RESUMO
Francisella tularensis induces the synthesis of prostaglandin E(2) (PGE(2)) by infected macrophages to alter host immune responses, thus providing a survival advantage to the bacterium. We previously demonstrated that PGE(2) synthesis by F. tularensis-infected macrophages requires cytosolic phospholipase A2 (cPLA(2)), cyclooxygenase 2 (COX-2), and microsomal prostaglandin E synthase 1 (mPGES1). During inducible PGE(2) synthesis, cPLA(2) hydrolyzes arachidonic acid (AA) from cellular phospholipids to be converted to PGE(2). However, in F. tularensis-infected macrophages we observed a temporal disconnect between Ser505-cPLA(2) phosphorylation (a marker of activation) and PGE(2) synthesis. These results suggested to us that cPLA(2) is not responsible for the liberation of AA to be converted into PGE(2) by F. tularensis-infected macrophages. Utilizing small-molecule inhibitors, we demonstrated that phospholipase D and diacylglycerol lipase were required for providing AA for PGE(2) biosynthesis. cPLA(2), on the other hand, was required for macrophage cytokine responses to F. tularensis. We also demonstrated for the first time that lipin-1 and PAP2a contribute to macrophage inflammation in response to F. tularensis. Our results identify both an alternative pathway for inducible PGE(2) synthesis and a role for lipid-modifying enzymes in the regulation of macrophage inflammatory function.
Assuntos
Dinoprostona/biossíntese , Francisella tularensis/imunologia , Lipase Lipoproteica/metabolismo , Macrófagos/metabolismo , Proteínas Nucleares/metabolismo , Fosfatidato Fosfatase/metabolismo , Fosfolipase D/metabolismo , Animais , Feminino , Humanos , Macrófagos/enzimologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Francisella tularensis, the causative agent of tularemia, modulates the host immune response to gain a survival advantage within the host. One mechanism of immune evasion is the ability of F. tularensis to induce the synthesis of the small lipid mediator prostaglandin E2 (PGE2), which alters the host T cell response making the host more susceptible to Francisella growth. PGE2 is synthesized by a tightly regulated biosynthetic pathway following stimulation. The synthesis of PGE2 begins with the liberation of arachidonic acid (AA) from membrane phospholipids by cytosolic phospholipase A2 (cPLA2). AA is subsequently converted to the unstable intermediate PGH2 by cyclooxygenase-2 (COX-2), and PGH2 undergoes an isomerization reaction to generate PGE2. Our objective was to identify F. tularensis-activated host signaling pathways that regulate the activity of the enzymes in the PGE2-biosynthetic pathway. In this study, we show that cPLA2, p38 mitogen-activated protein kinase (MAPK), and Janus kinase 3 (JAK3) signaling are necessary for F. tularensis-induced PGE2 production. Inhibition of JAK3 activity reduced the phosphorylation of cPLA2 and COX-2 protein levels. In addition, JAK3 regulates cPLA2 phosphorylation independent of transcription. Moreover, p38 MAPK activity is required for F. tularensis-induced COX-2 protein synthesis, but not for the phosphorylation of cPLA2. This research highlights a unique signaling axis in which JAK3 and p38 MAPK regulate the activity of multiple enzymes of the PGE2-biosynthetic pathway in macrophages infected with F. tularensis.
