RESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Lectinas/metabolismo , Mycobacterium marinum/genética , Factores de Transcripción/metabolismo , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Mycobacterium marinum/metabolismo , Mycobacterium marinum/patogenicidad , Factores de Transcripción/genética , Virulencia , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Mycobacterium tuberculosis is protected by an unusual and highly impermeable cell envelope that is critically important for the successful colonization of the host. The outermost surface of this cell envelope is formed by capsular polysaccharides that play an important role in modulating the initial interactions once the bacillus enters the body. Although the bioenzymatic steps involved in the production of the capsular polysaccharides are emerging, information regarding the ability of the bacterium to modulate the composition of the capsule is still unknown. Here, we study the mechanisms involved in regulation of mycobacterial capsule biosynthesis using a high throughput screen for gene products involved in capsular α-glucan production. Utilizing this approach we identified a group of mutants that all carried mutations in the ATP-binding cassette phosphate transport locus pst These mutants collectively exhibited a strong overproduction of capsular polysaccharides, including α-glucan and arabinomannan, suggestive of a role for inorganic phosphate (Pi) metabolism in modulating capsular polysaccharide production. These findings were corroborated by the observation that growth under low Pi conditions as well as chemical activation of the stringent response induces capsule production in a number of mycobacterial species. This induction is, in part, dependent on σ factor E. Finally, we show that Mycobacterium marinum, a model organism for M. tuberculosis, encounters Pi stress during infection, which shows the relevance of our findings in vivo.
Asunto(s)
Cápsulas Bacterianas/metabolismo , Embrión no Mamífero/metabolismo , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Mycobacterium marinum/efectos de los fármacos , Fosfatos/farmacología , Polisacáridos/metabolismo , Animales , Cápsulas Bacterianas/efectos de los fármacos , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/microbiología , Infecciones por Mycobacterium no Tuberculosas/microbiología , Pez CebraRESUMEN
Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a major pathogen responsible for 1.5 million deaths annually. This bacterium is characterized by a highly unusual and impermeable cell envelope, which plays a key role in mycobacterial survival and virulence. Although many studies have focused on the composition and functioning of the mycobacterial cell envelope, the capsular α-glucan has received relatively minor attention. Here we show that a murine monoclonal antibody (Mab) directed against glycogen cross-reacts with mycobacterial α-glucans, polymers of α(1-4)-linked glucose residues with α(1-6)-branch points. We identified the Mab epitope specificity by saturation transfer difference NMR and show that the α(1-4)-linked glucose residues are important in glucan-Mab interaction. The minimal epitope is formed by (linear) maltotriose. Notably, a Mycobacterium mutant lacking the branching enzyme GlgB does not react with the Mab; this suggests that the α(1-6)-branches form part of the epitope. These seemingly conflicting data can be explained by the fact that in the mutant the linear form of the α-glucan (amylose) is insoluble. This Mab was subsequently used to develop several techniques helpful in capsular α-glucan research. By using a capsular glucan-screening methodology based on this Mab we were able to identify several unknown genes involved in capsular α-glucan biogenesis. Additionally, we developed two methods for the detection of capsular α-glucan levels. This study therefore opens new ways to study capsular α-glucan and to identify possible targets for further research.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Cápsulas Bacterianas/metabolismo , Epítopos/inmunología , Glucógeno/inmunología , Glucógeno/metabolismo , Mycobacterium/metabolismo , Animales , Pared Celular/metabolismo , Elementos Transponibles de ADN/genética , Glucógeno/biosíntesis , Glucógeno/química , Espectroscopía de Resonancia Magnética , Ratones , Mutación , Mycobacterium/citología , Oligosacáridos/químicaRESUMEN
The causative agent of tuberculosis (TB), Mycobacterium tuberculosis, remains an important worldwide health threat. Although TB is one of the oldest infectious diseases of man, a detailed understanding of the mycobacterial mechanisms underlying pathogenesis remains elusive. Here, we studied the role of the α(1â2) mannosyltransferase MptC in mycobacterial virulence, using the Mycobacterium marinum zebrafish infection model. Like its M. tuberculosis orthologue, disruption of M. marinum mptC (mmar_3225) results in defective elongation of mannose caps of lipoarabinomannan (LAM) and absence of α(1â2)mannose branches on the lipomannan (LM) and LAM mannan core, as determined by biochemical analysis (NMR and GC-MS) and immunoblotting. We found that the M. marinum mptC mutant is strongly attenuated in embryonic zebrafish, which rely solely on innate immunity, whereas minor virulence defects were observed in adult zebrafish. Strikingly, complementation with the Mycobacterium smegmatis mptC orthologue, which restored mannan core branching but not cap elongation, was sufficient to fully complement the virulence defect of the mptC mutant in embryos. Altogether our data demonstrate that not LAM capping, but mannan core branching of LM/LAM plays an important role in mycobacterial pathogenesis in the context of innate immunity.
