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1.
Proc Natl Acad Sci U S A ; 117(6): 3167-3173, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-31980538

RESUMO

Pseudomonas aeruginosa strains with loss-of-function mutations in the transcription factor LasR are frequently encountered in the clinic and the environment. Among the characteristics common to LasR-defective (LasR-) strains is increased activity of the transcription factor Anr, relative to their LasR+ counterparts, in low-oxygen conditions. One of the Anr-regulated genes found to be highly induced in LasR- strains was PA14_42860 (PA1673), which we named mhr for microoxic hemerythrin. Purified P. aeruginosa Mhr protein contained the predicted di-iron center and bound molecular oxygen with an apparent Kd of ∼1 µM. Both Anr and Mhr were necessary for fitness in lasR+ and lasR mutant strains in colony biofilms grown in microoxic conditions, and the effects were more striking in the lasR mutant. Among genes in the Anr regulon, mhr was most closely coregulated with the Anr-controlled high-affinity cytochrome c oxidase genes. In the absence of high-affinity cytochrome c oxidases, deletion of mhr no longer caused a fitness disadvantage, suggesting that Mhr works in concert with microoxic respiration. We demonstrate that Anr and Mhr contribute to LasR- strain fitness even in biofilms grown in normoxic conditions. Furthermore, metabolomics data indicate that, in a lasR mutant, expression of Anr-regulated mhr leads to differences in metabolism in cells grown on lysogeny broth or artificial sputum medium. We propose that increased Anr activity leads to higher levels of the oxygen-binding protein Mhr, which confers an advantage to lasR mutants in microoxic conditions.


Assuntos
Proteínas de Bactérias/metabolismo , Hipóxia Celular/genética , Aptidão Genética/genética , Hemeritrina/metabolismo , Pseudomonas aeruginosa , Transativadores/metabolismo , Proteínas de Bactérias/genética , Hemeritrina/genética , Oxigênio/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/fisiologia , Transativadores/genética
2.
J Bacteriol ; 201(12)2019 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-30936375

RESUMO

Pseudomonas aeruginosa frequently resides among ethanol-producing microbes, making its response to the microbially produced concentrations of ethanol relevant to understanding its biology. Our transcriptome analysis found that genes involved in trehalose metabolism were induced by low concentrations of ethanol, and biochemical assays showed that levels of intracellular trehalose increased significantly upon growth with ethanol. The increase in trehalose was dependent on the TreYZ pathway but not other trehalose-metabolic enzymes (TreS or TreA). The sigma factor AlgU (AlgT), a homolog of RpoE in other species, was required for increased expression of the treZ gene and trehalose levels, but induction was not controlled by the well-characterized proteolysis of its anti-sigma factor, MucA. Growth with ethanol led to increased SpoT-dependent (p)ppGpp accumulation, which stimulates AlgU-dependent transcription of treZ and other AlgU-regulated genes through DksA, a (p)ppGpp and RNA polymerase binding protein. Ethanol stimulation of trehalose also required acylhomoserine lactone (AHL)-mediated quorum sensing (QS), as induction was not observed in a ΔlasR ΔrhlR strain. A network analysis using a model, eADAGE, built from publicly available P. aeruginosa transcriptome data sets (J. Tan, G. Doing, K. A. Lewis, C. E. Price, et al., Cell Syst 5:63-71, 2017, https://doi.org/10.1016/j.cels.2017.06.003) provided strong support for our model in which treZ and coregulated genes are controlled by both AlgU- and AHL-mediated QS. Consistent with (p)ppGpp- and AHL-mediated quorum-sensing regulation, ethanol, even when added at the time of culture inoculation, stimulated treZ transcript levels and trehalose production in cells from post-exponential-phase cultures but not in cells from exponential-phase cultures. These data highlight the integration of growth and cell density cues in the P. aeruginosa transcriptional response to ethanol.IMPORTANCEPseudomonas aeruginosa is often found with bacteria and fungi that produce fermentation products, including ethanol. At concentrations similar to those produced by environmental microbes, we found that ethanol stimulated expression of trehalose-biosynthetic genes and cellular levels of trehalose, a disaccharide that protects against environmental stresses. The induction of trehalose by ethanol required the alternative sigma factor AlgU through DksA- and SpoT-dependent (p)ppGpp. Trehalose accumulation also required AHL quorum sensing and occurred only in post-exponential-phase cultures. This work highlights how cells integrate cell density and growth cues in their responses to products made by other microbes and reveals a new role for (p)ppGpp in the regulation of AlgU activity.


