RESUMO
While the influence of water in Helicobacter pylori culturability and membrane integrity has been extensively studied, there are little data concerning the effect of this environment on virulence properties. Therefore, we studied the culturability of water-exposed H. pylori and determined whether there was any relation with the bacterium's ability to adhere, produce functional components of pathogenicity and induce inflammation and alterations in apoptosis in an experimental model of human gastric epithelial cells. H. pylori partially retained the ability to adhere to epithelial cells even after complete loss of culturability. However, the microorganism is no longer effective in eliciting in vitro host cell inflammation and apoptosis, possibly due to the non-functionality of the cag type IV secretion system. These H. pylori-induced host cell responses, which are lost along with culturability, are known to increase epithelial cell turnover and, consequently, could have a deleterious effect on the initial H. pylori colonisation process. The fact that adhesion is maintained by H. pylori to the detriment of other factors involved in later infection stages appears to point to a modulation of the physiology of the pathogen after water exposure and might provide the microorganism with the necessary means to, at least transiently, colonise the human stomach.
Assuntos
Aderência Bacteriana/fisiologia , Células Epiteliais/microbiologia , Helicobacter pylori/patogenicidade , Água , Antígenos de Bactérias/fisiologia , Proteínas de Bactérias/fisiologia , Sistemas de Secreção Bacterianos , Mucosa Gástrica/citologia , Helicobacter pylori/crescimento & desenvolvimento , Interações Hospedeiro-Patógeno , Humanos , Virulência/fisiologiaRESUMO
While the influence of water in Helicobacter pylori culturability and membrane integrity has been extensively studied, there are little data concerning the effect of this environment on virulence properties. Therefore, we studied the culturability of water-exposed H. pylori and determined whether there was any relation with the bacterium’s ability to adhere, produce functional components of pathogenicity and induce inflammation and alterations in apoptosis in an experimental model of human gastric epithelial cells. H. pylori partially retained the ability to adhere to epithelial cells even after complete loss of culturability. However, the microorganism is no longer effective in eliciting in vitro host cell inflammation and apoptosis, possibly due to the non-functionality of the cag type IV secretion system. These H. pylori-induced host cell responses, which are lost along with culturability, are known to increase epithelial cell turnover and, consequently, could have a deleterious effect on the initial H. pylori colonisation process. The fact that adhesion is maintained by H. pylori to the detriment of other factors involved in later infection stages appears to point to a modulation of the physiology of the pathogen after water exposure and might provide the microorganism with the necessary means to, at least transiently, colonise the human stomach.
Assuntos
Humanos , Aderência Bacteriana/fisiologia , Células Epiteliais/microbiologia , Helicobacter pylori/patogenicidade , Água , Antígenos de Bactérias/fisiologia , Sistemas de Secreção Bacterianos , Proteínas de Bactérias/fisiologia , Mucosa Gástrica/citologia , Interações Hospedeiro-Patógeno , Helicobacter pylori/crescimento & desenvolvimento , Virulência/fisiologiaRESUMO
BACKGROUND: Central nervous system (CNS) invasion by bacteria of the genus Brucella results in an inflammatory disorder called neurobrucellosis. We have recently demonstrated that B. abortus infects microglia and astrocytes, eliciting the production of a variety of pro-inflammatory cytokines which contribute to CNS damage. Matrix metalloproteinases (MMP) have been implicated in inflammatory tissue destruction in a range of pathological situations in the CNS. Increased MMP secretion is induced by pro-inflammatory cytokines in a variety of CNS diseases characterized by tissue-destructive pathology. METHODS: In this study, the molecular mechanisms that regulate MMP secretion from Brucella-infected astrocytes in vitro were investigated. MMP-9 was evaluated in culture supernatants by ELISA, zymography and gelatinolytic activity. Involvement of mitogen-activated protein kinases (MAPK) signaling pathways was evaluated by Western blot and using specific inhibitors. The role of TNF-α was evaluated by ELISA and by assays with neutralizing antibodies. RESULTS: B. abortus infection induced the secretion of MMP-9 from murine astrocytes in a dose-dependent fashion. The phenomenon was independent of bacterial viability and was recapitulated by L-Omp19, a B. abortus lipoprotein model, but not its LPS. B. abortus and L-Omp19 readily activated p38 and Erk1/2 MAPK, thus enlisting these pathways among the kinase pathways that the bacteria may address as they invade astrocytes. Inhibition of p38 or Erk1/2 significantly diminished MMP-9 secretion, and totally abrogated production of this MMP when both MAPK pathways were inhibited simultaneously. A concomitant abrogation of B. abortus- and L-Omp19-induced TNF-α production was observed when p38 and Erk1/2 pathways were inhibited, indicating that TNF-α could be implicated in MMP-9 secretion. MMP-9 secretion induced by B. abortus or L-Omp19 was completely abrogated when experiments were conducted in the presence of a TNF-α neutralizing antibody. MMP-9 activity was detected in cerebrospinal fluid (CSF) samples from patients suffering from neurobrucellosis. CONCLUSIONS: Our results indicate that the inflammatory response elicited by B. abortus in astrocytes would lead to the production of MMP-9 and that MAPK may play a role in this phenomenon. MAPK inhibition may thus be considered as a strategy to control inflammation and CNS damage in neurobrucellosis.
