RESUMO
The removal of mis-incorporated nucleotides by proofreading activity ensures DNA replication fidelity. Whereas the ε-exonuclease DnaQ is a well-established proofreader in the model organism Escherichia coli, it has been shown that proofreading in a majority of bacteria relies on the polymerase and histidinol phosphatase (PHP) domain of replicative polymerase, despite the presence of a DnaQ homolog that is structurally and functionally distinct from E. coli DnaQ. However, the biological functions of this type of noncanonical DnaQ remain unclear. Here, we provide independent evidence that noncanonical DnaQ functions as an additional proofreader for mycobacteria. Using the mutation accumulation assay in combination with whole-genome sequencing, we showed that depletion of DnaQ in Mycolicibacterium smegmatis leads to an increased mutation rate, resulting in AT-biased mutagenesis and increased insertions/deletions in the homopolymer tract. Our results showed that mycobacterial DnaQ binds to the ß clamp and functions synergistically with the PHP domain proofreader to correct replication errors. Furthermore, the loss of dnaQ results in replication fork dysfunction, leading to attenuated growth and increased mutagenesis on subinhibitory fluoroquinolones potentially due to increased vulnerability to fork collapse. By analyzing the sequence polymorphism of dnaQ in clinical isolates of Mycobacterium tuberculosis (Mtb), we demonstrated that a naturally evolved DnaQ variant prevalent in Mtb lineage 4.3 may enable hypermutability and is associated with drug resistance. These results establish a coproofreading model and suggest a division of labor between DnaQ and PHP domain proofreader. This study also provides real-world evidence that a mutator-driven evolutionary pathway may exist during the adaptation of Mtb.
Assuntos
Replicação do DNA , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , MutaçãoRESUMO
During its global dispersal, Mycobacterium tuberculosis (Mtb) has encountered varied geographic environments and host populations. Although local adaptation seems to be a plausible model for describing long-term host-pathogen interactions, genetic evidence for this model is lacking. Here, we analyzed 576 whole-genome sequences of Mtb strains sampled from different regions of high-altitude Tibet. Our results show that, after sequential introduction of a few ancestral strains, the Tibetan Mtb population diversified locally while maintaining strict separation from the Mtb populations on the lower altitude plain regions of China. The current population structure and estimated past population dynamics suggest that the modern Beijing sublineage strains, which expanded over most of China and other global regions, did not show an expansion advantage in Tibet. The mutations in the Tibetan strains showed a higher proportion of A > G/T > C transitions than strains from the plain regions, and genes encoding DNA repair enzymes showed evidence of positive selection. Moreover, the long-term Tibetan exclusive selection for truncating mutations in the thiol-oxidoreductase encoding sseA gene suggests that Mtb was subjected to local selective pressures associated with oxidative stress. Collectively, the population genomics of Mtb strains in the relatively isolated population of Tibet provides genetic evidence that Mtb has adapted to local environments.
Assuntos
Adaptação Biológica/genética , Adaptação Fisiológica/genética , Mycobacterium tuberculosis/genética , Aclimatação/genética , Altitude , Evolução Biológica , China , Genótipo , Mutação , Mycobacterium tuberculosis/metabolismo , Filogenia , Dinâmica Populacional/tendências , Seleção Genética/genética , Tibet/epidemiologiaRESUMO
Host-derived fatty acids are an important carbon source for pathogenic mycobacteria during infection. How mycobacterial cells regulate the catabolism of fatty acids to serve the pathogenicity, however, remains unknown. Here, we identified a TetR-family transcriptional factor, FdmR, as the key regulator of fatty acid catabolism in the pathogen Mycobacterium marinum by combining use of transcriptomics, chromatin immunoprecipitation followed by sequencing, dynamic 13C-based flux analysis, metabolomics, and lipidomics. An M. marinum mutant deficient in FdmR was severely attenuated in zebrafish larvae and adult zebrafish. The mutant showed defective growth but high substrate consumption on fatty acids. FdmR was identified as a long-chain acyl-coenzyme A (acyl-CoA)-responsive repressor of genes involved in fatty acid degradation and modification. We demonstrated that FdmR functions as a valve to direct the flux of exogenously derived fatty acids away from ß-oxidation toward lipid biosynthesis, thereby avoiding the overactive catabolism and accumulation of biologically toxic intermediates. Moreover, we found that FdmR suppresses degradation of long-chain acyl-CoAs endogenously synthesized through the type I fatty acid synthase. By modulating the supply of long-chain acyl-CoAs for lipogenesis, FdmR controls the abundance and chain length of virulence-associated lipids and mycolates and plays an important role in the impermeability of the cell envelope. These results reveal that despite the fact that host-derived fatty acids are used as an important carbon source, overactive catabolism of fatty acids is detrimental to mycobacterial cell growth and pathogenicity. This study thus presents FdmR as a potentially attractive target for chemotherapy.
