RESUMO
We report the heterologous expression, structure, and antimicrobial activity of a lasso peptide, ubonodin, encoded in the genome of Burkholderia ubonensis. The topology of ubonodin is unprecedented amongst lasso peptides, with 18 of its 28 amino acids found in the mechanically bonded loop segment. Ubonodin inhibits RNA polymerase in vitro and has potent antimicrobial activity against several pathogenic members of the Burkholderia genus, most notably B.â cepacia and B.â multivorans, causative agents of lung infections in cystic fibrosis patients.
Assuntos
Antibacterianos/farmacologia , Complexo Burkholderia cepacia/efeitos dos fármacos , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Descoberta de Drogas , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Antibacterianos/química , Complexo Burkholderia cepacia/classificação , Humanos , Proteínas Citotóxicas Formadoras de Poros/químicaRESUMO
Bdellovibrio spp. and Micavibrio spp. are Gram-negative predators that feed on other Gram-negative bacteria, making predatory bacteria potential alternatives to antibiotics for treating multi-drug resistant infections. While the ability of predatory bacteria to control bacterial infections in vitro is well documented, the in vivo effect of predators on a living host has yet to be extensively examined. In this study, respiratory and intravenous inoculations were used to determine the effects of predatory bacteria in mice. We found no reduction in mouse viability after intranasal or intravenous inoculation of B. bacteriovorus 109J, HD100 or M. aeruginosavorus. Introducing predators into the respiratory tract of mice provoked a modest inflammatory response at 1 hour post-exposure, but was not sustained at 24 hours, as measured by RT-qPCR and ELISA. Intravenous injection caused an increase of IL-6 in the kidney and spleen, TNF in the liver and CXCL-1/KC in the blood at 3 hours post-exposure, returning to baseline levels by 18 hours. Histological analysis of tissues showed no pathological changes due to predatory bacteria. Furthermore, qPCR detected predators were cleared from the host quickly and efficiently. This work addresses some of the safety concerns regarding the potential use of predatory bacteria as a live antibiotic.
Assuntos
Alphaproteobacteria/crescimento & desenvolvimento , Antibiose/fisiologia , Bdellovibrio/crescimento & desenvolvimento , Sistema Respiratório/microbiologia , Animais , Biofilmes/crescimento & desenvolvimento , Inflamação/metabolismo , Inflamação/microbiologia , Injeções Intravenosas/métodos , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Sistema Respiratório/metabolismoRESUMO
An understanding of anthrax toxins on the emerging immune system and blood production are significant to medicine. This study examined the effects of anthrax toxin on hematopoiesis and determined roles for cytokines. Anthrax holotoxin toxin is three components: protective antigen (PA) binds to the target cell and mediates the entry of lethal factor (LF) and edema factor (EF). Anthrax toxin dramatically inhibits signaling in immune cells. We first identified the cell subsets that interacted with the protective antigen (PA) and then studied the effects on hematopoietic progenitors in clonogenic assays: granulocytic-monocytic (CFU-GM) and late erythroid (CFU-E). Multi-color immunofluorescence with FITC-PA indicated its interaction with early and late myeloid cells. Clonogenic assays, in the presence or absence of holotoxin and individual toxin proteins resulted in significant suppression by hologenic toxic alone, despite the presence of growth-promoting cytokines. Antibodies to anthrax receptor (ATR1) reversed the suppressive effects, indicating specificity. Monomeric proteins showed different effects on myeloid and erythroid progenitors. Suppression was not due to cell death, based on undetectable active caspase 3. Cytokine array analyses with supernatants from toxin-stimulated stroma showed an increase in the hematopoietic suppressor, MIP-1α. This finding, in addition to our previous studies, showing an increase in IL-10, suggested indirect roles for cytokines in toxin-mediated hematopoietic suppression. The chemokine, SDF-1α was increased. Since SDF-1 is involved in the mobilization of hematopoietic cells, it is likely that anthrax holotoxin could induce cell exit from BM. In summary, anthrax holotoxin, but not individual toxins, exerted hematopoietic effects on myeloid and erythroid progenitors via specific receptor, partly through the induction of cytokines.
