSTRACT FROM AUTHOR]

Nontypeable Haemophilus influenzae (NTHi) is a major cause of community acquired pneumonia and exacerbation of chronic obstructive pulmonary disease. A current effort in NTHi vaccine development has focused on generating humoral responses and has been greatly impeded by antigenic variation among the numerous circulating NTHi strains. In this study, we showed that pulmonary immunization of mice with killed NTHi generated broad protection against lung infection by different strains. While passive transfer of immune antibodies protected only against the homologous strain, transfer of immune T cells conferred protection against both homologous and heterologous strains. Further characterization revealed a strong Th17 response that was cross-reactive with different NTHi strains. Responding Th17 cells recognized both cytosolic and membrane-associated antigens, while immune antibodies preferentially responded to surface antigens and were highly strain specific. We further identified several conserved proteins recognized by lung Th17 cells during NTHi infection. Two proteins yielding the strongest responses were tested as vaccine candidates by immunization of mice with purified proteins plus an adjuvant. Immunization induced antigen-specific Th17 cells that recognized different strains and, upon adoptive transfer, conferred protection. Furthermore, immunized mice were protected against challenge with not only NTHi strains but also a fully virulent, encapsulated strain. Together, these results show that the immune mechanism of cross-protection against pneumonia involves Th17 cells, which respond to a broad spectrum of antigens, including those that are highly conserved among NTHi strains. These mechanistic insights suggest that inclusion of Th17 antigens in subunit vaccines offers the advantage of inducing broad protection and complements the current antibody-based approaches. [ABSTRACT FROM AUTHOR]

The biosynthesis of plasmalogens in anaerobic bacteria differs fundamentally from that in animal cells. Firstly, the formation of the alk-1′-enyl ether bond in animal cells is oxygen dependent. Secondly, the first step in plasmalogen formation in animal cells is an acylation of dihydroxyacetone phosphate, which has been ruled out as a precursor in anaerobes. In bacteria the alk-1′-enyl ether bond is formed after the fully formed acyl glycerolipids are synthesized. Evidence will be presented for the conversion of the sn-1 acyl-linked chain to an O-alk-1′-enyl ether by an as yet unknown mechanism. [ABSTRACT FROM AUTHOR]

Author(s): Howard Goldfine (corresponding author); Dennis E. Vance (corresponding author) [illus. 1] Konrad Bloch, who died on 15 October 2000, was one of the small group of biochemists who first [...]

Abstract: 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. [Copyright &y& Elsevier]

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). [ABSTRACT FROM AUTHOR]

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 (cPLA2α). 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 (ΔhlyLM) induce similar levels of cyclooxygenase 2. Arachidonic acid release requires activation of MAPKs by WTLM and ΔhlyLM. The attenuated release of arachidonic acid that is observed in TLR2-/- and MyD88-/- macrophages infected with WTLM and ΔhlyLM correlates with diminished MAPK activation. WTLM but not ΔhlyLM increases intracellular calcium, which is implicated in regulation of cPLA2α. Prostaglandin E2, prostaglandin I2, and leukotriene C4 are produced by cPLA2α+/+ but not cPLA2α-/- macrophages in response to WTLM and ΔhlyLM. Tumor necrosis factor (TNF)-α production is significantly lower in cPLA2α+/+ than in cPLA2α-/- macrophages infected with WTLM and ΔhlyLM. Treatment of infected cPLA2α+/+ macrophages with the cyclooxygenase inhibitor indomethacin increases TNFα production to the level produced by cPLA2α-/- macrophages implicating prostaglandins in TNFα down-regulation. Therefore activation of cPLA2α in macrophages may impact immune responses to L. monocytogenes. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes phosphatidyl inositol-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 glycosylphosphatidyl inosito I (GPI)-anchored proteins. Previous crystal structure analysis has revealed that a small β-strand (Vb) is present in Bacillus cereus P1-PLC and is absent in the enzyme from L. monocytogenes. This Vb β-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 β-strand. Expression by L. monocytogenes of B. cereus PL-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 β-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 β-strand, thus leading to greatly reduced activity on GPI-anchored proteins. [ABSTRACT FROM AUTHOR]

We have identified phosphatidylethanolamine as one of the major phospholipids of Clostridium perfringens by two dimensional thin layer chromatography of the intact lipids and of their deacylation products and by liquid chromatography followed by mass spectrometry of the intact neutral phospholipid fraction. The principal fatty acids of phosphatidylethanolamine are myristic acid (14:0), lauric acid (12:0), and palmitic acid (16:0) and the major molecular species are 14:0,14:0 (26.3%); 12:0,14:0 (19.0%); 14:0,16:0 (22.4%) and 16:0,16:0 (17.6%). A similar distribution of molecular species was found in the other major phospholipid, O-alanyl phosphatidylglycerol. [Copyright &y& Elsevier]

Haemophilus influenzae obtains choline from either its growth medium or host cell membrane lipids and expresses it on its lipopolysaccharide (LPS) in the form of phosphorylcholine (ChoP), which contributes to its pathogenesis by mimicry of host cell molecules. Two genes ( licB and betT) revealed by whole genomic analysis as encoding potential choline transporters were tested for their role in LPS-ChoP synthesis. The betT gene in H. influenzae is similar to betT in Escherichia coli, which functions in choline transport for the generation of betaine in osmoprotection. The licB gene has homology to bacterial permeases including betT and is encoded in the lic1 locus, which is essential for the expression of LPS-ChoP. In the presence of high concentrations of choline, neither licB nor betT were necessary for expression of LPS-ChoP raising the possibility that other unidentified choline uptake mechanisms may exist in this species. However, under choline limiting conditions, including growth in human nasal airway surface fluid, the licB, but not betT, gene was required for choline transport and synthesis of LPS-ChoP suggesting that LicB functions as a high affinity choline permease. The betT, but not licB, gene was shown to function in osmoprotection in H. influenzae, similar to the role of betT in E. coli. Further analysis demonstrated growth condition dependent differences in the regulation of transcription of the licB and betT genes. We conclude that H. influenzae may have multiple mechanisms for choline uptake and distinct pathways for choline utilization in LPS-ChoP biosynthesis and osmoregulation. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

