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Biomedical subjects

V Deretic

Publications and source records attributed to V Deretic.

At least 19 recordsLinked to original sources
PubMed2002

Oxidative stress response genes in Mycobacterium tuberculosis: role of ahpC in resistance to peroxynitrite and stage-specific survival in macrophages.

The Mycobacterium tuberculosis ahpC gene, encoding the mycobacterial orthologue of alkylhydroperoxide reductase, undergoes an unusual regulatory cycle. The levels of AhpC alternate between stages of expression silencing in virulent strains grown as aerated cultures, secondary to a natural loss of the regulatory oxyR function in all strains of the tubercle bacillus, and expression activation in static bacilli by a yet undefined mechanism. The reasons for this unorthodox regulatory cycle controlling expression of an antioxidant factor are currently not known. In this work, M. tuberculosis H37Rv and Mycobacterium smegmatis mc(2)155 ahpC knockout mutants were tested for sensitivity to reactive nitrogen intermediates, in particular peroxynitrite, a highly reactive combinatorial product of reactive nitrogen and oxygen species, and sensitivity to bactericidal mechanisms in resting and activated macrophages. Both M. tuberculosis ahpC::Km(r) and M. smegmatis ahpC::Km(r) showed increased susceptibility to peroxynitrite. In contrast, inactivation of ahpC in M. tuberculosis did not cause increased sensitivity to donors of NO alone. M. tuberculosis ahpC::Km(r) also showed decreased survival in unstimulated macrophages, but the effect was no longer detectable upon IFNgamma activation. These studies establish a specific role for ahpC in antioxidant defences involving peroxynitrite and most likely additional cidal mechanisms in macrophages, with the regulatory cycle likely contributing to survival upon coming out of the stationary phase during dormancy (latent infection) or upon transmission to a new host.

Cell Line↗
PubMed2001

Molecular basis for defective glycosylation and Pseudomonas pathogenesis in cystic fibrosis lung.

The CFTR gene encodes a transmembrane conductance regulator, which is dysfunctional in patients with cystic fibrosis (CF). The mechanism by which defective CFTR (CF transmembrane conductance regulator) leads to undersialylation of plasma membrane glycoconjugates, which in turn promote lung pathology and colonization with Pseudomonas aeruginosa causing lethal bacterial infections in CF, is not known. Here we show by ratiometric imaging with lumenally exposed pH-sensitive green fluorescent protein that dysfunctional CFTR leads to hyperacidification of the trans-Golgi network (TGN) in CF lung epithelial cells. The hyperacidification of TGN, glycosylation defect of plasma membrane glycoconjugates, and increased P. aeruginosa adherence were corrected by incubating CF respiratory epithelial cells with weak bases. Studies with pharmacological agents indicated a role for sodium conductance, modulated by CFTR regulatory function, in determining the pH of TGN. These studies demonstrate the molecular basis for defective glycosylation of lung epithelial cells and bacterial pathogenesis in CF, and suggest a cure by normalizing the pH of intracellular compartments.

Acids↗
PubMed2001

Mycobacterium tuberculosis signal transduction system required for persistent infections.

It is estimated that nearly 2 billion people currently suffer from latent Mycobacterium tuberculosis infection. Although the key front-line antituberculosis drugs are effective in treating individuals with acute tuberculosis, these drugs are ineffective in eliminating M. tuberculosis during the persistent stages of latent infection. Consequently, therapeutics that directly target persistent bacilli are urgently needed. We have conducted a global analysis on a group of regulatory determinants that may play a role in M. tuberculosis virulence, and identified a two-component response regulator whose expression is required for entrance into and maintenance of persistent infection. Inactivation of this response regulator, Rv0981 (termed here mprA for mycobacterial persistence regulator), affected M. tuberculosis H37Rv growth in vivo in an organ- and infection stage-specific fashion. These results indicate that two-component systems are important for adaptation of the tubercle bacillus during stages of persistent infection.

Animals↗
PubMed2001

Role of phosphatidylinositol 3-kinase and Rab5 effectors in phagosomal biogenesis and mycobacterial phagosome maturation arrest.