Assuntos
Brucella abortus , Brucelose/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/fisiologia , Animais , Anticorpos Bloqueadores/farmacologia , Antígenos de Bactérias/fisiologia , Astrócitos/metabolismo , Astrócitos/microbiologia , Astrócitos/fisiologia , Proteínas da Membrana Bacteriana Externa/fisiologia , Citocinas/metabolismo , Gelatinases/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , Lipopolissacarídeos/farmacologia , Lipoproteínas/farmacologia , Lipoproteínas/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Cultura Primária de Células , Transdução de Sinais/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
Bacillus anthracis es un bacilo gram positivo del grupo Bacillus cereus, que posee un genoma extremadamente monomórfco y comparte gran similitud fsiológica y de estructura genética con B. cereus y Bacillus thuringiensis. En este artículo se describen nuevos métodos moleculares para la identifcación y tipifcación de B. anthracis, basados en repeticiones en tándem de número variable o en diferencias genéticas detectadas por secuenciación, desarrollados en los últimos años. Los aspectos moleculares de los factores de virulencia tradicionales, cápsula, antígeno protector, factor letal y factor edema se describen en profundidad, junto con factores de virulencia recientemente propuestos, como los sideróforos, petrobactina y bacilibactina, la adhesina de la capa S y la lipoproteína MntA. También se detalla la organización molecular de los megaplásmidos pXO1 y pXO2, incluyendo la isla de patogenicidad de pXO1. El esqueleto genético de estos plásmidos se ha encontrado en otras especies relacionadas, probablemente debido a eventos de transferencia lateral. Finalmente, se presentan los dos receptores celulares del antígeno protector, ANTXR1/TEM8 y ANTXR2/CMG2, esenciales en la interacción del patógeno con el hospedador. Los estudios moleculares realizados en los últimos años han permitido aumentar enormemente el conocimiento de los diferentes aspectos de este microorganismo y su relación con el hospedador, pero a la vez han abierto nuevos interrogantes sobre este notorio patógeno.
Bacillus anthracis, a gram-positive rod belonging to the Bacillus cereus group, has an extremely monomorphic genome, and presents high structural and physiological similarity with B. cereus and Bacillus thuringiensis. In this work, the new molecular methods for the identifcation and typing of B. anthracis developed in the last years, based on variable number tandem repeats or on genetic differences detected through sequencing, are described. The molecular aspects of traditional virulence factors: capsule, protective antigen, lethal factor and edema factor are described in depth, together with virulence factors recently proposed, such as the siderophores petrobactin and bacillibactin, the S-layer adhesin and the MntA lipoprotein. It is detailed the molecular organization of megaplasmids pXO1 and pXO2, including the pathogenicity island of pXO1. The genetic skeleton of these plasmids has been observed in related species, and this could be attributed to lateral gene transfer. Finally, the two anthrax toxin protective antigen receptors, ANTXR1/TEM8 and ANTXR2/CMG2, essential for the interaction of the pathogen with the host, are presented. The molecular studies performed in recent years have greatly increased knowledge in different aspects of this microorganism and its relationship with the host, but at the same time they have raised new questions about this noted pathogen.
