Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 16 de 16
Filtrar
1.
Mol Microbiol ; 113(1): 52-67, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31587365

RESUMO

The heparin-binding hemagglutinin (HBHA) is a multifunctional protein involved in adherence of Mycobacterium tuberculosis to non-phagocytic cells and in the formation of intracytosolic lipid inclusions. We demonstrate that the expression of hbhA is regulated by a transcriptional repressor, named HbhR, in Mycobacterium marinum. The hbhR gene, located upstream of hbhA, was identified by screening a transposon insertion library and detailed analysis of a mutant overproducing HBHA. HbhR was found to repress both hbhA and hbhR transcription by binding to the promoter regions of both genes. Complementation restored production of HBHA. RNA-seq analyses comparing the mutant and parental strains uncovered 27 genes, including hbhA, that were repressed and 20 genes activated by HbhR. Among the former, the entire locus of genes coding for a type-VII secretion system, including esxA, esxB and pe-ppe paralogs, as well as the gene coding for PspA, present in intracellular lipid vesicles, was identified, as was katG, a gene involved in the sensitivity to isoniazid. The latter category contains genes that play a role in diverse functions, such as metabolism and resistance to oxidative conditions. Thus, HbhR appears to be a master regulator, linking the transcriptional regulation of virulence, metabolic and antibiotic sensitivity genes in M. marinum.


Assuntos
Proteínas de Bactérias/metabolismo , Lectinas/metabolismo , Mycobacterium marinum/genética , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Mycobacterium marinum/metabolismo , Mycobacterium marinum/patogenicidade , Fatores de Transcrição/genética , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
2.
Front Immunol ; 14: 1254276, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37841236

RESUMO

Bordetella pertussis is a highly contagious respiratory pathogen responsible for whooping-cough or pertussis. Despite high vaccination coverage worldwide, this gram-negative bacterium continues to spread among the population. B. pertussis is transmitted by aerosol droplets from an infected individual to a new host and will colonize its upper respiratory tract. Alveolar macrophages (AMs) are effector cells of the innate immune system that phagocytose B. pertussis and secrete both pro-inflammatory and antimicrobial mediators in the lungs. However, understanding their role in B. pertussis pathogenesis at the molecular level is hampered by the limited number of primary AMs that can be collected in vivo. In order to decipher the regulation of innate response induced by B. pertussis infection, we used for the first time self-renewing, non-transformed cells, called Max Planck Institute (MPI) cells, which are phenotypically and functionally very close to pulmonary AMs. Using optimized infection conditions, we characterized the entry and the clearance of B. pertussis within MPI macrophages. We showed that under these conditions, MPI cells exhibit a pro-inflammatory phenotype with the production of TNF, IL-1ß, IL-6 and MIP-2α, similarly to primary AMs purified from broncho-alveolar fluids of mice. In addition, we explored the yet uncharacterized role of the signal transduction activator of transcription (STAT) proteins family in the innate immune response to B. pertussis infection and showed for the first time the parallel regulation of pro-inflammatory cytokines by STAT3 and STAT5 in MPI macrophages infected by B. pertussis. Altogether, this work highlights the interest of using MPI cells for experiments optimization and preliminary data acquisition to understand B. pertussis interaction with AMs, and thus significantly reduce the number of animals to be sacrificed.


Assuntos
Macrófagos Alveolares , Coqueluche , Animais , Camundongos , Macrófagos Alveolares/metabolismo , Bordetella pertussis , Fator de Transcrição STAT5/metabolismo , Citocinas/metabolismo
3.
Nat Med ; 10(9): 935-41, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15300244