Assuntos
Ácidos Graxos/metabolismo , Lipogênese/fisiologia , Mycobacterium marinum/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Lipólise , Metabolismo/fisiologia , Modelos Animais , Mycobacterium/metabolismo , Infecções por Mycobacterium não Tuberculosas/metabolismo , Infecções por Mycobacterium não Tuberculosas/fisiopatologia , Oxirredução , Fatores de Transcrição/metabolismo , Virulência/fisiologia , Peixe-Zebra/metabolismo , Peixe-Zebra/microbiologiaRESUMO
Mycobacterial arabinogalactan (AG) is an essential cell wall component of mycobacteria and a frequent structural and bio-synthetical target for anti-tuberculosis (TB) drug development. Here, we report that mycobacterial AG is recognized by galectin-9 and exacerbates mycobacterial infection. Administration of AG-specific aptamers inhibits cellular infiltration caused by Mycobacterium tuberculosis (Mtb) or Mycobacterium bovis BCG, and moderately increases survival of Mtb-infected mice or Mycobacterium marinum-infected zebrafish. AG interacts with carbohydrate recognition domain (CRD) 2 of galectin-9 with high affinity, and galectin-9 associates with transforming growth factor ß-activated kinase 1 (TAK1) via CRD2 to trigger subsequent activation of extracellular signal-regulated kinase (ERK) as well as induction of the expression of matrix metalloproteinases (MMPs). Moreover, deletion of galectin-9 or inhibition of MMPs blocks AG-induced pathological impairments in the lung, and the AG-galectin-9 axis aggravates the process of Mtb infection in mice. These results demonstrate that AG is an important virulence factor of mycobacteria and galectin-9 is a novel receptor for Mtb and other mycobacteria, paving the way for the development of novel effective TB immune modulators.
Assuntos
Mycobacterium tuberculosis , Peixe-Zebra , Animais , Galactanos , Galectinas/genética , CamundongosRESUMO
Nitrate is one of the major inorganic nitrogen sources for microbes. Many bacterial and archaeal lineages have the capacity to express assimilatory nitrate reductase (NAS), which catalyzes the rate-limiting reduction of nitrate to nitrite. Although a nitrate assimilatory pathway in mycobacteria has been proposed and validated physiologically and genetically, the putative NAS enzyme has yet to be identified. Here, we report the characterization of a novel NAS encoded by Mycolicibacterium smegmatis Msmeg_4206, designated NasN, which differs from the canonical NASs in its structure, electron transfer mechanism, enzymatic properties, and phylogenetic distribution. Using sequence analysis and biochemical characterization, we found that NasN is an NADPH-dependent, diflavin-containing monomeric enzyme composed of a canonical molybdopterin cofactor-binding catalytic domain and an FMN-FAD/NAD-binding, electron-receiving/transferring domain, making it unique among all previously reported hetero-oligomeric NASs. Genetic studies revealed that NasN is essential for aerobic M. smegmatis growth on nitrate as the sole nitrogen source and that the global transcriptional regulator GlnR regulates nasN expression. Moreover, unlike the NADH-dependent heterodimeric NAS enzyme, NasN efficiently supports bacterial growth under nitrate-limiting conditions, likely due to its significantly greater catalytic activity and oxygen tolerance. Results from a phylogenetic analysis suggested that the nasN gene is more recently evolved than those encoding other NASs and that its distribution is limited mainly to Actinobacteria and Proteobacteria. We observed that among mycobacterial species, most fast-growing environmental mycobacteria carry nasN, but that it is largely lacking in slow-growing pathogenic mycobacteria because of multiple independent genomic deletion events along their evolution.