Phagosomal biogenesis is a fundamental biological process of particular significance for the function of phagocytic and antigen-presenting cells. The precise mechanisms governing maturation of phagosomes into phagolysosomes are not completely understood. Here, we applied the property of pathogenic mycobacteria to cause phagosome maturation arrest in infected macrophages as a tool to dissect critical steps in phagosomal biogenesis. We report the requirement for 3-phosphoinositides and acquisition of Rab5 effector early endosome autoantigen (EEA1) as essential molecular events necessary for phagosomal maturation. Unlike the model phagosomes containing latex beads, which transiently recruited EEA1, mycobacterial phagosomes excluded this regulator of vesicular trafficking that controls membrane tethering and fusion processes within the endosomal pathway and is recruited to endosomal membranes via binding to phosphatidylinositol 3-phosphate (PtdIns[3]P). Inhibitors of phosphatidylinositol 3'(OH)-kinase (PI-3K) activity diminished EEA1 recruitment to newly formed latex bead phagosomes and blocked phagosomal acquisition of late endocytic properties, indicating that generation of PtdIns(3)P plays a role in phagosomal maturation. Microinjection into macrophages of antibodies against EEA1 and the PI-3K hVPS34 reduced acquisition of late endocytic markers by latex bead phagosomes, demonstrating an essential role of these Rab5 effectors in phagosomal biogenesis. The mechanism of EEA1 exclusion from mycobacterial phagosomes was investigated using mycobacterial products. Coating of latex beads with the major mycobacterial cell envelope glycosylated phosphatidylinositol lipoarabinomannan isolated from the virulent Mycobacterium tuberculosis H37Rv, inhibited recruitment of EEA1 to latex bead phagosomes, and diminished their maturation. These findings define the generation of phosphatidylinositol 3-phosphate and EEA1 recruitment as: (a) important regulatory events in phagosomal maturation and (b) critical molecular targets affected by M. tuberculosis. This study also identifies mycobacterial phosphoinositides as products with specialized toxic properties, interfering with discrete trafficking stages in phagosomal maturation.

Androstadienes↗
PubMed2001

CFTR and pseudomonas infections in cystic fibrosis.

Pseudomonas aeruginosa is a significant threat to human health as it is frequently recalcitrant to conventional antibacterial therapy. This ubiquitous gram-negative bacterium is notorious for its nutritional and ecological flexibility and its resistance to both antibiotic treatments and sanitary measures. These properties contribute to its prominence as a leading source of opportunistic nosocomial (hospital acquired) and a less appreciated, but significant cause of community acquired infections. P. aeruginosa remains a considerable problem for patients with burns, neutropenic individuals, and cystic fibrosis patients (CF). In this review, we will address the current issues in P. aeruginosa infections in CF. A major emphasis will be placed on the factors predisposing CF patients to colonization with P. aeruginosa.

Animals↗
PubMed2001

Regulation of catalase-peroxidase (KatG) expression, isoniazid sensitivity and virulence by furA of Mycobacterium tuberculosis.

Mycobacterium tuberculosis has two genes for ferric uptake regulator orthologues, one of which, furA, is situated immediately upstream of katG encoding catalase-peroxidase, a major virulence factor that also activates the prodrug isoniazid. This association suggested that furA might regulate katG and other genes involved in pathogenesis. Transcript mapping showed katG to be expressed from a strong promoter, with consensus -10 and -35 elements, preceding furA. No promoter activity was demonstrated downstream of the furA start codon, using different gene reporter systems, indicating that furA and katG are co-transcribed from a common regulatory region. The respective roles of these two genes in the isoniazid susceptibility and virulence of M. tuberculosis were assessed by combinatorial complementation of a Delta(furA-katG) strain that is heavily attenuated in a mouse model of tuberculosis. In the absence of furA, katG was upregulated, cells became hypersensitive to isoniazid, and full virulence was restored, indicating that furA regulates the transcription of both genes. When furA alone was introduced into the Delta(furA-katG) mutant, survival in mouse lungs was moderately increased, suggesting that FurA could regulate genes, other than katG, that are involved in pathogenesis. These do not include the oxidative stress genes ahpC and sodA, or those for siderophore production.