RESUMO

Although post-translational modifications of protein antigens may be important componenets of some B cell epitopes, the determinants of T cell immunity are generally nonmodified peptides. Here we show that methylation of the Mycobacterium tuberculosis heparin-binding hemagglutinin (HBHA) by the bacterium is essential for effective T cell immunity to this antigen in infected healthy humans and in mice. Methylated HBHA provides high levels of protection against M. tuberculosis challenge in mice, whereas nonmethylated HBHA does not. Protective immunity induced by methylated HBHA is comparable to that afforded by vaccination with bacille Calmette et Guérin, the only available anti-tuberculosis vaccine. Thus, post-translational modifications of proteins may be crucial for their ability to induce protective T cell-mediated immunity against infectious diseases such as tuberculosis.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Mycobacterium tuberculosis/metabolismo , Processamento de Proteína Pós-Traducional/imunologia , Linfócitos T/imunologia , Tuberculose/imunologia , Vacinação , Animais , Proteínas de Bactérias/imunologia , Bélgica , Testes Imunológicos de Citotoxicidade , Feminino , Humanos , Interferon gama/metabolismo , Proteínas de Membrana/imunologia , Metilação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Tuberculose/prevenção & controle
4.
J Proteome Res ; 9(12): 6467-78, 2010 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-20949974

RESUMO

Peptide microarrays are useful tools for the characterization of humoral responses against peptide antigens. The study of post-translational modifications requires the printing of appropriately modified peptides, whose synthesis can be time-consuming and expensive. We describe here a method named "chips from chips", which allows probing the presence of antibodies directed toward modified peptide antigens starting from unmodified peptide microarrays. The chip from chip concept is based on the modification of peptide microspots by simple chemical reactions. The starting peptide chip (parent chip) is covered by the reagent solution, thereby allowing the modification of specific residues to occur, resulting in the production of a modified peptide chip (daughter chip). Both parent and daughter chips can then be used for interaction studies. The method is illustrated using reductive methylation for converting lysines into dimethyllysines. The rate of methylation was studied using specific antibodies and fluorescence detection, or surface-assisted laser desorption ionization mass spectrometry. This later technique showed unambiguously the efficient methylation of the peptide probes. The method was then used to study the humoral response against the Mycobacterium tuberculosis heparin-binding hemagglutinin, a methylated surface-associated virulence factor and powerful diagnostic and protective antigen.


Assuntos
Espectrometria de Massas/métodos , Análise em Microsséries/métodos , Peptídeos/análise , Proteômica/métodos , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/sangue , Feminino , Lectinas/imunologia , Lectinas/metabolismo , Metilação , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mycobacterium tuberculosis/metabolismo , Peptídeos/metabolismo , Proteínas/imunologia , Proteínas/metabolismo , Reprodutibilidade dos Testes
5.
ACS Nano ; 13(4): 3992-4007, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30822386

RESUMO

Multi-drug-resistant tuberculosis (TB) is a major public health problem, concerning about half a million cases each year. Patients hardly adhere to the current strict treatment consisting of more than 10 000 tablets over a 2-year period. There is a clear need for efficient and better formulated medications. We have previously shown that nanoparticles made of cross-linked poly-ß-cyclodextrins (pßCD) are efficient vehicles for pulmonary delivery of powerful combinations of anti-TB drugs. Here, we report that in addition to being efficient drug carriers, pßCD nanoparticles are endowed with intrinsic antibacterial properties. Empty pßCD nanoparticles are able to impair Mycobacterium tuberculosis (Mtb) establishment after pulmonary administration in mice. pßCD hamper colonization of macrophages by Mtb by interfering with lipid rafts, without inducing toxicity. Moreover, pßCD provoke macrophage apoptosis, leading to depletion of infected cells, thus creating a lung microenvironment detrimental to Mtb persistence. Taken together, our results suggest that pßCD nanoparticles loaded or not with antibiotics have an antibacterial action on their own and could be used as a carrier in drug regimen formulations effective against TB.