Asunto(s)
Lipopolisacáridos/metabolismo , Mycobacterium marinum/inmunología , Mycobacterium marinum/patogenicidad , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Animales , Carga Bacteriana , Inmunidad Innata , Lipopolisacáridos/química , Manosa/química , Infecciones por Mycobacterium no Tuberculosas/inmunología , Mycobacterium marinum/genética , Mycobacterium smegmatis/patogenicidad , Mycobacterium tuberculosis/inmunología , Mycobacterium tuberculosis/patogenicidad , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Tuberculosis/inmunología , Pez Cebra/inmunología , Pez Cebra/microbiologíaRESUMEN
Mannose-capped lipoarabinomannan (ManLAM) is considered an important virulence factor of Mycobacterium tuberculosis. However, while mannose caps have been reported to be responsible for various immunosuppressive activities of ManLAM observed in vitro, there is conflicting evidence about their contribution to mycobacterial virulence in vivo. Therefore, we used Mycobacterium bovis BCG and M. tuberculosis mutants that lack the mannose cap of LAM to assess the role of ManLAM in the interaction of mycobacteria with the host cells, to evaluate vaccine-induced protection and to determine its importance in M. tuberculosis virulence. Deletion of the mannose cap did not affect BCG survival and replication in macrophages, although the capless mutant induced a somewhat higher production of TNF. In dendritic cells, the capless mutant was able to induce the upregulation of co-stimulatory molecules and the only difference we detected was the secretion of slightly higher amounts of IL-10 as compared to the wild type strain. In mice, capless BCG survived equally well and induced an immune response similar to the parental strain. Furthermore, the efficacy of vaccination against a M. tuberculosis challenge in low-dose aerosol infection models in mice and guinea pigs was not affected by the absence of the mannose caps in the BCG. Finally, the lack of the mannose cap in M. tuberculosis did not affect its virulence in mice nor its interaction with macrophages in vitro. Thus, these results do not support a major role for the mannose caps of LAM in determining mycobacterial virulence and immunogenicity in vivo in experimental animal models of infection, possibly because of redundancy of function.
Asunto(s)
Interacciones Huésped-Patógeno , Lipopolisacáridos/análisis , Manosa/análisis , Mycobacterium bovis/inmunología , Mycobacterium tuberculosis/inmunología , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/patología , Animales , Células Dendríticas/inmunología , Células Dendríticas/microbiología , Modelos Animales de Enfermedad , Cobayas , Macrófagos/microbiología , Ratones , Viabilidad Microbiana , Mycobacterium bovis/química , Mycobacterium bovis/genética , Mycobacterium bovis/crecimiento & desarrollo , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crecimiento & desarrollo , Tuberculosis Pulmonar/microbiología , Factores de Virulencia/análisisRESUMEN
Cyanovirin-N (CV-N) is a mannose-binding lectin that inhibits HIV-1 infection by blocking mannose-dependent target cell entry via C-type lectins. Like HIV-1, Mycobacterium tuberculosis expresses mannosylated surface structures and exploits C-type lectins to gain cell access. In this study, we investigated whether CV-N, like HIV-1, can inhibit M. tuberculosis infection. We found that CV-N specifically interacted with mycobacteria by binding to the mannose-capped lipoglycan lipoarabinomannan. Furthermore, CV-N competed with the C-type lectins DC-SIGN and mannose receptor for ligand binding and inhibited the binding of M. tuberculosis to dendritic cells but, unexpectedly, not to macrophages. Subsequent in vivo infection experiments in a mouse model demonstrated that, despite its activity, CV-N did not inhibit or delay M. tuberculosis infection. This outcome argues against a critical role for mannose-dependent C-type lectin interactions during the initial stages of murine M. tuberculosis infection and suggests that, depending on the circumstances, M. tuberculosis can productively infect cells using different modes of entry.