Assuntos
Coenzimas/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Metaloproteínas/metabolismo , Mycobacterium smegmatis/enzimologia , NAD/metabolismo , Nitrato Redutase/metabolismo , Nitratos/metabolismo , Pteridinas/metabolismo , Elétrons , Regulação Bacteriana da Expressão Gênica , Cofatores de Molibdênio , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crescimento & desenvolvimento , Nitrato Redutase/química , Nitrato Redutase/genética , Nitritos/metabolismo , Filogenia , Receptores de Neurotransmissores/metabolismoRESUMO
Growing evidence shows that generation of reactive oxygen species (ROS) derived from antibiotic-induced metabolic perturbation contribute to antibiotic lethality. However, our knowledge of the mechanisms by which antibiotic-induced oxidative stress actually kills cells remains elusive. Here, we show that oxidation of dCTP underlies ROS-mediated antibiotic lethality via induction of DNA double-strand breaks (DSBs). Deletion of mazG-encoded 5-OH-dCTP-specific pyrophosphohydrolase potentiates antibiotic killing of stationary-phase mycobacteria, but did not affect antibiotic efficacy in exponentially growing cultures. Critically, the effect of mazG deletion on potentiating antibiotic killing is associated with antibiotic-induced ROS and accumulation of 5-OH-dCTP. Independent lines of evidence presented here indicate that the increased level of DSBs observed in the ΔmazG mutant is a dead-end event accounting for enhanced antibiotic killing. Moreover, we provided genetic evidence that 5-OH-dCTP is incorporated into genomic DNA via error-prone DNA polymerase DnaE2 and repair of 5-OH-dC lesions via the endonuclease Nth leads to the generation of lethal DSBs. This work provides a mechanistic view of ROS-mediated antibiotic lethality in stationary phase and may have broad implications not only with respect to antibiotic lethality but also to the mechanism of stress-induced mutagenesis in bacteria.
Assuntos
Antibacterianos/farmacologia , Nucleotídeos de Desoxicitosina/metabolismo , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , DNA Bacteriano , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Humanos , Macrófagos , Oxirredução , Pirofosfatases/genética , Pirofosfatases/metabolismo , Espécies Reativas de OxigênioRESUMO
Generation of reactive oxygen species and reactive nitrogen species in phagocytes is an important innate immune response mechanism to eliminate microbial pathogens. It is known that deoxynucleotides (dNTPs), the precursor nucleotides to DNA synthesis, are one group of the significant targets for these oxidants and incorporation of oxidized dNTPs into genomic DNA may cause mutations and even cell death. Here we show that the mycobacterial dNTP pyrophosphohydrolase MazG safeguards the bacilli genome by degrading 5-OH-dCTP, thereby, preventing it from incorporation into DNA. Deletion of the (d)NTP pyrophosphohydrolase-encoding mazG in mycobacteria leads to a mutator phenotype both under oxidative stress and in the stationary phase of growth, resulting in increased CG to TA mutations. Biochemical analyses demonstrate that mycobacterial MazG can efficiently hydrolyze 5-OH-dCTP, an oxidized nucleotide that induces CG to TA mutation upon incorporation by polymerase. Moreover, chemical genetic analyses show that direct incorporation of 5-OH-dCTP into mazG-null mutant strain of Mycobacterium smegmatis (Msm) leads to a dose-dependent mutagenesis phenotype, indicating that 5-OH-dCTP is a natural substrate of mycobacterial MazG. Furthermore, deletion of mazG in Mycobacterium tuberculosis (Mtb) leads to reduced survival in activated macrophages and in the spleen of infected mice. This study not only characterizes the mycobacterial MazG as a novel pyrimidine-specific housecleaning enzyme that prevents CG to TA mutation by degrading 5-OH-dCTP but also reveals a genome-safeguarding mechanism for survival of Mtb in vivo.
Assuntos
Nucleotídeos de Desoxicitosina/metabolismo , Instabilidade Genômica/genética , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Pirofosfatases/fisiologia , Animais , Células Cultivadas , Feminino , Genoma Bacteriano , Camundongos , Camundongos Endogâmicos C57BL , Viabilidade Microbiana/genética , Mutação de Sentido Incorreto/genética , Organismos Geneticamente ModificadosRESUMO
IMPORTANCE: Rifamycins are a group of antibiotics with a wide antibacterial spectrum. Although the binding target of rifamycin has been well characterized, the mechanisms underlying the discrepant killing efficacy between gram-negative and gram-positive bacteria remain poorly understood. Using a high-throughput screen combined with targeted gene knockouts in the gram-negative model organism Escherichia coli, we established that rifampicin efficacy is strongly dependent on several cellular pathways, including iron acquisition, DNA repair, aerobic respiration, and carbon metabolism. In addition, we provide evidence that these pathways modulate rifampicin efficacy in a manner distinct from redox-related killing. Our findings provide insights into the mechanism of rifamycin efficacy and may aid in the development of new antimicrobial adjuvants.