Assuntos
Antituberculosos/uso terapêutico , Portadores de Fármacos/uso terapêutico , Mycobacterium tuberculosis/efeitos dos fármacos , Nanopartículas/uso terapêutico , Tuberculose/tratamento farmacológico , beta-Ciclodextrinas/uso terapêutico , Animais , Antituberculosos/administração & dosagem , Portadores de Fármacos/administração & dosagem , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/microbiologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Nanopartículas/administração & dosagem , beta-Ciclodextrinas/administração & dosagem
6.
PLoS Pathog ; 2(7): e65, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16839199

RESUMO

Pertussis is still among the principal causes of death worldwide, and its incidence is increasing even in countries with high vaccine coverage. Although all age groups are susceptible, it is most severe in infants too young to be protected by currently available vaccines. To induce strong protective immunity in neonates, we have developed BPZE1, a live attenuated Bordetella pertussis strain to be given as a single-dose nasal vaccine in early life. BPZE1 was developed by the genetic inactivation or removal of three major toxins. In mice, BPZE1 was highly attenuated, yet able to colonize the respiratory tract and to induce strong protective immunity after a single nasal administration. Protection against B. pertussis was comparable to that induced by two injections of acellular vaccine (aPV) in adult mice, but was significantly better than two administrations of aPV in infant mice. Moreover, BPZE1 protected against Bordetella parapertussis infection, whereas aPV did not. BPZE1 is thus an attractive vaccine candidate to protect against whooping cough by nasal, needle-free administration early in life, possibly at birth.


Assuntos
Vacinas Bacterianas/uso terapêutico , Bordetella pertussis/imunologia , Bordetella pertussis/patogenicidade , Coqueluche/imunologia , Coqueluche/prevenção & controle , Administração Intranasal , Fatores Etários , Animais , Animais Recém-Nascidos/imunologia , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Vacinas Bacterianas/administração & dosagem , Bordetella pertussis/genética , Relação Dose-Resposta a Droga , Feminino , Imunização/métodos , Camundongos , Camundongos Endogâmicos BALB C , Sistema Respiratório/microbiologia , Sistema Respiratório/patologia , Vacinas Acelulares/administração & dosagem , Vacinas Acelulares/uso terapêutico , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/uso terapêutico , Coqueluche/fisiopatologia
7.
Front Microbiol ; 9: 2258, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30333800

RESUMO

The heparin-binding hemagglutinin adhesin (HBHA) is an important virulence factor of Mycobacterium tuberculosis. It is a surface-displayed protein that serves as an adhesin for non-phagocytic cells and is involved in extra-pulmonary dissemination of the tubercle bacillus. It is also an important latency antigen useful for the diagnosis of latently M. tuberculosis-infected individuals. Using fluorescence time-lapse microscopy on mycobacteria that produce HBHA-green fluorescent protein chimera, we show here that HBHA can be found at two different locations and dynamically alternates between the mycobacterial surface and the interior of the cell, where it participates in the formation of intracytosolic lipid inclusions (ILI). Compared to HBHA-producing mycobacteria, HBHA-deficient mutants contain significantly lower amounts of ILI when grown in vitro or within macrophages, and the sizes of their ILI are significantly smaller. Lipid-binding assays indicate that HBHA is able to specifically bind to phosphatidylinositol and in particular to 4,5 di-phosphorylated phosphatidylinositol, but not to neutral lipids, the main constituents of ILI. HBHA derivatives lacking the C-terminal methylated, lysine-rich repeat region fail to bind to these lipids and these derivatives also fail to complement the phenotype of HBHA-deficient mutants. These studies indicate that HBHA is a moonlighting protein that serves several functions depending on its location. When surface exposed, HBHA serves as an adhesin, and when intracellularly localized, it participates in the generation of ILI, possibly as a cargo to transport phospholipids from the plasma membrane to the ILI in the process of being formed.