Assuntos
Antígenos de Bactérias/farmacologia , Toxinas Bacterianas/farmacologia , Citocinas/metabolismo , Hematopoese/efeitos dos fármacos , Adulto , Western Blotting , Células da Medula Óssea/citologia , Caspases/metabolismo , Citocinas/biossíntese , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/efeitos dos fármacos , Células Precursoras Eritroides/metabolismo , Citometria de Fluxo , Células Progenitoras de Granulócitos e Macrófagos/citologia , Células Progenitoras de Granulócitos e Macrófagos/efeitos dos fármacos , Células Progenitoras de Granulócitos e Macrófagos/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Modelos Biológicos , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Células Estromais/enzimologia , Adulto JovemRESUMO
Tuberculosis is the most prevalent infectious disease in the world. It is also believed that in developing countries, as many as 40 to 80% of individuals with AIDS are at risk of developing tuberculosis. In recent years there has been a significant increase in the incidence of tuberculosis due to the emergence of multi-drug resistant strains of Mycobacterium tuberculosis and due to increased numbers of highly susceptible immuno-compromised individuals arising from the AIDS pandemic. Extreme drug resistant tuberculosis raises the possibility that the current tuberculosis epidemic of mostly drug-susceptible tuberculosis will be replaced with a form of tuberculosis with severely restricted treatment options. This phenomenon would jeopardize the progress made in recent years to control tuberculosis globally and would also put at risk the plans to progress towards universal access to HIV prevention and treatment. Patients with extreme drug-resistant tuberculosis would have to be managed in the same way as tuberculosis patients before the antibiotic era. No new anti-tuberculosis drugs have been brought into the clinic in the past 40 years. Immuno-adjunctive therapy appears to be promising in improving outcome of clinical control of refractory mycobacterial infections, including multi-drug resistant- tuberculosis and Mycobacterium avium complex infection. The tripeptide, glutathione protects all cells against oxidizing agents, free radicals and reactive oxygen intermediates, either directly or through enzymatic action of glutathione peroxidases and glutathione-transferases. This review article is a compilation of our major research findings on the innate immune responses against Mycobacterium tuberculosis infection. We discuss in detail both the direct antimycobacterial effects as well as the immune enhancing effects of glutathione leading to the inhibition of growth of Mycobacterium tuberculosis. The article presents some new promising patents related to the control of Mycobacterium tuberculosis infection.
Assuntos
Glutationa/farmacologia , Glutationa/uso terapêutico , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose/tratamento farmacológico , Animais , Citocinas/metabolismo , Citometria de Fluxo , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Mycobacterium tuberculosis/imunologia , Fatores de Risco , Tuberculose/epidemiologia , Tuberculose/imunologia , Tuberculose/microbiologiaRESUMO
Two virulence factors produced by the periodontopathogen Aggregatibacter actinomycetemcomitans are leukotoxin, a secreted lipoprotein that kills human polymorphonuclear leukocytes and macrophages, and poly-N-acetylglucosamine (PGA), a surface polysaccharide that mediates intercellular adhesion, biofilm formation and detergent resistance. In this study we examined the roles of leukotoxin and PGA in protecting A. actinomycetemcomitans cells from killing by the human macrophage cell line THP-1. Monolayers of THP-1 cells were infected with single-cell suspensions of a wild-type A. actinomycetemcomitans strain, or of isogenic leukotoxin or PGA mutant strains. After 48h, viable bacteria were enumerated by dilution plating, macrophage morphology was evaluated microscopically, and macrophage viability was measured by a Trypan blue dye exclusion assay. The number of A. actinomycetemcomitans CFUs increased approximately twofold in wells infected with the wild-type strain, but decreased by approximately 70-90% in wells infected with the leukotoxin and PGA mutant strains. Infection with the wild-type or leukotoxin mutant strain caused a significant decrease in THP-1 cell viability, whereas infection with the PGA mutant strain did not result in any detectable changes in THP-1 viability. Pre-treatment of wild-type A. actinomycetemcomitans cells with the PGA-hydrolyzing enzyme dispersin B rendered them sensitive to killing by THP-1 cells. We concluded that both leukotoxin and PGA are necessary for evasion of macrophage killing by A. actinomycetemcomitans.
