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Emerg Microbes Infect ; 8(1): 734-748, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31130074


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.

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 264.7
Emerg Microbes Infect ; 8(1): 40-44, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30866758


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.

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-1
Proc Natl Acad Sci U S A ; 115(9): 2210-2215, 2018 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-29382762


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.

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 Oxigênio
Biomed Res Int ; 2014: 713071, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25371902


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.

Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Interferons/metabolismo , Macrófagos/imunologia , Macrófagos/microbiologia , Mycobacterium tuberculosis/imunologia , Tuberculose/genética , Sítios de Ligação , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Humanos , Inflamação/genética , Fatores de Transcrição/metabolismo , Transcriptoma/genética , Tuberculose/imunologia , Tuberculose/microbiologia
PLoS Pathog ; 9(12): e1003814, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24339782


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.

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 Modificados