RESUMO
Bordetella bronchiseptica PagP (PagPBB) is a lipid A palmitoyl transferase that is required for resistance to antibody-dependent complement-mediated killing in a murine model of infection. B. parapertussis contains a putative pagP homolog (encoding B. parapertussis PagP [PagPBPa]), but its role in the biosynthesis of lipid A, the membrane anchor of lipopolysaccharide (LPS), has not been investigated. Mass spectrometry analysis revealed that wild-type B. parapertussis lipid A consists of a heterogeneous mixture of lipid A structures, with penta- and hexa-acylated structures containing one and two palmitates, respectively. Through mutational analysis, we demonstrate that PagPBPa is required for the modification of lipid A with palmitate. While PagPBB transfers a single palmitate to the lipid A C-3' position, PagPBPa transfers palmitates to the lipid A C-2 and C-3' positions. The addition of two palmitate acyl chains is unique to B. parapertussis. Mutation of pagPBPa resulted in a mutant strain with increased sensitivity to antimicrobial peptide killing and decreased endotoxicity, as evidenced by reduced proinflammatory responses via Toll-like receptor 4 (TLR4) to the hypoacylated LPS. Therefore, PagP-mediated modification of lipid A regulates outer membrane function and may be a means to modify interactions between the bacterium and its human host during infection.
Assuntos
Aciltransferases/metabolismo , Bordetella parapertussis/enzimologia , Lipídeo A/metabolismo , Palmitatos/metabolismo , Aciltransferases/genética , Bordetella parapertussis/química , Bordetella parapertussis/genética , Análise Mutacional de DNA , Lipídeo A/química , Espectrometria de MassasRESUMO
PagL and LpxO are enzymes that modify lipid A. PagL is a 3-O deacylase that removes the primary acyl chain from the 3 position, and LpxO is an oxygenase that 2-hydroxylates specific acyl chains in the lipid A. pagL and lpxO homologues have been identified in the genome of Bordetella bronchiseptica, but in the current structure for B. bronchiseptica lipid A the 3 position is acylated and 2-OH acylation is not reported. We have investigated the role of B. bronchiseptica pagL and lpxO in lipid A biosynthesis. We report a different structure for wild-type (WT) B. bronchiseptica lipid A, including the presence of 2-OH-myristate, the presence of which is dependent on lpxO. We also demonstrate that the 3 position is not acylated in the major WT lipid A structures but that mutation of pagL results in the presence of 3-OH-decanoic acid at this position, suggesting that lipid A containing this acylation is synthesized but that PagL removes most of it from the mature lipid A. These data refine the structure of B. bronchiseptica lipid A and demonstrate that pagL and lpxO are involved in its biosynthesis.
Assuntos
Proteínas de Bactérias/metabolismo , Bordetella bronchiseptica/enzimologia , Bordetella bronchiseptica/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Lipídeo A/biossíntese , Lipídeo A/química , Oxigenases/metabolismo , Proteínas de Bactérias/genética , Bordetella bronchiseptica/genética , Hidrolases de Éster Carboxílico/genética , Ácidos Láuricos/análise , Miristatos/análise , Oxigenases/genéticaRESUMO
Levels of nonantigen-induced pro-inflammatory cytokines and prostaglandin in macrophages isolated from human leucocyte antigen (HLA)-matched type 1 diabetes mellitus patients, first-degree relatives and healthy controls were determined. We hypothesize that monocytes isolated from patients are sensitized or preactivated and therefore, have an altered response to in vitro stimulus compared with control groups as measured by levels of pro- and anti-inflammatory mediators. In this study, peripheral blood monocytes were differentiated to macrophages with macrophage-colony stimulating factor (M-CSF) to determine lipopolysaccharide (LPS)-stimulated tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, IL-12 and prostaglandin E-2 (PGE-2) secretion from hetero- or homozygous HLA DQB1*0201 and *0302 type 1 diabetes mellitus patients, first-degree relatives and homozygous HLA DQB1*0602 healthy controls. LPS-stimulated secretion of TNF-alpha, IL-1beta and IL-6 was immediate and markedly higher in the HLA-DQB1*0201/*0302 type 1 diabetes patients compared with all other groups including HLA-matched healthy first-degree relatives. In DQB1*0201/*0302 diabetes patients PGE-2 secretion was delayed but increased by LPS stimulation compared with HLA-matched healthy relatives. IL-12 was not detected at any condition. These data suggest that macrophages from DQB1*0201/*0302 type 1 diabetes patients are sensitized to secrete both cytokines and PGE-2 following nonantigenic stimulation. Sensitized macrophages may be important to high-risk DQB1*0201/*0302-associated type 1 diabetes.