Assuntos
Proteínas de Bactérias/metabolismo , Etanol/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Fator sigma/metabolismo , Trealose/biossíntese , Proteínas de Bactérias/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Regiões Promotoras Genéticas , Pseudomonas aeruginosa/metabolismo , Fatores de Transcrição , Transcrição Gênica , Trealose/análise
3.
Am J Pathol ; 187(4): 851-863, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28193481

RESUMO

Seasonal and pandemic influenza is a cause of morbidity and mortality worldwide. Most people infected with influenza virus display mild-to-moderate disease phenotypes and recover within a few weeks. Influenza is known to cause persistent alveolitis in animal models; however, little is known about the molecular pathways involved in this phenotype. We challenged C57BL/6 mice with influenza A/PR/8/34 and examined lung pathologic processes and inflammation, as well as transcriptomic and epigenetic changes at 21 to 60 days after infection. Influenza induced persistent parenchymal lung inflammation, alveolar epithelial metaplasia, and epithelial endoplasmic reticulum stress that were evident after the clearance of virus and resolution of morbidity. Influenza infection induced robust changes in the lung transcriptome, including a significant impact on inflammatory and extracellular matrix protein expression. Despite the robust changes in lung gene expression, preceding influenza (21 days) did not exacerbate secondary Staphylococcus aureus infection. Finally, we examined the impact of influenza on miRNA expression in the lung and found an increase in miR-155. miR-155 knockout mice recovered from influenza infection faster than controls and had decreased lung inflammation and endoplasmic reticulum stress. These data illuminate the dynamic molecular changes in the lung in the weeks after influenza infection and characterize the repair process, identifying a novel role for miR-155.


Assuntos
Epigênese Genética , Pulmão/metabolismo , Pulmão/virologia , Infecções por Orthomyxoviridae/genética , Transcriptoma/genética , Cicatrização/genética , Animais , Progressão da Doença , Estresse do Retículo Endoplasmático/genética , Epitélio/patologia , Perfilação da Expressão Gênica , Inflamação/patologia , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Pneumonia/etiologia , Pneumonia/microbiologia , Linfócitos T/imunologia , Fatores de Tempo
4.
Am J Physiol Lung Cell Mol Physiol ; 309(2): L158-67, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-26001778

RESUMO

Suppression of type 17 immunity by type I interferon (IFN) during influenza A infection has been shown to enhance susceptibility to secondary bacterial pneumonia. Although this mechanism has been described in coinfection with gram-positive bacteria, it is unclear whether similar mechanisms may impair lung defense against gram-negative infections. Furthermore, precise delineation of the duration of type I IFN-associated susceptibility to bacterial infection remains underexplored. Therefore, we investigated the effects of preceding influenza A virus infection on subsequent challenge with the gram-negative bacteria Escherichia coli or Pseudomonas aeruginosa and the temporal association between IFN expression with susceptibility to Staphylococcus aureus challenge in a mouse model of influenza and bacterial coinfection. Here we demonstrate that preceding influenza A virus led to increased lung E. coli and P. aeruginosa bacterial burden, which was associated with suppression of type 17 immunity and attenuation of antimicrobial peptide expression. Enhanced susceptibility to S. aureus coinfection ceased at day 14 of influenza infection, when influenza-associated type I IFN levels had returned to baseline levels, further suggesting a key role for type I IFN in coinfection pathogenesis. These findings further implicate type I IFN-associated suppression of type 17 immunity and antimicrobial peptide production as a conserved mechanism for enhanced susceptibility to both gram-positive and gram-negative bacterial coinfection during influenza infection.