Assuntos
Animais , Humanos , Antraz/microbiologia , Bacillus anthracis/fisiologia , Antraz/epidemiologia , Antraz/veterinária , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/fisiologia , Toxinas Bacterianas , Técnicas de Tipagem Bacteriana , Sequência de Bases , Bacillus anthracis/classificação , Bacillus anthracis/genética , Bacillus anthracis/patogenicidade , Bacillus/classificação , Cápsulas Bacterianas/fisiologia , DNA Bacteriano/genética , Ilhas Genômicas/fisiologia , Repetições Minissatélites , Dados de Sequência Molecular , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Plasmídeos , Polimorfismo de Nucleotídeo Único , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/fisiologia , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Virulência/genética , Virulência/fisiologia , ZoonosesRESUMO
Bacillus anthracis, a gram-positive rod belonging to the Bacillus cereus group, has an extremely monomorphic genome, and presents high structural and physiological similarity with B. cereus and Bacillus thuringiensis. In this work, the new molecular methods for the identification and typing of B. anthracis developed in the last years, based on variable number tandem repeats or on genetic differences detected through sequencing, are described. The molecular aspects of traditional virulence factors: capsule, protective antigen, lethal factor and edema factor are described in depth, together with virulence factors recently proposed, such as the siderophores petrobactin and bacillibactin, the S-layer adhesin and the MntA lipoprotein. It is detailed the molecular organization of megaplasmids pXO1 and pXO2, including the pathogenicity island of pXO1. The genetic skeleton of these plasmids has been observed in related species, and this could be attributed to lateral gene transfer. Finally, the two anthrax toxin protective antigen receptors, ANTXR1/TEM8 and ANTXR2/CMG2, essential for the interaction of the pathogen with the host, are presented. The molecular studies performed in recent years have greatly increased knowledge in different aspects of this microorganism and its relationship with the host, but at the same time they have raised new questions about this noted pathogen.
Assuntos
Antraz/microbiologia , Bacillus anthracis/fisiologia , Animais , Antraz/epidemiologia , Antraz/veterinária , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/fisiologia , Bacillus/classificação , Bacillus anthracis/classificação , Bacillus anthracis/genética , Bacillus anthracis/patogenicidade , Cápsulas Bacterianas/fisiologia , Toxinas Bacterianas , Técnicas de Tipagem Bacteriana , Sequência de Bases , DNA Bacteriano/genética , Ilhas Genômicas/fisiologia , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Proteínas dos Microfilamentos , Repetições Minissatélites , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Plasmídeos , Polimorfismo de Nucleotídeo Único , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/fisiologia , Receptores de Peptídeos , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Virulência/genética , Virulência/fisiologia , ZoonosesRESUMO
BACKGROUND: To date, the function of many hypothetical membrane proteins of Mycobacterium tuberculosis is still unknown and their involvement in pathogen-host interactions has not been yet clearly defined. In this study, the biological activity of peptides derived from the hypothetical membrane protein Rv0679c of M. tuberculosis and their involvement in pathogen-host interactions was assessed. Transcription of the Rv0679c gene was studied in 26 Mycobacterium spp. Strains. Antibodies raised against putative B-cell epitopes of Rv0679c were used in Western blot and immunoelectron microscopy assays. Synthetic peptides spanning the entire length of the protein were tested for their ability to bind to A549 and U937 cells. High-activity binding peptides (HABPs) identified in Rv0679c were tested for their ability to inhibit mycobacterial invasion into cells. RESULTS: The gene encoding Rv0679c was detected in all strains of the M. tuberculosis complex (MTC), but was only transcribed in M. tuberculosis H37Rv, M. tuberculosis H37Ra and M. africanum. Anti-Rv0679c antibodies specifically recognized the protein in M. tuberculosis H37Rv sonicate and showed its localization on mycobacterial surface. Four HABPs inhibited invasion of M. tuberculosis to target cells by up to 75%. CONCLUSIONS: The results indicate that Rv0679c HABPs and in particular HABP 30979 could be playing an important role during M. tuberculosis invasion of host cells, and therefore could be interesting research targets for studies aimed at developing strategies to control tuberculosis.