8.
Tuberculosis (Edinb) ; 86(3-4): 303-9, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16510310

RESUMO

Interactions of Mycobacterium tuberculosis with macrophages have long been recognized to be crucial to the pathogenesis of tuberculosis. The role of non-phagocytic cells is less well known. We have discovered a M. tuberculosis surface protein that interacts specifically with non-phagocytic cells, expresses hemagglutination activity and binds to sulfated glycoconjugates. It is therefore called heparin-binding hemagglutinin (HBHA). HBHA-deficient M. tuberculosis mutant strains are significantly impaired in their ability to disseminate from the lungs to other tissues, suggesting that the interaction with non-phagocytic cells, such as pulmonary epithelial cells, may play an important role in the extrapulmonary dissemination of the tubercle bacillus, one of the key steps that may lead to latency. Latently infected human individuals mount a strong T cell response to HBHA, whereas patients with active disease do not, suggesting that HBHA is a good marker for the immunodiagnosis of latent tuberculosis, and that HBHA-specific Th1 responses may contribute to protective immunity against active tuberculosis. Strong HBHA-mediated immuno-protection was shown in mouse challenge models. HBHA is a methylated protein and its antigenicity in latently infected subjects, as well as its protective immunogenicity strongly depends on the methylation pattern of HBHA. In both mice and man, the HBHA-specific IFN-gamma was produced by both the CD4(+) and the CD8(+) T cells. Furthermore, the HBHA-specific CD8(+) T cells expressed bactericidal and cytotoxic activities to mycobacteria-infected macrophages. This latter activity is most likely perforin mediated. Together, these observations strongly support the potential of methylated HBHA as an important component in future, acellular vaccines against tuberculosis.


Assuntos
Lectinas/imunologia , Mycobacterium tuberculosis/imunologia , Tuberculose/imunologia , Animais , Biomarcadores/análise , Células Epiteliais/imunologia , Humanos , Imunidade Celular , Interferon gama/biossíntese , Lectinas/análise , Camundongos , Subpopulações de Linfócitos T/imunologia , Tuberculose/diagnóstico , Tuberculose/microbiologia
9.
Nat Genet ; 45(2): 172-9, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23291586

RESUMO

Global spread and limited genetic variation are hallmarks of M. tuberculosis, the agent of human tuberculosis. In contrast, Mycobacterium canettii and related tubercle bacilli that also cause human tuberculosis and exhibit unusual smooth colony morphology are restricted to East Africa. Here, we sequenced and analyzed the whole genomes of five representative strains of smooth tubercle bacilli (STB) using Sanger (4-5× coverage), 454/Roche (13-18× coverage) and/or Illumina DNA sequencing (45-105× coverage). We show that STB isolates are highly recombinogenic and evolutionarily early branching, with larger genome sizes, higher rates of genetic variation, fewer molecular scars and distinct CRISPR-Cas systems relative to M. tuberculosis. Despite the differences, all tuberculosis-causing mycobacteria share a highly conserved core genome. Mouse infection experiments showed that STB strains are less persistent and virulent than M. tuberculosis. We conclude that M. tuberculosis emerged from an ancestral STB-like pool of mycobacteria by gain of persistence and virulence mechanisms, and we provide insights into the molecular events involved.


Assuntos
Adaptação Biológica/genética , Adaptação Biológica/imunologia , Evolução Molecular , Variação Genética , Genoma Bacteriano/genética , Mycobacterium tuberculosis/genética , Filogenia , Adaptação Biológica/fisiologia , Animais , Sequência de Bases , Análise por Conglomerados , Genômica , Sequências Repetidas Invertidas/genética , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/patogenicidade , Polimorfismo de Nucleotídeo Único/genética , Análise de Sequência de DNA , Especificidade da Espécie , Baço/virologia , Virulência
10.
Expert Rev Respir Med ; 4(3): 339-48, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20524917

RESUMO

Given that TB still constitutes a tremendous public health problem at the start of the 21st Century, it may come as a surprise that Bacillus Calmette-Guérin (BCG), developed nearly 100 years ago, is today still the only vaccine available against TB. Owing to its limited efficiency in controlling TB, much effort has been deployed to develop new, improved vaccines, with initial preclinical models showing encouraging results. However, since most individuals worldwide have been vaccinated with BCG, new vaccine developments have to be placed in that context. Consequently, several approaches explore the heterologous prime-boost strategy. In this strategy, BCG-primed immunity will be strengthened or prolonged by the administration of antigens present in BCG but formulated in a different manner; either as purified antigens in the presence of appropriate adjuvants, as DNA vaccines or as viral-encoded mycobacterial antigens.