Assuntos
Rifampina , Rifamicinas , Rifampina/farmacologia , Escherichia coli/genética , Antibacterianos/farmacologia , Testes de Sensibilidade MicrobianaRESUMO
The emergence of new Mycobacterium tuberculosis (Mtb) strains resistant to the key drugs currently used in the clinic for tuberculosis treatment can substantially reduce the probability of therapy success, causing the relevance and importance of studies on the development of novel potent antibacterial agents targeting different vulnerable spots of Mtb. In this study, 28,860 compounds from the library of bioactive molecules were screened to identify novel potential inhibitors of ß-ketoacyl-acyl carrier protein synthase I (KasA), one of the key enzymes involved in the biosynthesis of mycolic acids of the Mtb cell wall. In doing so, we used a structure-based virtual screening approach to drug repurposing that included high-throughput docking of the C171Q KasA enzyme with compounds from the library of bioactive molecules including the FDA-approved drugs and investigational drug candidates, assessment of the binding affinity for the docked ligand/C171Q KasA complexes, and molecular dynamics simulations followed by binding free energy calculations. As a result, post-modeling analysis revealed 6 top-ranking compounds exhibiting a strong attachment to the malonyl binding site of the enzyme, as evidenced by the values of binding free energy which are significantly lower than those predicted for the KasA inhibitor TLM5 used in the calculations as a positive control. In light of the data obtained, the identified compounds are suggested to form a good basis for the development of new antitubercular molecules of clinical significance with activity against the KasA enzyme of Mtb.Communicated by Ramaswamy H. Sarma.
RESUMO
Remove of mis-incorporated nucleotides ensures replicative fidelity. Although the ε-exonuclease DnaQ is a well-established proofreader in the model organism Escherichia coli, proofreading in mycobacteria relies on the polymerase and histidinol phosphatase (PHP) domain of replicative polymerase despite the presence of an alternative DnaQ homolog. Here, we show that depletion of DnaQ in Mycolicibacterium smegmatis results in increased mutation rate, leading to AT-biased mutagenesis and elevated insertions/deletions in homopolymer tract. We demonstrated that mycobacterial DnaQ binds to the b-clamp and functions synergistically with the PHP domain to correct replication errors. Further, we found that the mycobacterial DnaQ sustains replicative fidelity upon chromosome topological stress. Intriguingly, we showed that a naturally evolved DnaQ variant prevalent in clinical Mycobacterium tuberculosis isolates enables hypermutability and is associated with extensive drug resistance. These results collectively establish that the alternative DnaQ functions in proofreading, and thus reveal that mycobacteria deploy two proofreaders to maintain replicative fidelity.
RESUMO
Defence against oxidative stress is crucial for Mycobacterium tuberculosis to survive and replicate within macrophages. Mycobacteria have evolved multilayer antioxidant systems, including scavenging enzymes, iron homeostasis, repair pathways, and metabolic adaptation, for coping with oxidative stress. How these systems are coordinated to enable the physiological adaptation to different intensities of oxidative stress, however, remains unclear. To address this, we investigated the expression kinetics of the well-characterized antioxidant genes at bacteriostatic H2O2 concentrations ranging from 1 mM to 10 mM employing Mycolicibacterium smegmatis as a model. Our results showed that most of the selected genes were expressed in a H2O2 concentration-dependent manner, whereas a subset exhibited sustained induction or repression without dose-effect, reflecting H2O2 concentration-dependent physiological adaptations. Through analyzing the dynamics of the coordinated gene expression, we demonstrated that the expressions of the H2O2 scavenging enzymes, DNA damage response, and Fe-S cluster repair function were strikingly correlated to the intensity of oxidative stress. The sustained induction of mbtB, irtA, and dnaE2 indicated that mycobacteria might deploy increased iron acquisition and error-prone lesion bypass function as fundamental strategies to counteract oxidative damages, which are distinct from the defence tactics of Escherichia coli characterized by shrinking the iron pool and delaying the DNA repair. Moreover, the distinct gene expression kinetics among the tricarboxylic acid cycle, glyoxylate shunt, and methylcitrate cycle suggested that mycobacteria could dynamically redirect its metabolic fluxes according to the intensity of oxidative stress. This work defines the H2O2 concentration-dependent gene expression kinetics and provides unique insights into mycobacterial antioxidant defence strategies.