Assuntos
Antígenos de Bactérias/fisiologia , Proteínas de Bactérias/fisiologia , Interações Hospedeiro-Patógeno , Mycobacterium tuberculosis/patogenicidade , Fatores de Virulência/fisiologia , Anticorpos Antibacterianos/isolamento & purificação , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Western Blotting , Linhagem Celular , Células Epiteliais/microbiologia , Epitopos de Linfócito B/imunologia , Perfilação da Expressão Gênica , Humanos , Microscopia Imunoeletrônica , Monócitos/microbiologia , Ligação Proteica , Vacinas contra a Tuberculose/imunologia , Fatores de Virulência/imunologiaRESUMO
AIMS: Our objective was to evaluate the association between the MMP1-1607 single-nucleotide polymorphism (SNP), periodontopathogens and inflammatory cytokines with matrix metalloproteinase-1 (MMP-1) mRNA levels in vitro and in vivo. MATERIALS AND METHODS: This study investigated the influence of genetic (MMP1-1607 SNP), microbial (Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Actinobacillus actinomycetemcomitans) and inflammatory [tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta)] factors on the determination of MMP-1 mRNA levels in periodontal tissues of non-smoker chronic periodontitis (CP, N=178) and control (C, N=190) groups. The effects of single and repeated lipopolysaccharide (LPS) and inflammatory cytokine stimulation of macrophages with distinct MMP1-1607 SNP genotypes were also investigated. RESULTS: In healthy tissues, the MMP1-1607 2G allele was associated with higher MMP-1 levels while in CP MMP-1 levels were associated with the presence and load of periodontopathogens, and also with TNF-alpha and IL-1beta expression irrespective of the MMP1-1607 genotype. In vitro data demonstrate that in 2G macrophages low- and intermediate-dose LPS and TNF-alpha+IL-1beta stimulation was associated with increased MMP-1 expression, while strong and repeated stimulation resulted in higher MMP-1 levels irrespective of the MMP1-1607 genotype. CONCLUSION: Our data demonstrate a limited role for MMP1-1607 SNP in periodontitis, where the extensive chronic antigenic challenge exposure overcomes the genetic control and plays a major role in the determination of MMP-1 expression.
Assuntos
Bactérias Anaeróbias/fisiologia , Periodontite Crônica/enzimologia , Periodontite Crônica/microbiologia , Citocinas/metabolismo , Metaloproteinase 1 da Matriz/biossíntese , Metaloproteinase 1 da Matriz/genética , Adulto , Antígenos de Bactérias/fisiologia , Bactérias Anaeróbias/genética , Estudos de Casos e Controles , Periodontite Crônica/genética , DNA Bacteriano/análise , Feminino , Predisposição Genética para Doença , Humanos , Interleucina-1beta/metabolismo , Lipopolissacarídeos/fisiologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/análise , Análise de Regressão , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Currently available vaccines have limited effectiveness and therapeutic interventions are complicated by the difficulty in making an early diagnosis of leptospirosis. The genome of Leptospira interrogans was recently sequenced and comparative genomic analysis contributed to the identification of surface antigens, potential candidates for development of new vaccines and serodiagnosis. Lp49 is a membrane-associated protein recognized by antibodies present in sera from early and convalescent phases of leptospirosis patients. Its crystal structure was determined by single-wavelength anomalous diffraction using selenomethionine-labelled crystals and refined at 2.0 A resolution. Lp49 is composed of two domains and belongs to the all-beta-proteins class. The N-terminal domain folds in an immunoglobulin-like beta-sandwich structure, whereas the C-terminal domain presents a seven-bladed beta-propeller fold. Structural analysis of Lp49 indicates putative protein-protein binding sites, suggesting a role in Leptospira-host interaction. This is the first crystal structure of a leptospiral antigen described to date.
