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1.
Cell Host Microbe ; 27(1): 41-53.e6, 2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31862381

RESUMO

Many Gram-negative bacterial pathogens antagonize anti-bacterial immunity through translocated effector proteins that inhibit pro-inflammatory signaling. In addition, the intracellular pathogen Salmonella enterica serovar Typhimurium initiates an anti-inflammatory transcriptional response in macrophages through its effector protein SteE. However, the target(s) and molecular mechanism of SteE remain unknown. Here, we demonstrate that SteE converts both the amino acid and substrate specificity of the host pleiotropic serine/threonine kinase GSK3. SteE itself is a substrate of GSK3, and phosphorylation of SteE is required for its activity. Remarkably, phosphorylated SteE then forces GSK3 to phosphorylate the non-canonical substrate signal transducer and activator of transcription 3 (STAT3) on tyrosine-705. This results in STAT3 activation, which along with GSK3 is required for SteE-mediated upregulation of the anti-inflammatory M2 macrophage marker interleukin-4Rα (IL-4Rα). Overall, the conversion of GSK3 to a tyrosine-directed kinase represents a tightly regulated event that enables a bacterial virulence protein to reprogram innate immune signaling and establish an anti-inflammatory environment.


Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Macrófagos/microbiologia , Proteínas Serina-Treonina Quinases/metabolismo , Fator de Transcrição STAT3/metabolismo , Salmonella typhimurium , Animais , Proteínas de Bactérias/metabolismo , Células HEK293 , Células HeLa , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Interleucina-4/metabolismo , Ativação de Macrófagos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Tirosina Quinases/metabolismo , Salmonella typhimurium/imunologia , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidade , Virulência/imunologia
2.
J Biol Chem ; 293(39): 15316-15329, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-30049795

RESUMO

The closely related type III secretion system zinc metalloprotease effector proteins GtgA, GogA, and PipA are translocated into host cells during Salmonella infection. They then cleave nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) transcription factor subunits, dampening activation of the NF-κB signaling pathway and thereby suppressing host immune responses. We demonstrate here that GtgA, GogA, and PipA cleave a subset of NF-κB subunits, including p65, RelB, and cRel but not NF-κB1 and NF-κB2, whereas the functionally similar type III secretion system effector NleC of enteropathogenic and enterohemorrhagic Escherichia coli cleaved all five NF-κB subunits. Mutational analysis of NF-κB subunits revealed that a single nonconserved residue in NF-κB1 and NF-κB2 that corresponds to the P1' residue Arg-41 in p65 prevents cleavage of these subunits by GtgA, GogA, and PipA, explaining the observed substrate specificity of these enzymes. Crystal structures of GtgA in its apo-form and in complex with the p65 N-terminal domain explained the importance of the P1' residue. Furthermore, the pattern of interactions suggested that GtgA recognizes NF-κB subunits by mimicking the shape and negative charge of the DNA phosphate backbone. Moreover, structure-based mutational analysis of GtgA uncovered amino acids that are required for the interaction of GtgA with p65, as well as those that are required for full activity of GtgA in suppressing NF-κB activation. This study therefore provides detailed and critical insight into the mechanism of substrate recognition by this family of proteins important for bacterial virulence.


Assuntos
Escherichia coli/química , Metaloproteases/química , Infecções por Salmonella/genética , Salmonella enterica/química , Sequência de Aminoácidos/genética , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/patogenicidade , Células HeLa , Humanos , Imunidade Celular , Metaloproteases/genética , NF-kappa B/química , Conformação Proteica , Infecções por Salmonella/microbiologia , Salmonella enterica/genética , Salmonella enterica/patogenicidade , Transdução de Sinais , Relação Estrutura-Atividade , Fator de Transcrição RelA/química , Sistemas de Secreção Tipo III/química , Sistemas de Secreção Tipo III/genética , Zinco/química
3.
Nat Commun ; 7: 13292, 2016 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-27808091

RESUMO

Sensing bacterial products in the cytosol of mammalian cells by NOD-like receptors leads to the activation of caspase-1 inflammasomes, and the production of the pro-inflammatory cytokines interleukin (IL)-18 and IL-1ß. In addition, mouse caspase-11 (represented in humans by its orthologs, caspase-4 and caspase-5) detects cytosolic bacterial LPS directly. Activation of caspase-1 and caspase-11 initiates pyroptotic host cell death that releases potentially harmful bacteria from the nutrient-rich host cell cytosol into the extracellular environment. Here we use single cell analysis and time-lapse microscopy to identify a subpopulation of host cells, in which growth of cytosolic Salmonella Typhimurium is inhibited independently or prior to the onset of cell death. The enzymatic activities of caspase-1 and caspase-11 are required for growth inhibition in different cell types. Our results reveal that these proteases have important functions beyond the direct induction of pyroptosis and proinflammatory cytokine secretion in the control of growth and elimination of cytosolic bacteria.


Assuntos
Caspase 1/imunologia , Caspases/imunologia , Citosol/imunologia , Piroptose/imunologia , Infecções por Salmonella/imunologia , Salmonella typhimurium/imunologia , Células 3T3 , Animais , Caspase 1/genética , Caspase 1/metabolismo , Caspases/genética , Caspases/metabolismo , Caspases Iniciadoras , Citosol/enzimologia , Citosol/microbiologia , Modelos Animais de Doenças , Espaço Extracelular/microbiologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Inflamassomos/imunologia , Inflamassomos/metabolismo , Macrófagos/enzimologia , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Salmonella/microbiologia , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/patogenicidade , Análise de Célula Única , Imagem com Lapso de Tempo
4.
J Gen Virol ; 96(Pt 6): 1380-1388, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25701818

RESUMO

Hepatitis C virus (HCV) infects the liver and hepatocytes are the major cell type supporting viral replication. Hepatocytes and cholangiocytes derive from a common hepatic progenitor cell that proliferates during inflammatory conditions, raising the possibility that cholangiocytes may support HCV replication and contribute to the hepatic reservoir. We screened cholangiocytes along with a panel of cholangiocarcinoma-derived cell lines for their ability to support HCV entry and replication. While primary cholangiocytes were refractory to infection and lacked expression of several entry factors, two cholangiocarcinoma lines, CC-LP-1 and Sk-ChA-1, supported efficient HCV entry; furthermore, Sk-ChA-1 cells supported full virus replication. In vivo cholangiocarcinomas expressed all of the essential HCV entry factors; however, cholangiocytes adjacent to the tumour and in normal tissue showed a similar pattern of receptor expression to ex vivo isolated cholangiocytes, lacking SR-BI expression, explaining their inability to support infection. This study provides the first report that HCV can infect cholangiocarcinoma cells and suggests that these heterogeneous tumours may provide a reservoir for HCV replication in vivo.


Assuntos
Células Epiteliais/virologia , Hepacivirus/fisiologia , Tropismo Viral , Linhagem Celular Tumoral , Hepacivirus/crescimento & desenvolvimento , Humanos , Internalização do Vírus , Replicação Viral
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