Assuntos
Peróxido de Hidrogênio , Mycobacterium tuberculosis , Adaptação Fisiológica/genética , Expressão Gênica , Peróxido de Hidrogênio/farmacologia , Cinética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Estresse OxidativoRESUMO
Mycobacterial persistence mechanisms remain to be fully characterized. Screening a transposon insertion library of Mycobacterium marinum identified kdpA, whose inactivation reduced the fraction of persisters after exposure to rifampicin. kdpA encodes a transmembrane protein that is part of the Kdp-ATPase, an ATP-dependent high-affinity potassium (K+) transport system. We found that kdpA is induced under low K+ conditions and is required for pH homeostasis and growth in media with low concentrations of K+. The inactivation of the Kdp system in a kdpA insertion mutant caused hyperpolarization of the cross-membrane potential, increased proton motive force (PMF) and elevated levels of intracellular ATP. The KdpA mutant phenotype could be complemented with a functional kdpA gene or supplementation with high K+ concentrations. Taken together, our results suggest that the Kdp system is required for ATP homeostasis and persister formation. The results also confirm that ATP-mediated regulation of persister formation is a general mechanism in bacteria, and suggest that K+ transporters could play a role in the regulation of ATP levels and persistence. These findings could have implications for the development of new drugs that could either target persisters or reduce their presence.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium marinum/metabolismo , Potássio/metabolismo , Trifosfato de Adenosina/genética , Proteínas de Bactérias/genética , Proteínas de Transporte de Cátions/genética , Regulação Bacteriana da Expressão Gênica , Humanos , Mycobacterium marinum/genéticaRESUMO
The ability of Mycobacterium tuberculosis (Mtb) to adopt a slowly growing or nongrowing state within the host plays a critical role for the bacilli to persist in the face of a prolonged multidrug therapy, establish latency and sustain chronic infection. In our previous study, we revealed that genome maintenance via MazG-mediated elimination of oxidized dCTP contributes to the antibiotic tolerance of nongrowing Mtb. Here, we provide evidence that housecleaning of pyrimidine nucleotide pool via MazG coordinates metabolic adaptation of Mtb to nongrowing state. We found that the ΔmazG mutant fails to maintain a nongrowing and metabolic quiescence state under dormancy models in vitro. To investigate bacterial metabolic changes during infection, we employed RNA-seq to compare the global transcriptional response of wild-type Mtb and the ΔmazG mutant after infection of macrophages. Pathway enrichment analyses of the differentially regulated genes indicate that the deletion of mazG in Mtb not only results in DNA instability, but also perturbs pyrimidine metabolism, iron and carbon source uptake, catabolism of propionate and TCA cycle. Moreover, these transcriptional signatures reflect anticipatory metabolism and regulatory activities observed during cell cycle re-entry in the ΔmazG mutant. Taken together, these results provide evidence that pyrimidine metabolism is a metabolic checkpoint during mycobacterial adaptation to nongrowing state.
Assuntos
Perfilação da Expressão Gênica/métodos , Macrófagos/microbiologia , Mycobacterium tuberculosis/fisiologia , Nucleotídeos de Pirimidina/química , Pirofosfatases/genética , Adaptação Fisiológica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carbono/metabolismo , Quimioterapia Combinada , Regulação Bacteriana da Expressão Gênica , Humanos , Ferro/metabolismo , Mutação , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Pirimidinas/metabolismo , Pirofosfatases/metabolismo , Análise de Sequência de RNA/métodos , Células THP-1RESUMO
Many pathogens infect hosts through various immune evasion strategies. However, the molecular mechanisms by which pathogen proteins modulate and evade the host immune response remain unclear. Enterohemorrhagic Escherichia coli (EHEC) is a pathological strain that can induce mitogen-activated protein (MAP) kinase (Erk, Jnk and p38 MAPK) and NF-κB pathway activation and proinflammatory cytokine production, which then causes diarrheal diseases such as hemorrhagic colitis and hemolytic uremic syndrome. Transforming growth factor ß-activated kinase-1 (TAK1) is a key regulator involved in distinct innate immune signalling pathways. Here we report that EHEC translocated intimin receptor (Tir) protein inhibits the expression of EHEC-induced proinflammatory cytokines by interacting with the host tyrosine phosphatase SHP-1, which is dependent on the phosphorylation of immunoreceptor tyrosine-based inhibition motifs (ITIMs). Mechanistically, the association of EHEC Tir with SHP-1 facilitated the recruitment of SHP-1 to TAK1 and inhibited TAK1 phosphorylation, which then negatively regulated K63-linked polyubiquitination of TAK1 and downstream signal transduction. Taken together, these results suggest that EHEC Tir negatively regulates proinflammatory responses by inhibiting the activation of TAK1, which is essential for immune evasion and could be a potential target for the treatment of bacterial infection.