Assuntos
Antígenos de Bactérias/química , Leptospira/imunologia , Antígenos de Bactérias/fisiologia , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/imunologia , Sítios de Ligação , Cristalografia por Raios X , Leptospira/química , Leptospirose/imunologia , Leptospirose/terapia , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de ProteínaRESUMO
Serratia marcescens strains are ubiquitous bacteria isolated from environmental niches, such as soil, water, and air, and also constitute emergent nosocomial opportunistic pathogens. Among the numerous extracellular factors that S. marcescens is able to produce, the PhlA phospholipase is the only described exoprotein secreted by the flagellar apparatus while simultaneously being a member of the flagellar regulon. To gain insight into the regulatory mechanism that couples PhlA and flagellar expression, we conducted a generalized insertional mutagenesis and screened for PhlA-deficient strains. We found that three independent mutations in the wec cluster, which impaired the assembly of enterobacterial common antigen (ECA), provoked the inhibition of PhlA expression. Swimming and swarming assays showed that in these strains, motility was severely affected. Microscopic examination and flagellin immunodetection demonstrated that a strong defect in flagellum expression was responsible for the reduced motility in the wec mutant strains. Furthermore, we determined that in the ECA-defective strains, the transcriptional cascade that controls flagellar assembly was turned off due to the down-regulation of flhDC expression. These findings provide a new perspective on the physiological role of the ECA, providing evidence that in S. marcescens, its biosynthesis conditions the expression of the flagellar regulon.
Assuntos
Antígenos de Bactérias/fisiologia , Flagelos/fisiologia , Serratia marcescens/fisiologia , Antígenos de Bactérias/genética , Sequência de Bases , Primers do DNA , Elementos de DNA Transponíveis , DNA Bacteriano/genética , Humanos , Dados de Sequência Molecular , Mutagênese , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Serratia marcescens/genética , Serratia marcescens/isolamento & purificação , Infecções Urinárias/microbiologiaRESUMO
Rheumatic fever (RF) is an autoimmune disease which affects more than 20 million children in developing countries. It is triggered by Streptococcus pyogenes throat infection in untreated susceptible individuals. Carditis, the most serious manifestation of the disease, leads to severe and permanent valvular lesions, causing chronic rheumatic heart disease (RHD). We have been studying the mechanisms leading to pathological autoimmunity in RF/RHD for the last 15 years. Our studies allowed us a better understanding of the cellular and molecular pathogenesis of RHD, paving the way for the development of a safe vaccine for a post-infection autoimmune disease. We have focused on the search for protective T and B cell epitopes by testing 620 human blood samples against overlapping peptides spanning 99 residues of the C-terminal portion of the M protein, differing by one amino acid residue. We identified T and B cell epitopes with 22 and 25 amino acid residues, respectively. Although these epitopes were from different regions of the C-terminal portion of the M protein, they showed an identical core of 16 amino acid residues. Antibodies against the B cell epitope inhibited bacterial invasion/adhesion in vitro. Our results strongly indicated that the selected T and B cell epitopes could potentially be protective against S. pyogenes.
Assuntos
Cardiopatia Reumática/prevenção & controle , Infecções Estreptocócicas/complicações , Vacinas Estreptocócicas/administração & dosagem , Streptococcus pyogenes , Animais , Antígenos de Bactérias/fisiologia , Humanos , Febre Reumática/imunologia , Febre Reumática/prevenção & controle , Cardiopatia Reumática/etiologia , Cardiopatia Reumática/imunologia , Infecções Estreptocócicas/imunologia , Vacinas Estreptocócicas/imunologia , Streptococcus pyogenes/imunologia , Vacinas de Subunidades AntigênicasRESUMO
The pathogenesis of rheumatic fever (RF) is related to autoimmune humoral and cellular responses against human tissues triggered by Streptococcus pyogenes. CD4(+) T cells are the ultimate effectors of chronic heart lesions in rheumatic heart disease (RHD). Heart-infiltrating CD4(+) T cell clones are able to recognize heart tissue and streptococcal antigens by molecular mimicry. The streptococcal M5(81-103) region, an immunodominant region, was recognized by both intralesional and peripheral T cell clones (62% and 38%, respectively). Peripheral T lymphocytes from Brazilian patients with severe RHD preferentially recognized the M5(81-96) peptide, in the context of HLA-DR7(+) and DR53(+) molecules. HLA-DR7 seems to be related to the development of multiple valvular lesions in RHD patients from different countries. In addition, the fact that peripheral and intralesional T cells recognized the M5(81-103) region points to this region as one of the streptococcal triggers of autoimmune reactions in RHD. T cell repertoire analysis from peripheral and intralesional T cell lines derived from RHD patients showed several oligoclonal expansions of BV families. Major expansions were found in the heart lesions, suggesting that such T cell populations preferentially migrate from the periphery to the heart. Some cross-reactive intralesional T cell clones displayed the same T cell receptor (TCR) BVBJ and CDR3 sequences, showing a degenerate pattern of antigen recognition. Heart tissue-infiltrating cells from myocardium and valvular tissue produced TNF-alpha, IFN-gamma, IL-10, and IL-4, whereas few cells from valvular tissue produced IL-4, showing that the lack of regulation in the valves could be responsible for the permanent and progressive valvular lesions.