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Antígenos HLA-DQ , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Adolescente , Adulto , Estudos de Casos e Controles , Criança , Diabetes Mellitus Tipo 1/genética , Dinoprostona/biossíntese , Feminino , Antígenos HLA-DQ/genética , Cadeias beta de HLA-DQ , Heterozigoto , Homozigoto , Humanos , Técnicas In Vitro , Interleucina-1/biossíntese , Interleucina-6/biossíntese , Masculino , Pessoa de Meia-Idade , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Haemophilus influenzae type b capsular polysaccharide (PRP) conjugate vaccines, which are thought to induce T cell-dependent antibody production, induce protective responses after a single dose in individuals under 15 months of age. However, multiple doses of these vaccines are required to induce protective antibody responses in infants, with the exception of PRP conjugated to meningococcal outer membrane proteins (OMPC), which does so after a single dose. The basis for this difference is not fully understood, although others have proposed that OMPC and porins, the major protein component of OMPC, act as adjuvants or mitogens. In this report OMPC is shown to enhance CD40 ligand-mediated, T cell-dependent antibody production in mice. This paralleled the induction by OMPC of CD86, CD80 and CD40 costimulatory molecules on human neonatal and murine B cells and of Th1 cytokines. Neither porins nor lipopolysaccharide fully reproduced the effects of OMPC. These studies indicate that OMPC acts both as carrier and adjuvant, and thereby enhances T cell-dependent antibody responses in human infants.
Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Meningocócicas/imunologia , Neisseria meningitidis/imunologia , Polissacarídeos Bacterianos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Vacinas Conjugadas/imunologia , Adjuvantes Imunológicos , Adulto , Animais , Antígenos CD/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Antígeno B7-2 , Proteínas da Membrana Bacteriana Externa/administração & dosagem , Células Cultivadas , Citocinas/biossíntese , Citocinas/genética , Citometria de Fluxo , Vacinas Anti-Haemophilus/imunologia , Humanos , Imuno-Histoquímica , Recém-Nascido , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Glicoproteínas de Membrana/metabolismo , Vacinas Meningocócicas/administração & dosagem , Camundongos , Camundongos Endogâmicos , Monócitos/imunologia , Polissacarídeos Bacterianos/administração & dosagem , Toxoide Tetânico/imunologia , Regulação para Cima , Vacinas Conjugadas/administração & dosagemRESUMO
Resistance to innate immunity is essential for salmonellae pathogenesis. The salmonellae PhoP/PhoQ regulators sense host environments to promote remodeling of the bacterial envelope. This remodeling includes enzymes that modify lipopolysaccharide (LPS). Modified LPS promotes bacterial survival by increasing resistance to cationic antimicrobial peptides and by altered host recognition of LPS.
Assuntos
Imunidade Inata , Lipopolissacarídeos/metabolismo , Salmonella typhimurium/enzimologia , Salmonella typhimurium/patogenicidade , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Camundongos , Infecções por Salmonella/microbiologia , Salmonella typhimurium/genéticaRESUMO
The two-component regulatory system PmrA-PmrB confers resistance of Salmonella spp. to cationic antimicrobial peptides (AP) such as polymyxin (PM), bactericidal/permeability-increasing protein, and azurocidin. This resistance occurs by transcriptional activation of two loci termed pmrE and pmrHFIJKLM. Both pmrE and pmrHFIJKLM produce products required for the biosynthesis of lipid A with 4-aminoarabinose (Ara4N). Ara4N addition creates a more positively charged lipopolysaccharide (LPS) and thus reduces cationic AP binding. Experiments were conducted to further analyze the regulation of the pmrHFIJKLM operon and the role of this operon and the surrounding genomic region in LPS modification and antimicrobial peptide resistance. The pmrHFIJKLM genes are cotranscribed and over 3,000-fold regulated by PmrA-PmrB. The pmrHFIJKLM promoter bound PmrA, as determined by gel shift analysis, as did a 40-bp region of the PmrA-PmrB-regulated pmrCAB promoter. Construction of nonpolar mutations in the pmrHFIJKLM genes showed that all except pmrM were necessary for the Ara4N addition to lipid A and PM resistance. The flanking genes of the operon (pmrG and pmrD) were not necessary for PM resistance, but pmrD was shown to be regulated by the PhoP-PhoQ regulatory system. BALB/c mice inoculated with pmrA and pmrHFIJKLM mutant strains demonstrated virulence attenuation when the strains were administered orally but not when they were administered intraperitoneally, indicating that Ara4N addition may be important for resistance to host innate defenses within intestinal tissues.
