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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.

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]

A letter to the editor is presented on the involvement of ActA in maturation of the Listeria monocytogenes phagosome.

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]