Assuntos
Escherichia coli Êntero-Hemorrágica/patogenicidade , Infecções por Escherichia coli/fisiopatologia , Proteínas de Escherichia coli/metabolismo , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , MAP Quinase Quinase Quinases/antagonistas & inibidores , Receptores de Superfície Celular/metabolismo , Fatores de Virulência/metabolismo , Animais , Infecções por Escherichia coli/microbiologia , Células HEK293 , Humanos , Macrófagos Peritoneais , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Células RAW 264.7RESUMO
Lipids have been identified as important carbon sources for Mycobacterium tuberculosis (Mtb) to utilize in vivo. Thus gluconeogenesis bears a key role for Mtb to survive and replicate in host. A rate-limiting enzyme of gluconeogenesis, fructose 1, 6-bisphosphatase (FBPase) is encoded by the gene glpX. The functions of glpX were studied in M. marinum, a closely related species to Mtb. The glpX deletion strain (ΔglpX) displayed altered gluconeogenesis, attenuated virulence, and altered bacterial proliferation. Metabolic profiles indicate an accumulation of the FBPase substrate, fructose 1, 6-bisphosphate (FBP) and altered gluconeogenic flux when ΔglpX is cultivated in a gluconeogenic carbon substrate, acetate. In both macrophages and zebrafish, the proliferation of ΔglpX was halted, resulting in dramatically attenuated virulence. Intracellular ΔglpX exhibited an elongated morphology, which was also observed when ΔglpX was grown in a gluconeogenic carbon source. This elongated morphology is also supported by the observation of unseparated multi-nucleoid cell, indicating that a complete mycobacterial division in vivo is correlated with intact gluconeogenesis. Together, our results indicate that glpX has essential functions in gluconeogenesis, and plays an indispensable role in bacterial proliferation in vivo and virulence of M. marinum.
Assuntos
Divisão Celular , Frutose-Bifosfatase/genética , Frutose-Bifosfatase/metabolismo , Gluconeogênese , Mycobacterium marinum/citologia , Mycobacterium marinum/metabolismo , Animais , Antibacterianos/farmacologia , Proliferação de Células , Macrófagos/microbiologia , Mutação , Mycobacterium marinum/efeitos dos fármacos , Mycobacterium marinum/enzimologia , Peixe-Zebra/microbiologiaRESUMO
Network analysis of transcriptional signature typically relies on direct interaction between two highly expressed genes. However, this approach misses indirect and biological relevant interactions through a third factor (hub). Here we determine whether a hub-based network analysis can select an improved signature subset that correlates with a biological change in a stronger manner than the original signature. We have previously reported an interferon-related transcriptional signature (THP1r2Mtb-induced) from Mycobacterium tuberculosis (M. tb)-infected THP-1 human macrophage. We selected hub-connected THP1r2Mtb-induced genes into the refined network signature TMtb-iNet and grouped the excluded genes into the excluded signature TMtb-iEx. TMtb-iNet retained the enrichment of binding sites of interferon-related transcription factors and contained relatively more interferon-related interacting genes when compared to THP1r2Mtb-induced signature. TMtb-iNet correlated as strongly as THP1r2Mtb-induced signature on a public transcriptional dataset of patients with pulmonary tuberculosis (PTB). TMtb-iNet correlated more strongly in CD4(+) and CD8(+) T cells from PTB patients than THP1r2Mtb-induced signature and TMtb-iEx. When TMtb-iNet was applied to data during clinical therapy of tuberculosis, it resulted in the most pronounced response and the weakest correlation. Correlation on dataset from patients with AIDS or malaria was stronger for TMtb-iNet, indicating an involvement of TMtb-iNet in these chronic human infections. Collectively, the significance of this work is twofold: (1) we disseminate a hub-based approach in generating a biologically meaningful and clinically useful signature; (2) using this approach we introduce a new network-based signature and demonstrate its promising applications in understanding host responses to infections.