Assuntos
Infecções por Actinobacillus/imunologia , Aggregatibacter actinomycetemcomitans/imunologia , Exotoxinas/imunologia , Macrófagos/imunologia , Polissacarídeos/imunologia , Fatores de Virulência/imunologia , Infecções por Actinobacillus/microbiologia , Aggregatibacter actinomycetemcomitans/genética , Aggregatibacter actinomycetemcomitans/patogenicidade , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Exotoxinas/genética , Humanos , Macrófagos/microbiologia , Camundongos , Polissacarídeos/genética , Polissacarídeos Bacterianos/genética , Polissacarídeos Bacterianos/imunologia , Fatores de Virulência/genéticaRESUMO
It is becoming increasingly apparent that natural killer (NK) cells play a crucial role in innate defense mechanisms against Mycobacterium tuberculosis infection. Furthermore, NK cell functions are dependent on adequate levels of glutathione. In this study, we examined whether the NK cell-mediated growth control of intracellular M. tuberculosis is dependent on adequate levels of glutathione. We investigated the effects of glutathione both alone and in combination with interleukin-2 (IL-2) or IL-12 or both in modulating NK cell functions, such as cytolytic activity, activating receptor expression, induction of apoptosis, and cytokine synthesis. Our results strongly indicate that glutathione in combination with IL-2+IL-12 augments NK cell functions, leading to control M. tuberculosis infection.
Assuntos
Citocinas/metabolismo , Glutationa/metabolismo , Interleucina-12/metabolismo , Interleucina-2/metabolismo , Células Matadoras Naturais/imunologia , Monócitos/metabolismo , Mycobacterium tuberculosis/imunologia , Apoptose , Linhagem Celular , Citocinas/imunologia , Glutationa/farmacologia , Humanos , Imunidade Inata , Interleucina-12/imunologia , Interleucina-2/imunologia , Monócitos/imunologia , Mycobacterium tuberculosis/efeitos dos fármacos , Análise Serial de ProteínasRESUMO
Glutathione levels are significantly reduced in peripheral blood mononuclear cells and red blood cells isolated from tuberculosis patients. Treatment of blood cultures from tuberculosis patients with N-acetyl cysteine, a glutathione precursor, was associated with improved control of intracellular M. tuberculosis infection. N-acetyl-cysteine treatment decreased the levels of IL-10, IL-6, TNF-alpha and IL-1, in blood cultures derived from tuberculosis patients, favoring the host immune cells to successfully control M. tuberculosis replication.
Assuntos
Acetilcisteína/farmacologia , Glutationa/sangue , Leucócitos Mononucleares/imunologia , Ativação Linfocitária/imunologia , Tuberculose/sangue , Acetilcisteína/uso terapêutico , Antígenos de Bactérias/imunologia , Glutationa/metabolismo , Humanos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/imunologia , Projetos Piloto , Tuberculose/imunologiaRESUMO
The competition for L-arginine between the inducible nitric oxide synthase and arginase contributes to the outcome of several parasitic and bacterial infections. The acquisition of L-arginine, however, is important not only for the host cells but also for the intracellular pathogen. In this study we observe that strain AS-1, the Mycobacterium bovis BCG strain lacking the Rv0522 gene, which encodes an arginine permease, perturbs l-arginine metabolism in J774.1 murine macrophages. Infection with AS-1, but not with wild-type BCG, induced l-arginine uptake in J774.1 cells. This increase in L-arginine uptake was independent of activation with gamma interferon plus lipopolysaccharide and correlated with increased expression of the MCAT1 and MCAT2 cationic amino acid transport genes. AS-1 infection also enhanced arginase activity in resting J774.1 cells. Survival studies revealed that AS-1 survived better than BCG within resting J774.1 cells. Intracellular growth of AS-1 was further enhanced by inhibiting arginase and ornithine decarboxylase activities in J774.1 cells using L-norvaline and difluoromethylornithine treatment, respectively. These results suggest that the arginine-related activities of J774.1 macrophages are affected by the arginine transport capacity of the infecting BCG strain. The loss of Rv0522 gene-encoded arginine transport may have induced other cationic amino acid transport systems during intracellular growth of AS-1, allowing better survival within resting macrophages.