Assuntos
Proteínas de Bactérias/genética , Lipídeo A/fisiologia , Polimixina B/farmacologia , Regulon , Salmonella typhimurium/genética , Fatores de Transcrição/genética , Animais , Mapeamento Cromossômico , Resistência Microbiana a Medicamentos , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Regiões Promotoras Genéticas , Salmonella typhimurium/imunologia , Salmonella typhimurium/patogenicidade , Transcrição Gênica , VirulênciaRESUMO
In Escherichia coli, the waaP (rfaP) gene product was recently shown to be responsible for phosphorylation of the first heptose residue of the lipopolysaccharide (LPS) inner core region. WaaP was also shown to be necessary for the formation of a stable outer membrane. These earlier studies were performed with an avirulent rough strain of E. coli (to facilitate the structural chemistry required to properly define waaP function); therefore, we undertook the creation of a waaP mutant of Salmonella enterica serovar Typhimurium to assess the contribution of WaaP and LPS core phosphorylation to the biology of an intracellular pathogen. The S. enterica waaP mutant described here is the first to be both genetically and structurally characterized, and its creation refutes an earlier claim that waaP mutations in S. enterica must be leaky to maintain viability. The mutant was shown to exhibit characteristics of the deep-rough phenotype, despite its ability to produce a full-length core capped with O antigen. Further, phosphoryl modifications in the LPS core region were shown to be required for resistance to polycationic antimicrobials. The waaP mutant was significantly more sensitive to polymyxin in both wild-type and polymyxin-resistant backgrounds, despite the decreased negative charge of the mutant LPSs. In addition, the waaP mutation was shown to cause a complete loss of virulence in mouse infection models. Taken together, these data indicate that WaaP is a potential target for the development of novel therapeutic agents.
Assuntos
Glucosiltransferases/genética , Fosfotransferases (Aceptor do Grupo Álcool) , Polimixinas/farmacologia , Salmonella typhimurium/patogenicidade , Animais , Sequência de Carboidratos , Resistência Microbiana a Medicamentos , Proteínas de Escherichia coli , Lipopolissacarídeos/química , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mutação , Oligossacarídeos/química , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Análise de Sequência , Virulência/genéticaRESUMO
The PhoP-PhoQ two-component system is necessary for the virulence of Salmonella spp. and is responsible for regulating several modifications of the lipopolysaccharide (LPS). Mutagenesis of the transcriptional regulator phoP resulted in the identification of a mutant able to activate transcription of regulated genes approximately 100-fold in the absence of PhoQ. Sequence analysis showed two single-base alterations resulting in amino acid changes at positions 93 (S93N) and 203 (Q203R). These mutations were individually created, and although each resulted in a constitutive phenotype, the double mutant displayed a synergistic effect both in the induction of PhoP-activated gene expression and in resistance to antimicrobial peptides. The constitutive phoP gene was placed under the control of an arabinose-inducible promoter to examine the kinetics of PhoP-activated gene induction and the resultant modifications of LPS. Gene induction and 2-hydroxymyristate modification of the lipid A were shown to occur within minutes of the addition of arabinose and to peak at 4 h. As the first constitutive mutant of phoP identified, this allele will be invaluable to future genetic and biochemical studies of this and likely other regulatory systems.
Assuntos
Proteínas de Bactérias/genética , Lipopolissacarídeos/biossíntese , Proteínas de Membrana , Mutação Puntual , Salmonella typhimurium/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos , Regulação Bacteriana da Expressão Gênica , Lipídeo A/metabolismo , Dados de Sequência Molecular , Ácidos Mirísticos/metabolismo , Peptídeos , Fenótipo , Salmonella typhimurium/patogenicidade , Homologia de Sequência de Aminoácidos , Sorotipagem , Transativadores/genética , Ativação TranscricionalRESUMO
Cystic fibrosis (CF) patients develop chronic airway infections with Pseudomonas aeruginosa (PA). Pseudomonas aeruginosa synthesized lipopolysaccharide (LPS) with a variety of penta- and hexa-acylated lipid A structures under different environmental conditions. CF patient PA synthesized LPS with specific lipid A structures indicating unique recognition of the CF airway environment. CF-specific lipid A forms containing palmitate and aminoarabinose were associated with resistance to cationic antimicrobial peptides and increased inflammatory responses, indicating that they are likely to be involved in airway disease.