Assuntos
Vacina BCG/administração & dosagem , Esquemas de Imunização , Imunização Secundária , Tuberculose/prevenção & controle , Animais , Vacina BCG/imunologia , Química Farmacêutica , Desenho de Fármacos , Humanos , Tuberculose/imunologia
11.
Methods Mol Biol ; 669: 135-45, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20857363

RESUMO

Peptide microarrays are useful tools for characterizing the humoral response against methylated antigens. They are usually prepared by printing unmodified and methylated peptides on substrates such as functionalized microscope glass slides. The preferential capture of antibodies by methylated peptides suggests the specific recognition of methylated epitopes. However, unmodified peptide epitopes can be masked due to their interaction with the substrate. The accessibility of unmodified peptides and thus the specificity of the recognition of methylated peptide epitopes can be probed using the in situ methylation procedure described here. Alternately, the in situ methylation of peptide microarrays allows probing the presence of antibodies directed toward methylated epitopes starting from easy-to-make and cost-effective unmodified peptide libraries. In situ methylation was performed using formaldehyde in the presence of sodium cyanoborohydride and nickel chloride. This chemical procedure converts lysine residues into mono- or dimethyl lysines.


Assuntos
Anticorpos/imunologia , Lectinas/química , Lectinas/imunologia , Fragmentos de Peptídeos/química , Análise Serial de Proteínas/métodos , Alquilação , Aminas/química , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Boroidretos/química , Formaldeído/química , Vidro/química , Lisina/química , Metilação , Camundongos , Microscopia , Dados de Sequência Molecular , Mycobacterium tuberculosis , Níquel/química , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Impressão
12.
Microbes Infect ; 11(13): 995-1001, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19635582

RESUMO

Pulmonary tuberculosis remains a major health problem. Effective vaccination strategies are urgently needed. It was previously demonstrated that purified Mycobacterium bovis BCG Heparin Binding Haemagglutinin (HBHA) is able to induce in BALB/c mice protection levels against a Mycobacterium tuberculosis infection that are similar to those offered by BCG. Here we developed a heterologous prime/boost immunisation approach using a combination of BCG and HBHA in order to increase the protective immune response. We show that the time period between BCG priming and HBHA boosting strongly influences the efficacy of the boost. The optimized vaccine protocol consisting of a BCG administration followed 8 months later by boosting with HBHA resulted in an increase in the level of protection of up to 0.7log when compared to BCG alone. These results suggest an immunisation strategy where BCG is administered to neonates and is followed by subcutaneous HBHA boosting in young adults.


Assuntos
Proteínas de Bactérias/imunologia , Imunização Secundária , Proteínas de Membrana/imunologia , Mycobacterium bovis/imunologia , Vacinas contra a Tuberculose/farmacologia , Tuberculose/prevenção & controle , Animais , Proteínas de Bactérias/farmacologia , Células Cultivadas , Modelos Animais de Doenças , Esquema de Medicação , Feminino , Interferon gama/metabolismo , Interleucina-5/metabolismo , Proteínas de Membrana/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Baço/citologia , Estatísticas não Paramétricas , Tuberculose/imunologia , Vacinas contra a Tuberculose/imunologia
13.
Expert Opin Biol Ther ; 7(11): 1665-77, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17961090

RESUMO

Mycobacterium tuberculosis is one of the most successful human pathogens. It kills every year approximately 1.5 - 2 million people, and at present a third of the human population is estimated to be infected. Fortunately, only a relatively small proportion of the infected individuals will progress to active disease, and most will maintain a latent infection. Although a latent infection is clinically silent and not contagious, it can reactivate to cause highly contagious pulmonary tuberculosis, the most prevalent form of the disease in adults. Therefore, a thorough understanding of latency and reactivation may help to develop novel control strategies against tuberculosis. The most widely held view is that the mycobacteria are imprisoned in granulomatous structures during latency, where they can survive in a non-replicating, dormant form until reactivation occurs. However, there is no hard data to sustain that the reactivating mycobacteria are indeed those that laid dormant within the granulomas. In this review an alternative model, based on evidence from early studies, as well as recent reports is presented, in which the latent mycobacteria reside outside granulomas, within non-macrophage cell types throughout the infected body. Potential implications for new diagnostic and vaccine design are discussed.