Assuntos
Infecções por Escherichia coli/microbiologia , Vírus da Influenza A/patogenicidade , Infecções por Orthomyxoviridae/microbiologia , Pneumonia Bacteriana/microbiologia , Pneumonia/microbiologia , Receptor de Interferon alfa e beta/fisiologia , Infecções Estafilocócicas/microbiologia , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Coinfecção/imunologia , Coinfecção/microbiologia , Coinfecção/virologia , Suscetibilidade a Doenças , Escherichia coli/imunologia , Escherichia coli/patogenicidade , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/virologia , Vírus da Influenza A/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Pneumonia/imunologia , Pneumonia/virologia , Pneumonia Bacteriana/imunologia , Pneumonia Bacteriana/virologia , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/virologia , Staphylococcus aureus/imunologia , Staphylococcus aureus/patogenicidade
5.
J Infect Dis ; 209(6): 865-75, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24072844

RESUMO

Influenza A represents a significant cause of morbidity and mortality worldwide. Bacterial complications of influenza A confer the greatest risk to patients. TH17 pathway inhibition has been implicated as a mechanism by which influenza A alters bacterial host defense. Here we show that preceding influenza causes persistent Staphylococcus aureus infection and suppression of TH17 pathway activation in mice. Influenza does not inhibit S. aureus binding and uptake by phagocytic cells but instead attenuates S. aureus induced TH17 related antimicrobial peptides necessary for bacterial clearance in the lung. Importantly, exogenous lipocalin 2 rescued viral exacerbation of S. aureus infection and decreased free iron levels in the bronchoalveolar lavage from mice coinfected with S. aureus and influenza. These findings indicate a novel mechanism by which influenza A inhibits TH17 immunity and increases susceptibility to secondary bacterial pneumonia. Identification of new mechanisms in the pathogenesis of bacterial pneumonia could lead to future therapeutic targets.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/microbiologia , Pneumonia Estafilocócica/microbiologia , Staphylococcus aureus/imunologia , Análise de Variância , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/microbiologia , Líquido da Lavagem Broncoalveolar/virologia , Coinfecção/microbiologia , Coinfecção/virologia , Interações Hospedeiro-Patógeno/imunologia , Vírus da Influenza A/patogenicidade , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Pneumonia Estafilocócica/imunologia , Pneumonia Estafilocócica/virologia , Staphylococcus aureus/patogenicidade , Células Th17
6.
JCI Insight ; 3(7)2018 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-29618653

RESUMO

Secondary bacterial respiratory infections are commonly associated with both acute and chronic lung injury. Influenza complicated by bacterial pneumonia is an effective model to study host defense during pulmonary superinfection due to its clinical relevance. Multiprotein inflammasomes are responsible for IL-1ß production in response to infection and drive tissue inflammation. In this study, we examined the role of the inflammasome during viral/bacterial superinfection. We demonstrate that ASC-/- mice are protected from bacterial superinfection and produce sufficient quantities of IL-1ß through an apoptosis-associated speck-like protein containing CARD (ASC) inflammasome-independent mechanism. Despite the production of IL-1ß by ASC-/- mice in response to bacterial superinfection, these mice display decreased lung inflammation. A neutrophil elastase inhibitor blocked ASC inflammasome-independent production of IL-1ß and the IL-1 receptor antagonist, anakinra, confirmed that IL-1 remains crucial to the clearance of bacteria during superinfection. Delayed inhibition of NLRP3 during influenza infection by MCC950 decreases bacterial burden during superinfection and leads to decreased inflammatory cytokine production. Collectively, our results demonstrate that ASC augments the clearance of bacteria, but can also contribute to inflammation and mortality. ASC should be considered as a therapeutic target to decrease morbidity and mortality during bacterial superinfection.