Studies of the lipidomes of twenty-one species of clostridia have revealed considerable diversity. Even among those species now defined as Clostridium sensu stricto , which are related to Clostridium butyricum , the type species, lipid analysis has shown that a number of distinct clades have characteristic polar lipids. All species of Clostridium sensu stricto have phosphatidylethanolamine, phosphatidylglycerol and cardiolipin which are present as all acyl or alk-1′-enyl acyl (plasmalogen) species. In addition, almost every clade has specialized polar lipids. For example, the group closely related to Clostridium beijerinckii and several other solventogenic species has glycerol acetals of plasmenylethanolamine, which protects the membrane bilayer arrangement when the lipids are highly unsaturated or in the presence of solvents. The group related to Clostridium novyi has aminoacyl-phosphatidylglycerol, which protects these pathogens from cationic antimicrobial peptides (CAMPs) of innate immunity. Clostridium botulinum species, which fall into several groups, align with these clades, and have the same specific lipids. This review will present the current state of knowledge on clostridial lipids. • The diversity of lipidomes in clostridia • Clades have evolved lipids related to their physiology. • Several novel lipids have known properties. [ABSTRACT FROM AUTHOR]

R1775 --> The biosynthesis of phospholipids in bacteria begins with the acylation of sn‐glycerol‐3‐P. The second step is the acylation of the resulting 1‐O‐acyl‐glycerol‐3‐P catalyzed by PlsC to form phosphatidic acid. Clostridium beijerinckii has two genes annotated as plsC and a related third gene, plsD, of unknown function. We have used CRISPR‐Cas9 to delete each of these genes and examined the effects on phospholipid chain compositions. Deletion of Cbei_5028 (plsC) had no effect on the composition of the acyl or alk‐1‐enyl chains of the lipids. Deletion of Cbei_1741 (plsC) resulted in polar lipids with fewer unsaturated chains in diacyl lipids and plasmalogens. Deletion of Cbei_0579 (plsD) resulted in longer chains in all the lipids examined. It appears that Cbei_1741 (PlsC) is selective for unsaturated fatty acyl chains and that Cbei_0579 (PlsD) is a 1‐O‐acyl‐sn‐glycerol‐3‐P acyltransferase selective for shorter acyl chains. Strain characterization experiments demonstrated that ∆Cbei_1741 mutant had slightly increased tolerance to butanol compared to the wild type, possibly due to the more saturated chains in diacyl lipids and plasmalogens in the cell membrane. More dramatically, ∆Cbei_1741 mutant grew much faster and produced 40% more butanol (with more glucose consumed) than the wild type in a regular batch fermentation (at 35 oC). Comparatively, both ∆Cbei_5028 and ∆Cbei_0579 mutant strains produced slightly lower butanol than wild type under this condition. Interestingly, ∆Cbei_0579 produced 40% more butanol than the wild type when the fermentation was conducted at 20 oC. While at higher temperature (40 oC), the butanol yield in ∆Cbei_0579 reached 0.32 g/g (33% higher than in wild type) although the final butanol titer was actually slightly lower than in wild type. [ABSTRACT FROM AUTHOR]

A new phosphatidylglycerol acetal of cardiolipin plasmalogen has been isolated from Clostridium innocuum. The structure was derived from the results of quantitative group analyses, the identification of the products of acid hydrolysis, alkaline methanolysis, hydrolysis by a cardiolipin-specific phospholipase D and by one- and two-dimensional proton NMR. Two other minor ether phospholipids: the lyso form of the phosphatidylglycerol acetal of cardiolipin plasmalogen, and the phosphatidylglycerol acetal of plasmenylglycerol have been identified in C. innocuum lipid extracts. [ABSTRACT FROM AUTHOR]

We have studied the phospholipid composition of Rhizobium meliloti strains which do or do not contain the large, tumor-inducing (Ti) plasmid of Agrobacterium 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, and PC lower, in logarithmic phase cells. No significant differences were seen in the proportions of phospholipids in strains with or without the Ti plasmid. Qualitative examination of the phospholipids of A. tumefaciens with or without the Ti plasmid similarly revealed no differences. [ABSTRACT FROM AUTHOR]

Provides information on phospholipases which have been recognized as bacterial toxins. Virulence genes of Listeria monocytogenes; Importance of L. monocytogenes phospholipases for bacterial escape from a macrophage vacuole; Range of phospholipase C.

The article discusses the bacterial response to antimicrobial cationic peptides. Bacteria may meet different conditions in nature including changes in salinity, temperature, pH, and the presence of antibacterial molecules that changed either to kill them or hinder their growth. Some bacteria, in response, have changed modifications of their membrane lipids that enhance their survival.

Plasmalogens--phospholipids containing a characteristic vinyl ether group--are precursors of lipids important for cellular signaling such as arachidonic acid. Plasmalogen catabolism involves cleavage of the vinyl ether bond, but the identity of the corresponding enzyme that cleaves the sn-1 vinyl ether bond was unknown. New research shows that cytochrome c, with some help from another lipid, catalyzes the oxidative cleavage of this bond. This discovery, and the subsequent mechanistic dissection, provides exciting new directions for lipid signaling research. [ABSTRACT FROM AUTHOR]