Assuntos
Febre Reumática/etiologia , Cardiopatia Reumática/etiologia , Streptococcus pyogenes/imunologia , Linfócitos T/imunologia , Antígenos de Bactérias/fisiologia , Autoimunidade , Proteínas da Membrana Bacteriana Externa/fisiologia , Proteínas de Transporte/fisiologia , Citocinas/biossíntese , Humanos , Febre Reumática/imunologia , Cardiopatia Reumática/imunologiaRESUMO
On the Mycobacterium tuberculosis genome there are four mce operons, all of which are similar in sequence and organization, and code for putatively exported proteins. To investigate whether Mce proteins are essential for virulence, we generated knock-out mutants in mce1, mce2 and mce3 operons of M. tuberculosis and evaluated their ability to multiply in a mammalian host. The allelic replacement was confirmed in each mutant strain by Southern blotting. RT-PCR experiments demonstrated the lack of in vitro expression of mutated genes in Deltamce1 and Deltamce2 mutants. On the other hand, no expression of mce3 was detected in either the wild-type or mutant strains. Similar doubling time and growth characteristics in in vitro culture were observed for mutants and parental strains. The intratracheal route was used to infect BALB/c mice with the Deltamce3, Deltamce2 and Deltamce1 mutants. Ten weeks after infection, all mice infected with the Deltamce mutants survived, while those infected with the wild-type strain died. This long survival correlated with very low counts of colony-forming units (CFU) in the lungs. Deltamce1-infected mice developed very few and small granulomas, while animals infected with Deltamce3 or Deltamce2 mutants showed delayed granuloma formation. Mice infected with Deltamce1 did not develop pneumonia, while animals infected with Deltamce3 and Deltamce2 mutants showed small pneumonic patches. In spleens, bacterial counts of mutant strains were less reduced than in lungs, compared with those of wild-type. In contrast, no such attenuation was observed when the intraperitoneal route was used for infection. Moreover, Deltamce1 mutants appear to be more virulent in lungs after intraperitoneal inoculation. In conclusion, mce operons seem to affect the virulence of M. tuberculosis in mice, depending on the route of infection. Hypotheses are discussed to explain this last issue. Thus, mutants in these genes seem to be good candidates for vaccine testing.
Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Óperon/genética , Animais , Antígenos de Bactérias/fisiologia , Proteínas de Bactérias/fisiologia , Sequência de Bases , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mutação , Fatores de Tempo , Tuberculose Pulmonar/microbiologia , Virulência/genéticaRESUMO
At present, the genomes of various microorganisms have been completely sequenced, and many others are in progress. The availability of this level of information and the computational analysis of the described sequences have led to the development of new genomic areas such as: analysis in silico, comparative genomics, functional genomics, transcriptomics, proteomics, and pharmacogenomics. Microarray technology is a powerful tool for analyzing the expression profile of thousands of genes in a global way and can be applied to the study of various biological systems. Using the complete sequences for both the H. pylori and human genome that are available in the data bases, a number of researchers have revealed important information. Some of these data offer a glimpse into the great genetic diversity of H. pylori, the differential genetic expression between the strains that shows the complexity of the response of microorganisms to different conditions of development, and into the association of gene cluster expression with clinical outcome. Other groups have examined the global transcriptional response of gastric epithelial cells to H. pylori. The majority of these studies report an alteration in gene expression related to transcription functions, transduction signals, cell cycle regulation and differentiation, development factors, proliferation/apoptosis balance, expression of membrane proteins, and inflammatory response.