Assuntos
Dinoprostona/metabolismo , Francisella tularensis/metabolismo , Janus Quinase 3/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Fosfolipases A2 Citosólicas/metabolismo , Fosforilação/genética , Animais , Ácido Araquidônico/genética , Ácido Araquidônico/metabolismo , Linhagem Celular , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Citosol/metabolismo , Citosol/microbiologia , Dinoprostona/genética , Feminino , Janus Quinase 3/genética , Camundongos , Camundongos Endogâmicos C57BL , Fosfolipases A2 Citosólicas/genética , Transdução de Sinais/genética , Transcrição Gênica/genética , Tularemia/genética , Tularemia/metabolismo , Tularemia/microbiologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Streptococcus agalactiae (group B Streptococcus [GBS]) remains a leading cause of invasive infections in neonates and has emerged as a pathogen of the immunocompromised and elderly populations. The virulence mechanisms of GBS are relatively understudied and are still poorly understood. Previous evidence indicated that the GBS cspA gene is necessary for full virulence and the cleavage of fibrinogen. The predicted cspA product displays homology to members of the extracellular cell envelope protease family. CXC chemokines, many of which can recruit neutrophils to sites of infection, are important signaling peptides of the immune system. In this study, we purified CspA and demonstrated that it readily cleaved the CXC chemokines GRO-alpha, GRO-beta, GRO-gamma, neutrophil-activating peptide 2 (NAP-2), and granulocyte chemotactic protein 2 (GCP-2) but did not cleave interleukin-8. CspA did not cleave a panel of other test substrates, suggesting that it possesses a certain degree of specificity. CXC chemokines also underwent cleavage by whole GBS cells in a cspA-dependent manner. CspA abolished the abilities of three representative CXC chemokines, GRO-gamma, NAP-2, and GCP-2, to attract and activate neutrophils. Genetic and biochemical evidence indicated that CspA is a serine protease with S575 at its active site. D180 was also implicated as part of the signature serine protease catalytic triad, and both S575 and D180 were required for both N-terminal and C-terminal autocatalytic processing of CspA.
Assuntos
Proteínas de Bactérias/imunologia , Quimiocinas/imunologia , Serina Endopeptidases/imunologia , Infecções Estreptocócicas/imunologia , Streptococcus agalactiae/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Domínio Catalítico , Células Cultivadas , Quimiocinas/genética , Quimiotaxia , Humanos , Ativação de Neutrófilo , Neutrófilos/imunologia , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/isolamento & purificação , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/química , Streptococcus agalactiae/genéticaRESUMO
Group B Streptococcus (GBS) is major cause of invasive disease in newborn infants and the leading cause of neonatal meningitis. To gain access to the central nervous system (CNS), GBS must not only subvert host defenses in the bloodstream but also invade and survive within brain microvascular endothelial cells (BMEC), the principal cell layer composing the blood-brain barrier (BBB). While several GBS determinants that contribute to the invasion of BMEC have been identified, little is known about the GBS factors that are required for intracellular survival and ultimate disease progression. In this study we sought to identify these factors by screening a random GBS mutant library in an in vitro survival assay. One mutant was identified which contained a disruption in a two-component regulatory system homologous to CiaR/CiaH, which is present in other streptococcal pathogens. Deletion of the putative response regulator, ciaR, in GBS resulted in a significant decrease in intracellular survival within neutrophils, murine macrophages, and human BMEC, which was linked to increased susceptibility to killing by antimicrobial peptides, lysozyme, and reactive oxygen species. Furthermore, competition experiments with mice showed that wild-type GBS had a significant survival advantage over the GBS DeltaciaR mutant in the bloodstream and brain. Microarray analysis comparing gene expression between wild-type and DeltaciaR mutant GBS bacteria revealed several CiaR-regulated genes that may contribute to stress tolerance and the subversion of host defenses by GBS. Our results identify the GBS CiaR response regulator as a crucial factor in GBS intracellular survival and invasive disease pathogenesis.
Assuntos
Proteínas de Bactérias/imunologia , Imunidade Inata , Proteínas Quinases/imunologia , Infecções Estreptocócicas/imunologia , Streptococcus agalactiae/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Linhagem Celular , Células Cultivadas , Células Endoteliais/microbiologia , Regulação Bacteriana da Expressão Gênica , Humanos , Macrófagos/microbiologia , Masculino , Camundongos , Dados de Sequência Molecular , Neutrófilos/microbiologia , Proteínas Quinases/química , Proteínas Quinases/genética , Alinhamento de Sequência , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/genética , Streptococcus agalactiae/imunologia , Streptococcus agalactiae/patogenicidade , VirulênciaRESUMO
BACKGROUND: Streptococcus agalactiae (group B Streptococcus; GBS) is a significant bacterial pathogen of neonates and an emerging pathogen of adults. Though transcriptional regulators are abundantly encoded on the GBS genome, their role in GBS pathogenesis is poorly understood. The mtaR gene encodes a putative LysR-type transcriptional regulator that is critical for the full virulence of GBS. Previous studies have shown that an mtaR- mutant transports methionine at reduced rates and grows poorly in normal human plasma not supplemented with methionine. The decreased virulence of the mtaR mutant was correlated with a methionine transport defect; however, no MtaR-regulated genes were identified. RESULTS: Microarray analysis of wild-type GBS and an mtaR mutant revealed differential expression of 12 genes, including 1 upregulated and 11 downregulated genes in the mtaR mutant. Among the downregulated genes, we identified a cluster of cotranscribed genes encoding a putative methionine transporter (metQ1NP) and peptidase (pdsM). The expression of four genes potentially involved in arginine transport (artPQ) and arginine biosynthesis (argGH) was downregulated and these genes localized to two transcriptional units. The virulence factor cspA, which encodes an extracellular protease, was downregulated. Additionally, the SAN_1255 locus, which putatively encodes a protein displaying similarity to plasminogen activators, was downregulated. CONCLUSION: To our knowledge, this is the first study to describe the global influence of MtaR on GBS gene expression. This study implicates the metQ1NP genes as encoding the MtaR-regulated methionine transporter, which may provide a mechanistic explanation for the methionine-dependent growth defect of the mtaR mutant. In addition to modulating the expression of genes involved in metabolism and amino acid transport, inactivation of mtaR affected the expression of other GBS genes implicated in pathogenesis. These findings suggest the possibility that MtaR may play a multifaceted role in GBS pathogenesis by regulating the expression of numerous genes.