Assuntos
Fibrose Cística/microbiologia , Lipídeo A/biossíntese , Lipídeo A/química , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/metabolismo , Sistema Respiratório/microbiologia , Acilação , Peptídeos Catiônicos Antimicrobianos , Arabinose/análogos & derivados , Arabinose/análise , Arabinose/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Células Cultivadas , Fibrose Cística/complicações , Resistência Microbiana a Medicamentos , Humanos , Lactente , Interleucina-8/biossíntese , Lipopolissacarídeos/química , Lipopolissacarídeos/imunologia , Magnésio/farmacologia , Mutação , Palmitatos/análise , Palmitatos/metabolismo , Peptídeos/farmacologia , Polimixinas/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , VirulênciaRESUMO
Bacterial pathogens regulate the expression of virulence factors in response to environmental signals. In the case of salmonellae, many virulence factors are regulated via PhoP/PhoQ, a two-component signal transduction system that is repressed by magnesium and calcium in vitro. PhoP/PhoQ-activated genes promote intracellular survival within macrophages, whereas PhoP-repressed genes promote entrance into epithelial cells and macrophages by macropinocytosis and stimulate epithelial cell cytokine production. PhoP-activated genes include those that alter the cell envelope through structural alterations of lipopolysaccharide and lipid A, the bioactive component of lipopolysaccharide. PhoP-activated changes in the bacterial envelope likely promote intracellular survival by increasing resistance to host cationic antimicrobial peptides and decreasing host cell cytokine production.
Assuntos
Bactérias/imunologia , Bactérias/patogenicidade , Animais , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Sequência de Carboidratos , Genes Bacterianos , Humanos , Lipopolissacarídeos/química , Lipopolissacarídeos/imunologia , Dados de Sequência Molecular , Fagocitose , Salmonella/genética , Salmonella/imunologia , Salmonella/patogenicidade , Transdução de Sinais , VirulênciaRESUMO
The Mason-Pfizer monkey virus (MPMV) genome contains a cis-acting element that serves to facilitate nucleocytoplasmic export of intron-containing RNA. This element, known as the constitutive transport element (CTE), has been mapped to a 154-nt region close to the 3' end of the MPMV genome. The CTE contains a degenerate direct repeat of approximately 70 nt. We have probed the secondary structure of the CTE using double-strand- and single-strand-specific ribonucleases and chemical modification agents. A mutational analysis was also performed to confirm critical features of the CTE structure, as well as to identify regions that contain sequence-specific information required for function. Our results indicate that the CTE forms a long stem structure that contains a 9-nt terminal hairpin loop as well as two identical 16-nt inner loops. The inner loop sequences are rotated 180 degrees relative to each other within the structure. The mutational analysis shows that primary sequences in the loop regions are important for function, suggesting that they may contain binding sites for cellular proteins involved in RNA export. Interestingly, sequences with significant homology to the inner loop regions are found in the genomes of spumaviruses and mouse intracisternal A particles.
Assuntos
Vírus dos Macacos de Mason-Pfizer/genética , Conformação de Ácido Nucleico , RNA Viral/química , Sequência de Bases , Dados de Sequência Molecular , Mutagênese , Sondas RNA , RNA Viral/genética , RNA Viral/metabolismoRESUMO
A common feature of gene expression in all retroviruses is that unspliced, intron-containing RNA is exported to the cytoplasm despite the fact that cellular RNAs which contain introns are usually restricted to the nucleus. In complex retroviruses, the export of intron-containing RNA is mediated by specific viral regulatory proteins (e.g., human immunodeficiency virus type 1 [HIV-1] Rev) that bind to elements in the viral RNA. However, simpler retroviruses do not encode such regulatory proteins. Here we show that the genome of the simpler retrovirus Mason-Pfizer monkey virus (MPMV) contains an element that serves as an autonomous nuclear export signal for intron-containing RNA. This element is essential for MPMV replication; however, its function can be complemented by HIV-1 Rev and the Rev-responsive element. The element can also facilitate the export of cellular intron-containing RNA. These results suggest that the MPMV element mimics cellular RNA transport signals and mediates RNA export through interaction with endogenous cellular factors.