Asunto(s)
Proteínas Bacterianas/fisiología , Proteínas Portadoras/fisiología , Lectinas Tipo C/antagonistas & inhibidores , Manosa/metabolismo , Mycobacterium tuberculosis/inmunología , Mycobacterium tuberculosis/metabolismo , Tuberculosis/inmunología , Animales , Proteínas Bacterianas/administración & dosificación , Proteínas Portadoras/administración & dosificación , Línea Celular , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/microbiología , Modelos Animales de Enfermedad , Humanos , Lectinas Tipo C/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/microbiología , Manosa/fisiología , Ratones , Ratones Endogámicos C57BL , Monocitos/inmunología , Monocitos/metabolismo , Monocitos/microbiología , Mycobacterium tuberculosis/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Unión Proteica/inmunología , Resultado del Tratamiento , Tuberculosis/microbiología , Tuberculosis/prevención & controlRESUMEN
ESX-5 is a mycobacterial type VII protein secretion system responsible for transport of numerous PE and PPE proteins. It is involved in the induction of host cell death and modulation of the cytokine response in vitro. In this work, we studied the effects of ESX-5 in embryonic and adult zebrafish using Mycobacterium marinum. We found that ESX-5-deficient M. marinum was slightly attenuated in zebrafish embryos. Surprisingly, the same mutant showed highly increased virulence in adult zebrafish, characterized by increased bacterial loads and early onset of granuloma formation with rapid development of necrotic centres. This early onset of granuloma formation was accompanied by an increased expression of pro-inflammatory cytokines and tissue remodelling genes in zebrafish infected with the ESX-5 mutant. Experiments using RAG-1-deficient zebrafish showed that the increased virulence of the ESX-5 mutant was not dependent on the adaptive immune system. Mixed infection experiments with wild-type and ESX-5 mutant bacteria showed that the latter had a specific advantage in adult zebrafish and outcompeted wild-type bacteria. Together our experiments indicate that ESX-5-mediated protein secretion is used by M. marinum to establish a moderate and persistent infection.
Asunto(s)
Eliminación de Gen , Interacciones Huésped-Patógeno , Mycobacterium marinum/genética , Mycobacterium marinum/patogenicidad , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Pez Cebra/microbiología , Animales , Carga Bacteriana , Citocinas/biosíntesis , Perfilación de la Expresión Génica , Granuloma/patología , Necrosis/patología , VirulenciaRESUMEN
Maintenance of cell-wall integrity in Mycobacterium tuberculosis is essential and is the target of several antitubercular drugs. For example, ethambutol targets arabinogalactan and lipoarabinomannan (LAM) biosynthesis through the inhibition of several arabinofuranosyltransferases. Apart from their role in cell-wall integrity, mycobacterial LAMs also exhibit important immunomodulatory activities. Here we report the isolation and detailed structural characterization of a unique LAM molecule derived from Mycobacterium smegmatis deficient in the arabinofuranosyltransferase AftC (AftC-LAM). This mutant LAM expresses a severely truncated arabinan domain completely devoid of 3,5-Araf-branching residues, revealing an intrinsic involvement of AftC in the biosynthesis of LAM. Furthermore, we found that ethambutol efficiently inhibits biosynthesis of the AftC-LAM arabinan core, unambiguously demonstrating the involvement of the arabinofuranosyltransferase EmbC in early stages of LAM-arabinan biosynthesis. Finally, we demonstrate that AftC-LAM exhibits an enhanced proinflammatory activity, which is due to its ability to activate Toll-like receptor 2 (TLR2). Overall, our efforts further describe the mechanism of action of an important antitubercular drug, ethambutol, and demonstrate a role for specific arabinofuranosyltransferases in LAM biosynthesis. In addition, the availability of sufficient amounts of chemically defined wild-type and isogenic truncated LAMs paves the way for further investigations of the structure-function relationship of TLR2 activation by mycobacterial lipoglycans.