Assuntos
Proteínas de Bactérias/metabolismo , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Streptococcus agalactiae/genética , Fatores de Transcrição/metabolismo , Arginina/metabolismo , Proteínas de Bactérias/genética , Mapeamento Cromossômico , Genes Bacterianos , Metionina/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Bacteriano/genética , Streptococcus agalactiae/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica , Fatores de Virulência/metabolismoRESUMO
The heterologous expression of streptococcal genes in common Gram-negative hosts may be complicated by low-level expression, toxicity to the host, formation of inclusion bodies, and mislocalization of the encoded proteins. Biochemical study of the Streptococcus agalactiae virulence-associated cell-envelope protease (CEP) CspA, as well as other CEPs, has been limited by the lack of effective expression systems. In this study, we present a simple strategy to express cspA as a catalytically active exoprotein. A recombinant allele of cspA, cspADeltaCWA, was engineered to eliminate the dispensable cell-wall anchor. The cspADeltaCWA allele was expressed in the Gram-positive organism, Lactococcus lactis, using an established, plasmid-based, nisin-inducible expression system. After induction, nearly all of the exoprotein observable by SDS-PAGE corresponded to CspADeltaCWA. CspADeltaCWA-containing medium exhibited similar fibrinolytic activity as whole cells of GBS, indicating the recombinant protein was active. Characterization of CspADeltaCWA indicated that like some other CEPs, it is N-terminally processed, exists predominantly as a dimer, and has the ability to cleave itself at its C-terminus. Taken together, this work presents an efficient strategy for expression of cspA that could be applied to other streptococcal proteins that are not amenable to expression using common Gram-negative hosts.
Assuntos
Clonagem Molecular/métodos , Lactococcus lactis/genética , Proteínas Recombinantes/genética , Serina Endopeptidases/genética , Streptococcus agalactiae/genética , Fatores de Virulência/genética , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Lactococcus lactis/metabolismo , Proteínas Recombinantes/metabolismo , Solubilidade , Fatores de Virulência/metabolismoRESUMO
The S. cerevisiae Yck2 protein is a plasma membrane-associated member of the casein kinase 1 protein kinase family that, with its homolog Yck1p, is required for bud morphogenesis, cytokinesis, endocytosis and other cellular processes. Membrane localization of Yckp is critical for its function, since soluble mutants do not provide sufficient biological activity to sustain normal growth. Yck2p has neither a predicted signal sequence nor obvious transmembrane domain to achieve its plasma membrane localization, but has a C-terminal -Cys-Cys sequence that is likely to be palmitoylated. We demonstrate here that Yck2p is targeted through association with vesicular intermediates of the classical secretory pathway. Yck2p lacking C-terminal Cys residues fails to associate with any membrane, whereas substitution of these residues with a farnesyl transferase signal sequence allows sec-dependent plasma membrane targeting and biological function, suggesting that modification is required for interaction with early secretory membranes but that targeting does not require a particular modification. Deletion analysis within the 185 residue C-terminus indicates that the final 28 residues are critical for membrane association, and additional sequences just upstream are required for proper plasma membrane targeting.