Assuntos
Íntrons , Vírus dos Macacos de Mason-Pfizer/genética , RNA Viral/genética , RNA Viral/metabolismo , Animais , Sequência de Bases , Transporte Biológico , Células COS , Núcleo Celular/virologia , Mapeamento Cromossômico , Citoplasma/virologia , Produtos do Gene env/biossíntese , Produtos do Gene gag/biossíntese , Produtos do Gene rev/genética , Produtos do Gene rev/fisiologia , HIV-1/genética , Humanos , Vírus dos Macacos de Mason-Pfizer/fisiologia , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Splicing de RNA , RNA Mensageiro/metabolismo , RNA Viral/química , Replicação Viral/genética , Produtos do Gene rev do Vírus da Imunodeficiência HumanaRESUMO
The constitutive transport elements (CTEs) of type D retroviruses are cis-acting elements that promote nuclear export of incompletely spliced mRNAs. Unlike the Rev response element (RRE) of human immunodeficiency virus type 1 (HIV-1), CTEs depend entirely on factors encoded by the host cell genome. We show that an RNA comprised almost entirely of the CTE of Mason-Pfizer monkey virus (CTE RNA) is exported efficiently from Xenopus oocyte nuclei. The CTE RNA and an RNA containing the RRE of HIV-1 (plus Rev) have little effect on export of one another, demonstrating differences in host cell requirements of these two viral mRNA export pathways. Surprisingly, even very low amounts of CTE RNA block export of normal mRNAs, apparently through the sequestration of cellular mRNA export factors. Export of a CTE-containing lariat occurs when wild-type CTE, but not a mutant form, is inserted into the pre-mRNA. The CTE has two symmetric structures, either of which supports export and the titration of mRNA export factors, but both of which are required for maximal inhibition of mRNA export. Two host proteins bind specifically to the CTE but not to non-functional variants, making these proteins candidates for the sequestered mRNA export factors.
Assuntos
Vírus dos Macacos de Mason-Pfizer/genética , RNA Mensageiro/metabolismo , Animais , Sequência de Bases , Núcleo Celular/fisiologia , Feminino , Produtos do Gene rev/metabolismo , HIV-1/genética , Humanos , Vírus dos Macacos de Mason-Pfizer/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Oócitos/fisiologia , Splicing de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , Tetra-Hidrofolato Desidrogenase/biossíntese , Transcrição Gênica , Xenopus laevis , Produtos do Gene rev do Vírus da Imunodeficiência HumanaRESUMO
The invasion of HEp-2 cells by Salmonella typhimurium was studied under various conditions. Anaerobiosis was shown to markedly affect the internalization of bacterial cells by HEp-2 cells. Anaerobically grown bacteria incubated with HEp-2 cells under anaerobic conditions markedly stimulated the rate of invasion. Anaerobiosis may therefore be a controlling factor in the invasion process. Cells obtained during the logarithmic phase of growth invaded at much higher rates than cells obtained during the stationary phase of growth. The presence of mannose-sensitive type 1 fimbriae on the bacterial surface also promoted invasion, and these fimbriae appear to play a role as an accessory virulence factor.
Assuntos
Epitélio/microbiologia , Fímbrias Bacterianas/fisiologia , Salmonella typhimurium/patogenicidade , Anaerobiose , Linhagem Celular , Humanos , Técnicas In Vitro , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/metabolismoRESUMO
A number of dietary lectins have been shown to resist proteolytic digestion. These lectins interact with the small intestinal mucosa causing structural and functional changes. Concomitant to these changes, bacterial overgrowth was reported and a possible interaction between lectins and bacteria in the small intestine was postulated. The aim of this study was to investigate the effect of various lectins on adherence of Salmonella typhimurium to both isolated small intestinal enterocytes and ligated intestinal loops. Isolated intestinal cells or ligated intestinal loops were incubated with [3H] adenine-S. typhimurium in the presence or absence of concanavalin A, phytohemagglutinin, peanut agglutinin, and wheat germ agglutinin. Only concanavalin A promoted the adherence of various strains of nonfimbriated S. typhimurium to isolated viable intestinal cells. Other lectins showed no effect on the adherence. In situ studies showed that bacterial binding was increased in concanavalin A-treated intestinal loops, supporting the significance of the experiments in vitro. These data suggest that lectins may act by promoting bacterial adherence to the small intestine, thereby facilitating colonization and infection, and leading to bacterial overgrowth.