Assuntos
Helicobacter pylori/genética , Análise de Sequência com Séries de Oligonucleotídeos , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiologia , Gastrite/microbiologia , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Variação Genética , Genoma Bacteriano , Ilhas Genômicas/genética , Infecções por Helicobacter/microbiologia , Helicobacter pylori/patogenicidade , Humanos , Concentração de Íons de Hidrogênio , Neoplasias Gástricas/etiologia , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologia , Células Tumorais Cultivadas/metabolismo , Virulência/genéticaRESUMO
Previously, it was reported that the oxidative capacity and ability to grow on carbon sources such as pyruvate and glucose were severely diminished in the Rhizobium etli phaC::OmegaSm(r)/Sp(r) mutant CAR1, which is unable to synthesize poly-beta-hydroxybutyric acid (PHB) (M. A. Cevallos, S. Encarnación, A. Leija, Y. Mora, and J. Mora, J. Bacteriol. 178:1646-1654, 1996). By random Tn5 mutagenesis of the phaC strain, we isolated the mutants VEM57 and VEM58, both of which contained single Tn5 insertions and had recovered the ability to grow on pyruvate or glucose. Nucleotide sequencing of the region surrounding the Tn5 insertions showed that they had interrupted an open reading frame designated aniA based on its high deduced amino acid sequence identity to the aniA gene product of Sinorhizobium meliloti. R. etli aniA was located adjacent to and divergently transcribed from genes encoding the PHB biosynthetic enzymes beta-ketothiolase (PhaA) and acetoacetyl coenzyme A reductase (PhaB). An aniA::Tn5 mutant (VEM5854) was constructed and found to synthesize only 40% of the wild type level of PHB. Both VEM58 and VEM5854 produced significantly more extracellular polysaccharide than the wild type. Organic acid excretion and levels of intracellular reduced nucleotides were lowered to wild-type levels in VEM58 and VEM5854, in contrast to those of strain CAR1, which were significantly elevated. Proteome analysis of VEM58 showed a drastic alteration of protein expression, including the absence of a protein identified as PhaB. We propose that the aniA gene product plays an important role in directing carbon flow in R. etli.
Assuntos
Antígenos de Bactérias/fisiologia , Proteínas da Membrana Bacteriana Externa/fisiologia , Proteínas de Bactérias/biossíntese , Rhizobium/metabolismo , Aciltransferases/fisiologia , Sequência de Aminoácidos , Antígenos de Bactérias/química , Proteínas da Membrana Bacteriana Externa/química , Clonagem Molecular , Elementos de DNA Transponíveis , Teste de Complementação Genética , Glicogênio/metabolismo , Hidroxibutiratos/metabolismo , Dados de Sequência Molecular , Família Multigênica , Poliésteres/metabolismo , Polissacarídeos Bacterianos/metabolismo , Proteoma , Ácido Pirúvico/metabolismo , Rhizobium/genética , SimbioseRESUMO
The Rhizobium etli poly-beta-hydroxybutyrate synthase (PhaC) mutant SAM100 grows poorly with pyruvate as the carbon source. The inactivation of aniA, encoding a global carbon flux regulator, in SAM100 restores growth of the resulting double mutant (VEM58) on pyruvate. Pyruvate carboxylase (PYC) activity, pyc gene transcription, and holoenzyme content, which were low in SAM100, were restored in strain VEM58. The genetically engineered overexpression of PYC in SAM100 also allowed its growth on pyruvate. The possible relation between AniA, pyc transcription, and reduced-nucleotide levels is discussed.