Assuntos
Inflamassomos/imunologia , Influenza Humana/imunologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia , Superinfecção/imunologia , Animais , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/imunologia , Modelos Animais de Doenças , Furanos/farmacologia , Furanos/uso terapêutico , Compostos Heterocíclicos de 4 ou mais Anéis , Humanos , Indenos , Inflamassomos/efeitos dos fármacos , Inflamassomos/genética , Inflamassomos/metabolismo , Influenza Humana/mortalidade , Influenza Humana/patologia , Influenza Humana/virologia , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Proteína Antagonista do Receptor de Interleucina 1/uso terapêutico , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Pulmão/imunologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Interleucina-1/antagonistas & inibidores , Receptores de Interleucina-1/imunologia , Receptores de Interleucina-1/metabolismo , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/mortalidade , Infecções Estafilocócicas/patologia , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Sulfonas , Superinfecção/microbiologia , Superinfecção/mortalidade , Superinfecção/patologia
7.
Front Immunol ; 9: 2151, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30337919

RESUMO

Influenza is a common respiratory virus that infects between 5 and 20% of the US population and results in 30,000 deaths annually. A primary cause of influenza-associated death is secondary bacterial pneumonia. We have previously shown that influenza induces type I interferon (IFN)-mediated inhibition of Type 17 immune responses, resulting in exacerbation of bacterial burden during influenza and Staphylococcus aureus super-infection. In this study, we investigated the role of STAT2 signaling during influenza and influenza-bacterial super-infection in mice. Influenza-infected STAT2-/- mice had increased morbidity, viral burden, and inflammation when compared to wild-type mice. Despite an exaggerated inflammatory response to influenza infection, we found increased bacterial control and survival in STAT2 deficient mice during influenza-MRSA super-infection compared to controls. Further, we found that increased bacterial clearance during influenza-MRSA super-infection is not due to rescue of Type 17 immunity. Absence of STAT2 was associated with increased accumulation of M1, M2 and M1/M2 co-expressing macrophages during influenza-bacterial super-infection. Neutralization of IFNγ (M1) and/or Arginase 1 (M2) impaired bacterial clearance in Stat2-/- mice during super-infection, demonstrating that pulmonary macrophages expressing a mixed M1/M2 phenotype promote bacterial control during influenza-bacterial super-infection. Together, these results suggest that the STAT2 signaling is involved in suppressing macrophage activation and bacterial control during influenza-bacterial super-infection. Further, these studies reveal novel mechanistic insight into the roles of macrophage subpopulations in pulmonary host defense.


Assuntos
Influenza Humana/imunologia , Macrófagos Alveolares/imunologia , Pneumonia Estafilocócica/imunologia , Fator de Transcrição STAT2/metabolismo , Superinfecção/imunologia , Animais , Transplante de Medula Óssea , Embrião de Galinha , Modelos Animais de Doenças , Feminino , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/diagnóstico , Influenza Humana/microbiologia , Influenza Humana/mortalidade , Ativação de Macrófagos/imunologia , Macrófagos Alveolares/metabolismo , Masculino , Células-Tronco Mesenquimais , Staphylococcus aureus Resistente à Meticilina/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pneumonia Estafilocócica/diagnóstico , Pneumonia Estafilocócica/microbiologia , Pneumonia Estafilocócica/mortalidade , Cultura Primária de Células , Fator de Transcrição STAT2/genética , Fator de Transcrição STAT2/imunologia , Índice de Gravidade de Doença , Transdução de Sinais/imunologia , Superinfecção/diagnóstico , Superinfecção/microbiologia , Superinfecção/mortalidade , Quimeras de Transplante
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