Assuntos
Mycobacterium tuberculosis/fisiologia , Vacinas contra a Tuberculose/administração & dosagem , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/prevenção & controle , Antígenos de Bactérias/imunologia , Humanos
14.
Microbiology (Reading) ; 153(Pt 3): 659-666, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17322185

RESUMO

Mycobacterium tuberculosis survives and multiplies inside macrophages of its host by modulating the expression of several genes essential for in vivo survival. An in vivo expression system has been developed, based on green fluorescent protein and kanamycin resistance, to identify M. tuberculosis genes which appear to be up-regulated in infected macrophages. A promoter-trap shuttle vector, pLL192, was constructed, containing a streptomycin resistance gene as selection marker and an artificial bicistronic operon composed of the promoterless green fluorescent protein (gfp) gene, followed by the kanamycin resistance gene. A unique BamHI site upstream of the gfp gene allowed for insertion of promoter libraries. The vector was validated by the use of known regulated or constitutive M. tuberculosis promoters. In addition, an M. tuberculosis genomic DNA library was inserted into pLL192 and then introduced into Mycobacterium bovis BCG. The recombinant BCG cells were then used to infect the J774A.1 murine macrophage-like cell line in the presence of kanamycin. Several recombinant BCG cells were thereby selected that were resistant to kanamycin within infected macrophages, but were sensitive to kanamycin when grown in vitro. The kanamycin resistance phenotype was paralleled by the fluorescence phenotype. After nucleotide sequencing, the corresponding genes were identified as mce1A, PE_PGRS63(RV3097c), Rv2232, Rv1026, Rv1635c, viuB, Rv2231(cobC) and Rv0997. Real-time PCR analysis using RNA isolated at various time points from M. tuberculosis and M. bovis BCG grown in vitro and within macrophages, confirmed the up-regulation of these genes. The level of up-regulation varied from 2- to 40-fold in macrophages compared to growth in vitro.


Assuntos
Adaptação Fisiológica/genética , Perfilação da Expressão Gênica/métodos , Genes Bacterianos , Macrófagos/microbiologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/imunologia , Animais , Linhagem Celular , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Resistência a Canamicina , Camundongos , Mycobacterium bovis/genética , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , RNA Bacteriano/análise , RNA Mensageiro/análise , Análise de Sequência de DNA , Regulação para Cima
15.
J Bacteriol ; 184(3): 654-65, 2002 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11790734

RESUMO

In the plant-pathogenic bacterium Erwinia chrysanthemi production of pectate lyases, the main virulence determinant, is modulated by a complex network involving several regulatory proteins. One of these regulators, PecS, also controls the synthesis of a blue pigment identified as indigoidine. Since production of this pigment is cryptic in the wild-type strain, E. chrysanthemi ind mutants deficient in indigoidine synthesis were isolated by screening a library of Tn5-B21 insertions in a pecS mutant. These ind mutations were localized close to the regulatory pecS-pecM locus, immediately downstream of pecM. Sequence analysis of this DNA region revealed three open reading frames, indA, indB, and indC, involved in indigoidine biosynthesis. No specific function could be assigned to IndA. In contrast, IndB displays similarity to various phosphatases involved in antibiotic synthesis and IndC reveals significant homology with many nonribosomal peptide synthetases (NRPS). The IndC product contains an adenylation domain showing the signature sequence DAWCFGLI for glutamine recognition and an oxidation domain similar to that found in various thiazole-forming NRPS. These data suggest that glutamine is the precursor of indigoidine. We assume that indigoidine results from the condensation of two glutamine molecules that have been previously cyclized by intramolecular amide bond formation and then dehydrogenated. Expression of ind genes is strongly derepressed in the pecS background, indicating that PecS is the main regulator of this secondary metabolite synthesis. DNA band shift assays support a model whereby the PecS protein represses indA and indC expression by binding to indA and indC promoter regions. The regulatory link, via pecS, between indigoidine and virulence factor production led us to explore a potential role of indigoidine in E. chrysanthemi pathogenicity. Mutants impaired in indigoidine production were unable to cause systemic invasion of potted Saintpaulia ionantha. Moreover, indigoidine production conferred an increased resistance to oxidative stress, indicating that indigoidine may protect the bacteria against the reactive oxygen species generated during the plant defense response.