Assuntos
Aciltransferases/fisiologia , Antígenos de Bactérias/fisiologia , Proteínas da Membrana Bacteriana Externa/fisiologia , Ácido Pirúvico/metabolismo , Rhizobium/metabolismo , Ciclo do Ácido Cítrico , Mutação , Piruvato Carboxilase/genética , Piruvato Carboxilase/metabolismo , Rhizobium/genética , Transcrição GênicaRESUMO
BACKGROUND: This study was carried out with the aim of detecting possible differences between proteins secreted by fresh wild isolates of Mycobacterium tuberculosis and from a reference strain of this microorganism, H37Rv TMCC 102. MATERIALS AND METHODS: This reference strain of M. tuberculosis has been in our laboratory for over 10 years, where it has been maintained by serial subcultures in PBY and Lowenstein-Jensen media. Patterns of protein secretion and recognition by sera derived from both tuberculosis patients and normal individuals were analyzed by electrophoresis and Western blotting. RESULTS: No major qualitative differences were observed among the several strains studied with respect to protein patterns or recognition of these proteins by test sera. Normal sera were found to react with almost all antigens recognized by tuberculosis sera, but with less intensity. However, a small protein of 14.5 kDa, secreted by both the wild and reference strains of M. tuberculosis, was recognized by 32 of the 40 tuberculous patient sera tested (80%), and was not recognized by any of the 40 serum samples derived from healthy individuals. CONCLUSIONS: This small protein seems to be a potentially important antigen for the serological diagnosis of tuberculosis and/or for use in the follow-up of patients who received treatment.
Assuntos
Antígenos de Bactérias/fisiologia , Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/imunologia , Estudos de Casos e Controles , Humanos , Padrões de ReferênciaRESUMO
Background. This study was carried out with the aim of detecting possible differences between proteins secreted by fresh wild isolates of Mycrobacterium tuberculosis and from a reference strain of this microorganism, H37Rv TMCC 102. Materials and Methods. This reference strain of M tuberculosis has been in our laboratory for over 10 years, where it has been maintained by serial subcultures in PBY and Lo-wenstein-Jensen media. Patterns of protein secretion and recognition by sera derive from both tuberculosis patients and normal individuals were analysed by electrophoresis and Western blotting. Results. No major qualitative differences were observed among the several strains studied with respect to protein patterns or recognition of these proteins by test sera. Normal sera were found to react with almost all antigens recognized by tuberculosis sera, but with less intensity. However, a small protein of 14.5 kDa, secreted by both the wild and reference strain of M. tuberculosis, was recognized by 32 of the 40 tuberculous patient sera tested (80 percent), and was not recognized by any of the 40 serum samples derived from healthy individuals. Conclusions. This small protein seems to be a potentially important antigen for the serological diagnosis of tuberculosis and/or for use in the follow-up patients who received treatment
Assuntos
Humanos , Antígenos de Bactérias/fisiologia , Mycobacterium tuberculosis/imunologia , Proteínas de Bactérias , Estudos de Casos e Controles , Padrões de ReferênciaRESUMO
It has proved difficult to vaccinate effectively against tuberculosis with mycobacterial components or even with whole dead mycobacteria; protection was always inferior to that obtained with the live attenuated vaccine known as bacillus Calmette-Guérin (BCG). We have found that this may no longer be the case. Expression of the gene for a single mycobacterial antigen (Mycobacterium leprae hsp65) in adult BALB/c mice resulted in substantial cell-mediated protection against challenge with M. tuberculosis, but only when it was generated as an endogenous antigen within antigen-presenting cells. CD4 and CD8 T cells cloned from spleens of immunized mice passively transferred protection to non-immunized mice, and CD8 cells selectively lysed macrophages infected with M. tuberculosis. The ability of the clones to protect recipient mice against challenge infection was most strongly associated with specific cytotoxic capacity and secondarily with IFN-gamma production. Three modes of expressing the gene have been tested: a) expression from a retroviral vector (pZIPNeoSV) in implanted J774 tumor cells, b) expression from the same vector via bone marrow cells transfected in vitro and used to reconstitute irradiated mice, and c) in a preliminary experiment, from cytomegalovirus (CMV) immediate-early and hydroxymethylglutaryl Co-A reductase promoters injected as plasmid DNA into muscle.