Assuntos
Arginina/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Mycobacterium bovis , Tuberculose Bovina/metabolismo , Tuberculose Bovina/microbiologia , Sistemas de Transporte de Aminoácidos Básicos/genética , Animais , Proteínas de Bactérias/genética , Transportador 1 de Aminoácidos Catiônicos/genética , Transportador 1 de Aminoácidos Catiônicos/metabolismo , Transportador 2 de Aminoácidos Catiônicos/genética , Transportador 2 de Aminoácidos Catiônicos/metabolismo , Bovinos , Camundongos , Mutação , Mycobacterium bovis/crescimento & desenvolvimentoRESUMO
Intracellular levels of glutathione are depleted in patients with acquired immunodeficiency syndrome in whom the risk of tuberculosis, particularly disseminated disease is many times that of healthy individuals. In this study, we examined the role of glutathione in immunity against tuberculosis infection in samples derived from healthy and human immunodeficiency virus infected subjects. Our studies confirm that glutathione levels are reduced in peripheral blood mononuclear cells and in red blood cells isolated from human immunodeficiency virus-infected subjects (CD4>400/cumm). Furthermore, treatment of blood cultures from human immunodeficiency virus infected subjects with N-acetyl cysteine, a glutathione precursor, caused improved control of intracellular M. tuberculosis infection. N-acetyl cysteine treatment decreased the levels of IL-1, TNF-alpha, and IL-6, and increased the levels of IFN-gamma in blood cultures derived from human immunodeficiency virus-infected subjects, promoting the host immune responses to contain M. tuberculosis infection successfully.
RESUMO
beta-Defensins are a family of small cationic peptides involved in the innate response to microbial infection. Although their role in microbial killing is well established, the mechanisms through which this occurs remain largely undefined. Here, using protein array technology, we describe a role for human beta-defensins in the induction of an inflammatory cytokine response by human peripheral blood mononuclear cells (PBMCs). Human beta-defensins 1, 2, and 3 were examined for induction of an array of cytokines and chemokines. Some cytokines, such as interleukin 8 (IL-8) and monocyte chemoattractant protein 1, were up-regulated by all three defensins, while others, such as IL-6 and IL-10, were induced more selectively. It was notable that each defensin induced a unique pattern of cytokines. This report documents, for the first time, an analysis of the composite cytokine response of human PBMCs to beta-defensins. The induction or up-regulation of a number of cytokines involved in the adaptive immune response suggests a possible role for these defensins in linking innate and acquired immunity.
Assuntos
Citocinas/biossíntese , Leucócitos Mononucleares/efeitos dos fármacos , beta-Defensinas/farmacologia , Células Cultivadas , Humanos , Interleucina-10/biossíntese , Interleucina-10/genética , Interleucina-6/biossíntese , Interleucina-6/genética , Interleucina-8/biossíntese , Interleucina-8/genética , Leucócitos Mononucleares/imunologia , RNA Mensageiro/análiseRESUMO
Glutathione is a tripeptide and antioxidant, synthesized at high levels by cells during the production of reactive oxygen and nitrogen intermediates. Glutathione also serves as a carrier molecule for nitric oxide in the form of S-nitrosoglutathione. Previous studies from this laboratory have shown that glutathione and S-nitrosoglutathione are directly toxic to mycobacteria. Glutathione is not transported into the cells as a tripeptide. Extracellular glutathione is converted to a dipeptide due to the action of transpeptidase, and the dipeptide is then transported into the bacterial cells. The processing of glutathione and S-nitrosoglutathione is brought about by the action of the enzyme gamma-glutamyl transpeptidase. The function of gamma-glutamyl transpeptidase is to cleave glutathione and S-nitrosoglutathione to the dipeptide (Cys-Gly), which is then transported into the bacterium by the multicomponent ABC transporter dipeptide permease. We have created a mutant strain of Mycobacterium tuberculosis lacking this metabolic enzyme. We investigated the sensitivity of this strain to glutathione and S-nitrosoglutathione compared to that of the wild-type bacteria. In addition, we examined the role of glutathione and/or S-nitrosoglutathione in controlling the growth of intracellular M. tuberculosis inside mouse macrophages.
Assuntos
Glutationa/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , gama-Glutamiltransferase/genética , Animais , Anti-Infecciosos/farmacologia , Farmacorresistência Bacteriana , Glutationa/metabolismo , Macrófagos Peritoneais/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , S-Nitrosoglutationa/metabolismo , S-Nitrosoglutationa/farmacologiaRESUMO
The role of tumor necrosis factor-alpha (TNF-alpha) in controlling growth of Mycobacterium tuberculosis in murine peritoneal macrophages infected in vitro was studied. TNF-alpha was shown to be required but not sufficient, and the amount of TNF-alpha produced by the infected cells did not correlate with the extent of growth control. In this system, TNF-alpha-dependent control of growth of the avirulent strain H37Ra was independent of inducible nitric oxide synthase (iNOS) and interferon-gamma (IFN-gamma), as shown by the infection of macrophages from selected gene-disrupted mice. TNF-alpha-mediated bacteriostasis of H37Ra in the infected macrophages was associated with increased expression of selected Th1-type cytokines and chemokines. In contrast, growth of the virulent strain H37Rv in macrophages involved upregulation by infected cells of Th2-type cytokines, including interleukin-5 (IL-5), IL-10, and IL-13. Taken together, these results suggest that the particular nature of macrophage activation and the cytokine and chemokine response to infection with different M. tuberculosis strains determine the ability of the cells to control the growth of the intracellular bacilli.
Assuntos
Quimiocinas/imunologia , Citocinas/imunologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/microbiologia , Mycobacterium tuberculosis/imunologia , Animais , Células Cultivadas , Quimiocinas/genética , Citocinas/genética , Técnicas In Vitro , Ativação de Macrófagos , Macrófagos Peritoneais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Fator de Necrose Tumoral alfa/imunologiaRESUMO
We demonstrate that Mycobacterium tuberculosis grown in vitro is sensitive to glutathione and its derivative S-nitrosoglutathione. Furthermore, our infection studies with J774.1 macrophages indicate that glutathione is essential for the control of the intracellular growth of M. tuberculosis. This study indicates the important role of glutathione in the control of macrophages by M. tuberculosis.
Assuntos
Glutationa/farmacologia , Macrófagos/imunologia , Macrófagos/microbiologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , S-Nitrosoglutationa/farmacologia , Animais , Linhagem Celular , Glutationa/metabolismo , Humanos , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos , CamundongosRESUMO
L-arginine uptake systems in macrophages play a role in regulating nitric oxide synthesis via the inducible L-arginine nitric oxide pathway. This paper describes the association of L-arginine transport with nitric oxide production in human peripheral blood monocyte-derived macrophages and in peritoneal macrophages from control and inducible nitric oxide synthase knock out C57BL6 mice. Experiments performed with human macrophages suggested that little or no nitric oxide was produced in human macrophages in vitro and that human macrophages exhibit a different arginine transport-specific response to stimuli compared with rodent macrophages. We conclude that increased L-arginine transport in both human and murine macrophages is dependent on the requirement for intracellular nitric oxide.
Assuntos
Arginina/metabolismo , Macrófagos/metabolismo , Óxido Nítrico/fisiologia , Animais , Transporte Biológico , Células Cultivadas , Humanos , Ativação de Macrófagos , Macrófagos/microbiologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mycobacterium bovis/crescimento & desenvolvimento , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/fisiologia , Óxido Nítrico Sintase Tipo II , Especificidade da EspécieRESUMO
Reactive oxygen and nitrogen intermediates are important antimicrobial defense mechanisms of macrophages and other phagocytic cells. While reactive nitrogen intermediates have been shown to play an important role in tuberculosis control in the murine system, their role in human disease is not clearly established. Glutathione, a tripeptide and antioxidant, is synthesized at high levels by cells during reactive oxygen intermediate and nitrogen intermediate production. Glutathione has been recently shown to play an important role in apoptosis and to regulate antigen-presenting-cell functions. Glutathione also serves as a carrier molecule for nitric oxide, in the form of S-nitrosoglutathione. Previous work from this laboratory has shown that glutathione and S-nitrosoglutathione are directly toxic to mycobacteria. A mutant strain of Mycobacterium bovis BCG, defective in the transport of small peptides such as glutathione, is resistant to the toxic effect of glutathione and S-nitrosoglutathione. Using the peptide transport mutant as a tool, we investigated the role of glutathione and S-nitrosoglutathione in animal and human macrophages in controlling intracellular mycobacterial growth.
Assuntos
Glutationa/fisiologia , Macrófagos/imunologia , Macrófagos/microbiologia , Mycobacterium bovis/patogenicidade , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , Células Cultivadas , Humanos , Macrófagos Peritoneais/microbiologia , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/microbiologia , Mycobacterium bovis/genética , Mycobacterium bovis/crescimento & desenvolvimento , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo IIRESUMO
Using a Mycobacterium bovis BCG mutant (AS1) lacking a Bacillus subtilis L-arginine transporter homolog, we demonstrate here that the interaction between intracellular mycobacteria and the macrophage with respect to L-arginine transport and metabolism is quite complex. Intracellular AS1 stimulates macrophage L-arginine transport and accumulates 2.5-fold more (3)H label derived from L-arginine than does the wild type. These studies suggest that the accumulation of (3)H label reflects the acquisition of metabolites of L-arginine produced by the macrophage.