Asunto(s)
Proteínas Bacterianas/metabolismo , Lipopolisacáridos/inmunología , Mycobacterium smegmatis/metabolismo , Pentosiltransferasa/metabolismo , Antígenos Bacterianos/química , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Antituberculosos/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Línea Celular , Línea Celular Tumoral , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Etambutol/farmacología , Humanos , Interleucina-8/metabolismo , Lipopolisacáridos/química , Lipopolisacáridos/metabolismo , Espectroscopía de Resonancia Magnética , Mutación , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium smegmatis/genética , Pentosiltransferasa/química , Pentosiltransferasa/genética , Receptor Toll-Like 2/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The cell envelope of mycobacteria, a group of Gram positive bacteria, is composed of a plasma membrane and a Gram-negative-like outer membrane containing mycolic acids. In addition, the surface of the mycobacteria is coated with an ill-characterized layer of extractable, non-covalently linked glycans, lipids and proteins, collectively known as the capsule, whose occurrence is a matter of debate. By using plunge freezing cryo-electron microscopy technique, we were able to show that pathogenic mycobacteria produce a thick capsule, only present when the cells were grown under unperturbed conditions and easily removed by mild detergents. This detergent-labile capsule layer contains arabinomannan, alpha-glucan and oligomannosyl-capped glycolipids. Further immunogenic and proteomic analyses revealed that Mycobacterium marinum capsule contains high amounts of proteins that are secreted via the ESX-1 pathway. Finally, cell infection experiments demonstrated the importance of the capsule for binding to cells and dampening of pro-inflammatory cytokine response. Together, these results show a direct visualization of the mycobacterial capsular layer as a labile structure that contains ESX-1-secreted proteins.
Asunto(s)
Cápsulas Bacterianas/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Macrófagos/microbiología , Mycobacterium/metabolismo , Mycobacterium/ultraestructura , Cápsulas Bacterianas/ultraestructura , Proteínas de la Membrana Bacteriana Externa/ultraestructura , Microscopía por Crioelectrón , Citocinas/metabolismo , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Microscopía Inmunoelectrónica , Mycobacterium bovis/metabolismo , Mycobacterium bovis/ultraestructura , Mycobacterium marinum/metabolismo , Mycobacterium marinum/ultraestructura , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/ultraestructura , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/ultraestructuraRESUMEN
Mycobacterium tuberculosis possesses a variety of immunomodulatory factors that influence the host immune response. When the bacillus encounters its target cell, the outermost components of its cell envelope are the first to interact. Mycobacteria, including M. tuberculosis, are surrounded by a loosely attached capsule that is mainly composed of proteins and polysaccharides. Although the chemical composition of the capsule is relatively well studied, its biological function is only poorly understood. The aim of this study was to further assess the functional role of the mycobacterial capsule by identifying host receptors that recognize its constituents. We focused on alpha-glucan, which is the dominant capsular polysaccharide. Here we demonstrate that M. tuberculosis alpha-glucan is a novel ligand for the C-type lectin DC-SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin). By using related glycogen structures, we show that recognition of alpha-glucans by DC-SIGN is a general feature and that the interaction is mediated by internal glucosyl residues. As for mannose-capped lipoarabinomannan, an abundant mycobacterial cell wall-associated glycolipid, binding of alpha-glucan to DC-SIGN stimulated the production of immunosuppressive IL-10 by LPS-activated monocyte-derived dendritic cells. By using specific inhibitors, we show that this IL-10 induction was DC-SIGN-dependent and also required acetylation of NF-kappaB. Finally, we demonstrate that purified M. tuberculosis alpha-glucan, in contrast to what has been reported for fungal alpha-glucan, was unable to activate TLR2.
Asunto(s)
Cápsulas Bacterianas/inmunología , Moléculas de Adhesión Celular/inmunología , Células Dendríticas/inmunología , Glucanos/inmunología , Lectinas Tipo C/inmunología , Lipopolisacáridos/inmunología , Mycobacterium tuberculosis/inmunología , Receptores de Superficie Celular/inmunología , Células Cultivadas , Células Dendríticas/efectos de los fármacos , Células Dendríticas/microbiología , Humanos , Interleucina-10/biosíntesis , Interleucina-10/inmunología , Lipopolisacáridos/metabolismo , FN-kappa B/inmunología , FN-kappa B/metabolismo , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 2/metabolismoRESUMEN
Helicobacter pylori causes gastritis, which has been attributed to the development of H. pylori-specific T cells during infection. However, the mechanism underlying innate immune detection leading to the priming of T cells is not fully understood, as H. pylori evades TLR detection. Here, we report that H. pylori metabolites modified from host cholesterol exacerbate gastritis through the interaction with C-type lectin receptors. Cholesteryl acyl α-glucoside (αCAG) and cholesteryl phosphatidyl α-glucoside (αCPG) were identified as noncanonical ligands for Mincle (Clec4e) and DCAR (Clec4b1). During chronic infection, H. pylori-specific T cell responses and gastritis were ameliorated in Mincle-deficient mice, although bacterial burdens remained unchanged. Furthermore, a mutant H. pylori strain lacking αCAG and αCPG exhibited an impaired ability to cause gastritis. Thus H. pylori-specific modification of host cholesterol plays a pathophysiological role that exacerbates gastric inflammation by triggering C-type lectin receptors.
Asunto(s)
Colesterol/metabolismo , Gastritis/metabolismo , Infecciones por Helicobacter/metabolismo , Helicobacter pylori/metabolismo , Lectinas Tipo C/metabolismo , Proteínas de la Membrana/metabolismo , Receptores Inmunológicos/metabolismo , Animales , Colesterol/genética , Enfermedad Crónica , Gastritis/genética , Gastritis/microbiología , Infecciones por Helicobacter/genética , Lectinas Tipo C/genética , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Receptores Inmunológicos/genéticaRESUMEN
The human gastric pathogen Helicobacter pylori spontaneously switches lipopolysaccharide (LPS) Lewis (Le) antigens on and off (phase-variable expression), but the biological significance of this is unclear. Here, we report that Le+ H. pylori variants are able to bind to the C-type lectin DC-SIGN and present on gastric dendritic cells (DCs), and demonstrate that this interaction blocks T helper cell (Th)1 development. In contrast, Le- variants escape binding to DCs and induce a strong Th1 cell response. In addition, in gastric biopsies challenged ex vivo with Le+ variants that bind DC-SIGN, interleukin 6 production is decreased, indicative of increased immune suppression. Our data indicate a role for LPS phase variation and Le antigen expression by H. pylori in suppressing immune responses through DC-SIGN.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Helicobacter pylori/fisiología , Lectinas Tipo C/fisiología , Lipopolisacáridos/farmacología , Receptores de Superficie Celular/fisiología , Células TH1/inmunología , Células Th2/inmunología , Fucosiltransferasas/fisiología , Humanos , Interleucina-10/biosíntesis , Interleucina-6/biosíntesis , Antígenos del Grupo Sanguíneo de Lewis/fisiología , Antígeno Lewis X/fisiología , Glicoproteínas de Membrana/fisiología , Receptores Toll-LikeRESUMEN
Autoimmune gastritis and Helicobacter pylori-associated gastric atrophy develop through similar mechanisms involving the proton pump H+,K+-adenosine triphosphatase as autoantigen. Here, we report that H. pylori-infected patients with gastric autoimmunity harbor in vivo-activated gastric CD4+ T cells that recognize both H+, K+-adenosine triphosphatase and H. pylori antigens. We characterized the submolecular specificity of such gastric T cells and identified cross-reactive epitopes from nine H. pylori proteins. Cross-reactive H. pylori peptides induced T cell proliferation and expression of T helper type 1 functions. We suggest that in genetically susceptible individuals, H. pylori infection can activate cross-reactive gastric T cells leading to gastric autoimmunity via molecular mimicry.
Asunto(s)
Antígenos Bacterianos/inmunología , Enfermedades Autoinmunes/microbiología , Gastritis Atrófica/inmunología , ATPasa Intercambiadora de Hidrógeno-Potásio/inmunología , Helicobacter pylori/inmunología , Imitación Molecular , Adulto , Autoantígenos/inmunología , Reacciones Cruzadas , Epítopos de Linfocito T/inmunología , Femenino , Mucosa Gástrica/inmunología , Gastritis Atrófica/microbiología , Antígenos HLA-DQ/inmunología , Antígenos HLA-DR/inmunología , Infecciones por Helicobacter/inmunología , Humanos , Activación de Linfocitos , Persona de Mediana Edad , Estómago/inmunología , Linfocitos T/inmunologíaRESUMEN
ESX-5 is one of the five type VII secretion systems found in mycobacteria. These secretion systems are also known as ESAT-6-like secretion systems. Here, we have determined the secretome of ESX-5 by a proteomic approach in two different strains of Mycobacterium marinum. Comparison of the secretion profile of wild-type strains and their ESX-5 mutants showed that a number of PE_PGRS and PPE-MPTR proteins are dependent on ESX-5 for transport. The PE and PPE protein families are unique to mycobacteria, are highly expanded in several pathogenic species, such as Mycobacterium tuberculosis and M. marinum, and certain family members are cell surface antigens associated with virulence. Using a monoclonal antibody directed against the PGRS domain we showed that nearly all PE_PGRS proteins that are recognized by this antibody are missing in the supernatant of ESX-5 mutants. In addition to PE_PGRS and PPE proteins, the ESX-5 secretion system is responsible for the secretion of a ESAT-6-like proteins. Together, these data show that ESX-5 is probably a major secretion pathway for mycobacteria and that this system is responsible for the secretion of recently evolved PE_PGRS and PPE proteins.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de la Membrana/metabolismo , Mycobacterium marinum/metabolismo , Proteoma , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Clonación Molecular , ADN Bacteriano/genética , Genes Bacterianos , Prueba de Complementación Genética , Genoma Bacteriano , Proteínas de la Membrana/genética , Mycobacterium marinum/genética , Vías SecretorasRESUMEN
Lipoarabinomannan (LAM) is a major glycolipid in the mycobacterial cell envelope. LAM consists of a mannosylphosphatidylinositol (MPI) anchor, a mannan core and a branched arabinan domain. The termini of the arabinan branches can become substituted with one to three α(1â2)-linked mannosyl residues, the mannose cap, producing ManLAM. ManLAM has been associated with a range of different immunomodulatory properties of Mycobacterium tuberculosis during infection of the host. In some of these effects, the presence of the mannose cap on ManLAM appears to be crucial for its activity. So far, in the biosynthesis of the mannose cap on ManLAM, two enzymes have been reported to be involved: a mannosyltransferase that adds the first mannosyl residue of the mannose caps to the arabinan domain of LAM, and another mannosyltransferase that elongates the mannose cap up to three mannosyl residues. Here, we report that a third gene is involved, MMAR_2380, which is the Mycobacterium marinum orthologue of Rv1565c. MMAR_2380 encodes a predicted transmembrane acyltransferase. In M. marinum ΔMMAR_2380, the LAM arabinan domain is still intact, but the mutant LAM lacks the mannose cap. Additional effects of mutation of MMAR_2380 on LAM were observed: a higher degree of branching of both the arabinan domain and the mannan core, and a decreased incorporation of [1,2-(14)C]acetate into the acyl chains in mutant LAM as compared with the wild-type form. This latter effect was also observed for related lipoglycans, i.e. lipomannan (LM) and phosphatidylinositol mannosides (PIMs). Furthermore, the mutant strain showed increased aggregation in liquid cultures as compared with the wild-type strain. All phenotypic traits of M. marinum ΔMMAR_2380, the deficiency in the mannose cap on LAM and changes at the cell surface, could be reversed by complementing the mutant strain with MMAR_2380. Strikingly, membrane preparations of the mutant strain still showed enzymic activity for the arabinan mannose-capping mannosyltransferase similar to that of the wild-type strain. Although the exact function of MMAR_2380 remains unknown, we show that the protein is essential for the presence of a mannose cap on LAM.