Assuntos
Dickeya chrysanthemi/genética , Genes Bacterianos , Piperidonas/metabolismo , Doenças das Plantas/microbiologia , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Sequência de Bases , Betaproteobacteria/genética , Relação Dose-Resposta a Droga , Regulação Bacteriana da Expressão Gênica , Peróxido de Hidrogênio/farmacologia , Magnoliopsida/microbiologia , Dados de Sequência Molecular , Fases de Leitura Aberta , Estresse Oxidativo , Peptídeo Sintases/genética , Pigmentos Biológicos , Sequências Reguladoras de Ácido Nucleico , Proteínas Repressoras/metabolismo , Mapeamento por Restrição , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transcrição Gênica
16.
J Biol Chem ; 279(29): 30158-67, 2004 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-15140891

RESUMO

In Erwinia chrysanthemi, production of pectic enzymes is modulated by a complex network involving several regulators. One of them, PecS, which belongs to the MarR family, also controls the synthesis of various other virulence factors, such as cellulases and indigoidine. Here, the PecS consensus-binding site is defined by combining a systematic evolution of ligands by an exponential enrichment approach and mutational analyses. The consensus consists of a 23-base pair palindromic-like sequence (C(-11)G(-10)A(-9)N(-8)W(-7)T(-6)C(-5)G(-4)T(-3)A(-2))T(-1)A(0)T(1)(T(2)A(3)C(4)G(5)A(6)N(7)N(8)N(9)C(10)G(11)). Mutational experiments revealed that (i) the palindromic organization is required for the binding of PecS, (ii) the very conserved part of the consensus (-6 to 6) allows for a specific interaction with PecS, but the presence of the relatively degenerated bases located apart significantly increases PecS affinity, (iii) the four bases G, A, T, and C are required for efficient binding of PecS, and (iv) the presence of several binding sites on the same promoter increases the affinity of PecS. This consensus is detected in the regions involved in PecS binding on the previously characterized target genes. This variable consensus is in agreement with the observation that the members of the MarR family are able to bind various DNA targets as dimers by means of a winged helix DNA-binding motif. Binding of PecS on a promoter region containing the defined consensus results in a repression of gene transcription in vitro. Preliminary scanning of the E. chrysanthemi genome sequence with the consensus revealed the presence of strong PecS-binding sites in the intergenic region between fliE and fliFGHIJKLMNOPQR which encode proteins involved in the biogenesis of flagellum. Accordingly, PecS directly represses fliE expression. Thus, PecS seems to control the synthesis of virulence factors required for the key steps of plant infection.


Assuntos
Proteínas de Bactérias/fisiologia , Dickeya chrysanthemi/patogenicidade , Proteínas Repressoras/fisiologia , Motivos de Aminoácidos , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , DNA/química , Análise Mutacional de DNA , Primers do DNA/química , RNA Polimerases Dirigidas por DNA/química , Escherichia coli/metabolismo , Cinética , Ligantes , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Oligonucleotídeos/química , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Permanganato de Potássio/farmacologia , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , RNA/química , Proteínas Repressoras/metabolismo , Transcrição Gênica , Virulência
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA