RESUMO
The pathogen Mycobacterium tuberculosis employs a range of ESX-1 substrates to manipulate the host and build a successful infection. Although the importance of ESX-1 secretion in virulence is well established, the characterization of its individual components and the role of individual substrates is far from complete. Here, we describe the functional characterization of the Mycobacterium marinum accessory ESX-1 proteins EccA1, EspG1 and EspH, i.e. proteins that are neither substrates nor structural components. Proteomic analysis revealed that EspG1 is crucial for ESX-1 secretion, since all detectable ESX-1 substrates were absent from the cell surface and culture supernatant in an espG1 mutant. Deletion of eccA1 resulted in minor secretion defects, but interestingly, the severity of these secretion defects was dependent on the culture conditions. Finally, espH deletion showed a partial secretion defect; whereas several ESX-1 substrates were secreted in normal amounts, secretion of EsxA and EsxB was diminished and secretion of EspE and EspF was fully blocked. Interaction studies showed that EspH binds EspE and therefore could function as a specific chaperone for this substrate. Despite the observed differences in secretion, hemolytic activity was lost in all M. marinum mutants, implying that hemolytic activity is not strictly correlated with EsxA secretion. Surprisingly, while EspH is essential for successful infection of phagocytic host cells, deletion of espH resulted in a significantly increased virulence phenotype in zebrafish larvae, linked to poor granuloma formation and extracellular outgrowth. Together, these data show that different sets of ESX-1 substrates play different roles at various steps of the infection cycle of M. marinum.
Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium marinum/metabolismo , Mycobacterium marinum/patogenicidade , Sistemas de Secreção Tipo VII/genética , Fatores de Virulência/fisiologia , Animais , Proteínas de Bactérias/genética , Células Cultivadas , Embrião não Mamífero , Larva , Camundongos , Mycobacterium marinum/genética , Células RAW 264.7 , Ovinos , Sistemas de Secreção Tipo VII/metabolismo , Virulência/genética , Fatores de Virulência/genética , Peixe-Zebra/embriologia , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Central nervous system (CNS) infection by Mycobacterium tuberculosis is one of the most devastating complications of tuberculosis, in particular in early childhood. In order to induce CNS infection, M. tuberculosis needs to cross specialised barriers protecting the brain. How M. tuberculosis crosses the blood-brain barrier (BBB) and enters the CNS is not well understood. Here, we use transparent zebrafish larvae and the closely related pathogen Mycobacterium marinum to answer this question. We show that in the early stages of development, mycobacteria rapidly infect brain tissue, either as free mycobacteria or within circulating macrophages. After the formation of a functionally intact BBB, the infiltration of brain tissue by infected macrophages is delayed, but not blocked, suggesting that crossing the BBB via phagocytic cells is one of the mechanisms used by mycobacteria to invade the CNS. Interestingly, depletion of phagocytic cells did not prevent M. marinum from infecting the brain tissue, indicating that free mycobacteria can independently cause brain infection. Detailed analysis showed that mycobacteria are able to cause vasculitis by extracellular outgrowth in the smaller blood vessels and by infecting endothelial cells. Importantly, we could show that this second mechanism is an active process that depends on an intact ESX-1 secretion system, which extends the role of ESX-1 secretion beyond the macrophage infection cycle.
Assuntos
Barreira Hematoencefálica/microbiologia , Infecções do Sistema Nervoso Central/patologia , Interações Hospedeiro-Patógeno , Infecções por Mycobacterium não Tuberculosas/patologia , Mycobacterium marinum/crescimento & desenvolvimento , Animais , Encéfalo/microbiologia , Modelos Animais de Doenças , Macrófagos/microbiologia , Peixe-ZebraRESUMO
Mycobacteria produce a capsule layer, which consists of glycan-like polysaccharides and a number of specific proteins. In this study, we show that, in slow-growing mycobacteria, the type VII secretion system ESX-5 plays a major role in the integrity and stability of the capsule. We have identified PPE10 as the ESX-5 substrate responsible for this effect. Mutants in esx-5 and ppe10 both have impaired capsule integrity as well as reduced surface hydrophobicity. Electron microscopy, immunoblot and flow cytometry analyses demonstrated reduced amounts of surface localized proteins and glycolipids, and morphological differences in the capsular layer. Since capsular proteins secreted by the ESX-1 system are important virulence factors, we tested the effect of the mutations that cause capsular defects on virulence mechanisms. Both esx-5 and ppe10 mutants of Mycobacterium marinum were shown to be impaired in ESX-1-dependent hemolysis. In agreement with this, the ppe10 and esx5 mutants showed reduced recruitment of ubiquitin in early macrophage infection and intermediate attenuation in zebrafish embryos. These results provide a pivotal role for the ESX-5 secretion system and its substrate PPE10, in the capsular integrity of pathogenic mycobacteria. These findings open up new roads for research on the mycobacterial capsule and its role in virulence and immune modulation.
Assuntos
Cápsulas Bacterianas/metabolismo , Infecções por Mycobacterium não Tuberculosas/metabolismo , Mycobacterium marinum/patogenicidade , Sistemas de Secreção Tipo VII/metabolismo , Virulência/fisiologia , Animais , Linhagem Celular , Cromatografia em Camada Fina , Modelos Animais de Doenças , Citometria de Fluxo , Humanos , Immunoblotting , Microscopia Eletrônica , Mycobacterium marinum/metabolismo , Fatores de Virulência/metabolismo , Peixe-ZebraRESUMO
The interaction of environmental bacteria with unicellular eukaryotes is generally considered a major driving force for the evolution of intracellular pathogens, allowing them to survive and replicate in phagocytic cells of vertebrate hosts. To test this hypothesis on a genome-wide level, we determined for the intracellular pathogen Mycobacterium marinum whether it uses conserved strategies to exploit host cells from both protozoan and vertebrate origin. Using transposon-directed insertion site sequencing (TraDIS), we determined differences in genetic requirements for survival and replication in phagocytic cells of organisms from different kingdoms. In line with the general hypothesis, we identified a number of general virulence mechanisms, including the type VII protein secretion system ESX-1, biosynthesis of polyketide lipids, and utilization of sterols. However, we were also able to show that M. marinum contains an even larger set of host-specific virulence determinants, including proteins involved in the modification of surface glycolipids and, surprisingly, the auxiliary proteins of the ESX-1 system. Several of these factors were in fact counterproductive in other hosts. Therefore, M. marinum contains different sets of virulence factors that are tailored for specific hosts. Our data imply that although amoebae could function as a training ground for intracellular pathogens, they do not fully prepare pathogens for crossing species barriers.
Assuntos
Genoma Bacteriano , Viabilidade Microbiana , Mutagênese Insercional , Mycobacterium marinum/genética , Mycobacterium marinum/fisiologia , Fatores de Virulência/metabolismo , Acanthamoeba castellanii/microbiologia , Animais , Elementos de DNA Transponíveis , Dictyostelium/microbiologia , Humanos , Mycobacterium marinum/crescimento & desenvolvimento , Fagócitos/microbiologia , Virulência , Fatores de Virulência/genéticaRESUMO
The pathogenicity of mycobacteria is closely associated with their ability to export virulence factors. For this purpose, mycobacteria possess different protein secretion systems, including the accessory Sec translocation pathway, SecA2. Although this pathway is associated with intracellular survival and virulence, the SecA2-dependent effector proteins remain largely undefined. In this work, we studied a Mycobacterium marinumâ secA2 mutant with an impaired capacity to initiate granuloma formation in zebrafish embryos. By comparing the proteomic profile of cell envelope fractions from the secA2 mutant with wild type M. marinum, we identified putative SecA2-dependent substrates. Immunoblotting procedures confirmed SecA2-dependent membrane localization for several of these proteins, including the virulence factor protein kinase G (PknG). Interestingly, phenotypical defects of the secA2 mutant are similar to those described for ΔpknG, including phagosomal maturation. Overexpression of PknG in the secA2 mutant restored its localization to the cell envelope. Importantly, PknG-overexpression also partially restored the virulence of the secA2 mutant, as indicated by enhanced infectivity in zebrafish embryos and restored inhibition of phagosomal maturation. These results suggest that SecA2-dependent membrane localization of PknG is an important determinant for M. marinum virulence.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mycobacterium marinum/metabolismo , Fatores de Virulência/metabolismo , Animais , Elementos de DNA Transponíveis , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Immunoblotting , Mutagênese Insercional , Infecções por Mycobacterium/microbiologia , Mycobacterium marinum/patogenicidade , Especificidade por Substrato , Peixe-ZebraRESUMO
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.
Assuntos
Lipopolissacarídeos/metabolismo , Mycobacterium marinum/imunologia , Mycobacterium marinum/patogenicidade , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Animais , Carga Bacteriana , Imunidade Inata , Lipopolissacarídeos/química , Manose/química , Infecções por Mycobacterium não Tuberculosas/imunologia , Mycobacterium marinum/genética , Mycobacterium smegmatis/patogenicidade , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/patogenicidade , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Tuberculose/imunologia , Peixe-Zebra/imunologia , Peixe-Zebra/microbiologiaRESUMO
Finding new anti-tuberculosis compounds with convincing in vivo activity is an ongoing global challenge to fight the emergence of multidrug-resistant Mycobacterium tuberculosis isolates. In this study, we exploited the medium-throughput capabilities of the zebrafish embryo infection model with Mycobacterium marinum as a surrogate for M. tuberculosis. Using a representative set of clinically established drugs, we demonstrate that this model could be predictive and selective for antibiotics that can be administered orally. We further used the zebrafish infection model to screen 240 compounds from an anti-tuberculosis hit library for their in vivo activity and identified 14 highly active compounds. One of the most active compounds was the tetracyclic compound TBA161, which was studied in more detail. Analysis of resistant mutants revealed point mutations in aspS (rv2572c), encoding an aspartyl-tRNA synthetase. The target was genetically confirmed, and molecular docking studies propose the possible binding of TBA161 in a pocket adjacent to the catalytic site. This study shows that the zebrafish infection model is suitable for rapidly identifying promising scaffolds with in vivo activity.
Assuntos
Aspartato-tRNA Ligase , Mycobacterium tuberculosis , Tuberculose , Animais , Simulação de Acoplamento Molecular , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia , Peixe-ZebraRESUMO
The cell-envelope of Mycobacterium tuberculosis plays a key role in bacterial virulence and antibiotic resistance. Little is known about the molecular mechanisms of regulation of cell-envelope formation. Here, we elucidate functional and structural properties of RNase AS, which modulates M. tuberculosis cell-envelope properties and strongly impacts bacterial virulence in vivo. The structure of RNase AS reveals a resemblance to RNase T from Escherichia coli, an RNase of the DEDD family involved in RNA maturation. We show that RNase AS acts as a 3'-5'-exoribonuclease that specifically hydrolyzes adenylate-containing RNA sequences. Also, crystal structures of complexes with AMP and UMP reveal the structural basis for the observed enzyme specificity. Notably, RNase AS shows a mechanism of substrate recruitment, based on the recognition of the hydrogen bond donor NH2 group of adenine. Our work opens a field for the design of drugs able to reduce bacterial virulence in vivo.
Assuntos
Mycobacterium tuberculosis/patogenicidade , Ribonucleases/química , Ribonucleases/metabolismo , Adenina , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Embrião não Mamífero/microbiologia , Exorribonucleases/química , Técnicas de Inativação de Genes , Ligação de Hidrogênio , Modelos Moleculares , Mutação , Mycobacterium marinum/genética , Mycobacterium marinum/patogenicidade , Mycobacterium tuberculosis/enzimologia , Poli A/metabolismo , Multimerização Proteica , Ribonucleases/genética , Especificidade por Substrato , Uridina Monofosfato/química , Uridina Monofosfato/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/microbiologiaRESUMO
The light chain of activated factor IX comprises multiple interactions between both epidermal growth factor-like domains that contribute to enzymatic activity and binding of factor IXa to its cofactor factor VIIIa. To investigate the association between factor IXa-specific properties and surface-exposed structure elements, chimeras were constructed in which the interconnection between the modules Leu(84)-Thr(87) and the factor IX-specific loop Asn(89)-Lys(91) were exchanged for corresponding regions of factor X and factor VII. In absence of factor VIIIa, all chimeras displayed normal enzymatic activity. In the presence of factor VIIIa, replacement of loop Asn(89)-Lys(91) resulted in a minor reduction in factor IXa activity. However, chimeras with substitutions or insertions in the spacer between the epidermal growth factor-like domains showed a major defect in response to factor VIIIa. Of these chimeras, some displayed a normal response to isolated factor VIII A2 domain as a cofactor in factor X activation. Surprisingly, chimeras containing elongated inter-domain spacers from factor X or VII displayed reduced response to both complete factor VIIIa and the isolated A2 domain. Moreover, these chimeras still displayed effective association with immobilized A2 domain as assessed by surface plasmon resonance. We conclude that both sequence and length of the junction Leu(84)-Thr(87) between both epidermal growth factor-like domains contribute to the enhancement of factor IXa enzymatic activity that occurs upon assembly with factor VIIIa.
Assuntos
Fator de Crescimento Epidérmico/metabolismo , Fator IX/metabolismo , Fator VIIIa/metabolismo , Amidas/metabolismo , Sequência de Aminoácidos , Fator de Crescimento Epidérmico/química , Fator IX/química , Fator VIIIa/química , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Low-density lipoprotein receptor-related protein (LRP) is an endocytic receptor that binds multiple distinct ligands, including blood coagulation factor VIII (FVIII). FVIII is a heterodimeric multidomain protein that consists of a heavy chain (domains A1, a1, A2, a2, and B) and a light chain (domains a3, A3, C1, and C2). Both chains contribute to high-affinity interaction with LRP. One LRP-interactive region has previously been located in the C2 domain, but its affinity is low in comparison with that of the entire FVIII light chain. We now have compared a variety of FVIII light chain derivatives with the light chain of its homolog FVa for LRP binding. In surface plasmon resonance studies employing LRP cluster II, the FVa and FVIII light chains proved different in that only FVIII displayed high-affinity binding. Because the FVIII a3-A3-C1 fragment was effective in associating with LRP, this region was explored for structural elements that are exposed but not conserved in FV. Competition studies using synthetic peptides suggested that LRP binding involves the FVIII-specific region Lys(1804)-Ala(1834) in the A3 domain. In line with this observation, LRP binding was inhibited by a recombinant antibody fragment that specifically binds to the FVIII sequence Glu(1811)-Lys(1818). The role of this sequence in LRP binding was further tested using a FVIII/FV chimera in which sequence Glu(1811)-Lys(1818) was replaced with the corresponding sequence of FV. Although this chimera still displayed residual binding to LRP cluster II, its affinity was reduced. This suggests that multiple sites in FVIII contribute to high-affinity LRP binding, one of which is the FVIII A3 domain region Glu(1811)-Lys(1818). This suggests that LRP binding to the FVIII A3 domain involves the same structural elements that also contribute to the assembly of FVIII with FIXa.