Asunto(s)
Aciltransferasas/metabolismo , Lipopolisacáridos/biosíntesis , Manosa/biosíntesis , Mycobacterium marinum/enzimología , Acilación , Aciltransferasas/genética , Genes Bacterianos , Prueba de Complementación Genética , Lipopolisacáridos/química , Manosa/química , Manosiltransferasas/metabolismo , Mutación , Mycobacterium marinum/genéticaRESUMEN
The C-type lectin dendritic cell (DC)-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) is the major receptor on DCs for mycobacteria of the Mycobacterium tuberculosis complex. Recently, we have shown that although the mannose caps of the mycobacterial surface glycolipid lipoarabinomannan (ManLAM) are essential for the binding to DC-SIGN, genetic removal of these caps did not diminish the interaction of whole mycobacteria with DC-SIGN and DCs. Here we investigated the role of the structurally related glycolipids phosphatidylinositol mannosides (PIMs) as possible ligands for DC-SIGN. In a binding assay with both synthetic and natural PIMs, DC-SIGN exhibited a high affinity for hexamannosylated PIM(6), which contains terminal alpha(1-->2)-linked mannosyl residues identical to the mannose cap on ManLAM, but not for di- and tetramannosylated PIM(2) and PIM(4), respectively. To determine the role of PIM(6) in the binding of whole mycobacteria to DC-SIGN, a mutant strain of M. bovis bacillus Calmette-Guérin deficient in the production of PIM(6) (Delta pimE) was created, as well as a double knockout deficient in the production of both PIM(6) and the mannose caps on LAM (Delta pimE Delta capA). Compared to the wild-type strain, both mutant strains bound similarly well to DC-SIGN and DCs. Furthermore, the wild-type and mutant strains induced comparable levels of interleukin-10 and interleukin-12p40 when used to stimulate DCs. Hence, we conclude that, like ManLAM, PIM(6) represents a bona fide DC-SIGN ligand but that other, as-yet-unknown, ligands dominate in the interaction between mycobacteria and DCs.
Asunto(s)
Adhesión Bacteriana , Moléculas de Adhesión Celular/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/microbiología , Lectinas Tipo C/metabolismo , Mycobacterium tuberculosis/inmunología , Fosfatidilinositoles/metabolismo , Receptores de Superficie Celular/metabolismo , Células Cultivadas , Eliminación de Gen , Humanos , Interleucina-10/metabolismo , Subunidad p40 de la Interleucina-12/metabolismo , Mycobacterium bovis/genética , Mycobacterium bovis/metabolismo , Unión ProteicaRESUMEN
Dectin-2 is a C-type lectin involved in the recognition of several pathogens such as Aspergillus fumigatus, Candida albicans, Schistosoma mansonii, and Mycobacterium tuberculosis that triggers Th17 immune responses. Identifying pathogen ligands and understanding the molecular basis of their recognition is one of the current challenges. Purified M. tuberculosis mannose-capped lipoarabinomannan (ManLAM) was shown to induce signaling via Dectin-2, an activity that requires the (α1 â 2)-linked mannosides forming the caps. Here, using isogenic M. tuberculosis mutant strains, we demonstrate that ManLAM is a bona fide and actually the sole ligand mediating bacilli recognition by Dectin-2, although M. tuberculosis produces a variety of cell envelope mannoconjugates, such as phosphatidyl-myo-inositol hexamannosides, lipomannan or manno(lipo)proteins, that bear (α1 â 2)-linked mannosides. In addition, we found that Dectin-2 can recognize lipoglycans from other bacterial species, such as Saccharotrix aerocolonigenes or the human opportunistic pathogen Tsukamurella paurometabola, suggesting that lipoglycans are prototypical Dectin-2 ligands. Finally, from a structure/function relationship perspective, we show, using lipoglycan variants and synthetic mannodendrimers, that dimannoside caps and multivalent interaction are required for ligand binding to and signaling via Dectin-2. Better understanding of the molecular basis of ligand recognition by Dectin-2 will pave the way for the rational design of potent adjuvants targeting this receptor.
Asunto(s)
Lectinas Tipo C/metabolismo , Lipopolisacáridos/metabolismo , Mycobacterium tuberculosis/metabolismo , Animales , Membrana Celular/metabolismo , Humanos , Ligandos , Lipopolisacáridos/química , Ratones Endogámicos C57BL , Transducción de SeñalRESUMEN
We have developed a multiplexed and miniaturized TB serological assay with the aim of identifying (combinations of) antigens that maximally discriminate between TB and non-TB patients. It features a microarray accommodating 54 TB antigens, less than 1 microl serum consumption and an indirect immunofluorescence detection protocol. With a panel of 20 TB and 80 non-TB sera we ranked combinations of TB antigens with respect to sensitivity and specificity of TB detection by means of logistic step-forward regression analysis. The highest-ranking TB antigen combination had an area-under-the-curve of the receiver-operator-characteristics (ROC) of 0.95. We also identified an antigen that on its own provided good specificity and sensitivity of TB detection (Ara6-BSA; area-under-the-ROC curve: 0.90). These area-under-the-ROC curve values are exceptionally high for a serological TB assay. We conclude that TB antigen microarrays permit rapid identification of TB antigens that, either alone or in combination, discriminate maximally between TB and non-TB patients and that such identification provides an excellent starting point for developing point-of-care diagnostic assays.
Asunto(s)
Antígenos Bacterianos/inmunología , Sueros Inmunes/inmunología , Mycobacterium tuberculosis/inmunología , Pruebas Serológicas/métodos , Tuberculosis/sangre , Tuberculosis/inmunología , Anticuerpos Antibacterianos/inmunología , Especificidad de Anticuerpos , Conformación de Carbohidratos , Secuencia de Carbohidratos , Humanos , Datos de Secuencia Molecular , Oligosacáridos/química , Oligosacáridos/metabolismo , Análisis por Matrices de Proteínas , Curva ROC , Sensibilidad y Especificidad , Tuberculosis/diagnóstico , Tuberculosis/microbiologíaRESUMEN
Pathogens can induce autoreactive T cells to initiate autoimmune disease by several mechanisms. Pathogen-induced inflammation results in the enhanced presentation of self antigens, which causes the expansion of the activated autoreactive T cells that are required for disease onset. Alternatively, a pathogen might express antigens with epitopes that are structurally similar to epitopes of autoantigens, resulting in a mechanism of molecular mimicry. This is the case for Helicobacter pylori-associated human autoimmune gastritis, in which the activated CD4+ Th1 cells that infiltrate the gastric mucosa cross-recognize the epitopes of self gastric parietal cell H(+)K(+)-ATPase and of various H. pylori proteins. Therefore, in genetically susceptible individuals, H. pylori infection can start or worsen gastric autoimmunity, leading to atrophic gastritis.
Asunto(s)
Enfermedades Autoinmunes/microbiología , Gastritis/microbiología , ATPasa Intercambiadora de Hidrógeno-Potásio/inmunología , Helicobacter pylori/inmunología , Imitación Molecular , Animales , Autoantígenos/inmunología , Enfermedades Autoinmunes/enzimología , Autoinmunidad , Gastritis/enzimología , Infecciones por Helicobacter/inmunología , Humanos , Células Parietales Gástricas/enzimología , Células TH1/inmunologíaRESUMEN
The gastric mucosal pathogen Helicobacter pylori induces autoantibodies directed against the gastric proton pump H+,K+-ATPase in 20-30% of infected patients. The presence of these autoantibodies is associated with severity of gastritis, increased atrophy, and apoptosis in the corpus mucosa, and patients with these autoantibodies infected with H. pylori display histopathological and clinical features that are similar to those of autoimmune gastritis (AIG). This review will focus on the T helper cell responses, cytokines, and adhesion molecules involved in corpus mucosal atrophy in chronic H. pylori gastritis and in AIG, and the role of H. pylori in the onset of AIG.