Animals↗
PubMed2001

Mycobacterial FurA is a negative regulator of catalase-peroxidase gene katG.

In several bacteria, the catalase-peroxidase gene katG is under positive control by oxyR, a transcriptional regulator of the peroxide stress response. The Mycobacterium tuberculosis genome also contains sequences corresponding to oxyR, but this gene has been inactivated in the tubercle bacillus because of the presence of multiple mutations and deletions. Thus, M. tuberculosis katG and possibly other parts of the oxidative stress response in this organism are either not regulated or are controlled by a factor different from OxyR. The mycobacterial FurA is a homologue of the ferric uptake regulator Fur and is encoded by a gene located immediately upstream of katG. Here, we examine the possibility that FurA regulates katG expression. Inactivation of furA on the Mycobacterium smegmatis chromosome, a mycobacterial species that also lacks an oxyR homologue, resulted in derepression of katG, concomitant with increased resistance of the furA mutant to H2O2. In addition, M. smegmatis furA::Km(r) was more sensitive to the front-line antituberculosis agent isonicotinic acid hydrazide (INH) compared with the parental furA+ strain. The phenotypic manifestations were specific, as the mutant strain did not show altered sensitivity to organic peroxides, and both H2O2 and INH susceptibility profiles were complemented by the wild-type furA+ gene. We conclude that FurA is a second regulator of oxidative stress response in mycobacteria and that it negatively controls katG. In species lacking a functional oxyR, such as M. tuberculosis and M. smegmatis, FurA appears to be a dominant regulator affecting mycobacterial physiology and intracellular survival.

Bacterial Proteins↗
PubMed2001

Silencing of oxidative stress response in Mycobacterium tuberculosis: expression patterns of ahpC in virulent and avirulent strains and effect of ahpC inactivation.

Intracellular pathogens such as Mycobacterium tuberculosis are able to survive in the face of antimicrobial products generated by the host cell in response to infection. The product of the alkyl hydroperoxide reductase gene (ahpC) of M. tuberculosis is thought to be involved in protecting the organism against both oxidative and nitrosative stress encountered within the infected macrophage. Here we report that, contrary to expectations, ahpC expression in virulent strains of M. tuberculosis and Mycobacterium bovis grown in vitro is repressed, often below the level of detection, whereas expression in the avirulent vaccine strain M. bovis BCG is constitutively high. The repression of the ahpC gene of the virulent strains is independent of the naturally occurring lesions of central regulator oxyR. Using a green fluorescence protein vector (gfp)-ahpC reporter construct we present data showing that repression of ahpC of virulent M. tuberculosis also occurred during growth inside macrophages, whereas derepression in BCG was again seen under identical conditions. Inactivation of ahpC on the chromosome of M. tuberculosis by homologous recombination had no effect on its growth during acute infection in mice and did not affect in vitro sensitivity to H2O2. However, consistent with AhpC function in detoxifying organic peroxides, sensitivity to cumene hydroperoxide exposure was increased in the ahpC::Km(r) mutant strain. The preservation of a functional ahpC gene in M. tuberculosis in spite of its repression under normal growth conditions suggests that, while AhpC does not play a significant role in establishing infection, it is likely to be important under certain, as yet undefined conditions. This is supported by the observation that repression of ahpC expression in vitro was lifted under conditions of static growth.

Animals↗
PubMed2001

Mapping of Mycobacterium tuberculosis katG promoters and their differential expression in infected macrophages.

Intracellular pathogenic bacteria, including Mycobacterium tuberculosis, frequently have multitiered defense mechanisms ensuring their survival in host phagocytic cells. One such defense determinant in M. tuberculosis is the katG gene, which encodes an enzyme with catalase, peroxidase, and peroxynitritase activities. KatG is considered to be important for protection against reactive oxygen and nitrogen intermediates produced by phagocytic cells. However, KatG also activates the front-line antituberculosis drug isoniazid, hence rendering M. tuberculosis exquisitely sensitive to this compound. In this context, katG expression represents a double-edged sword, as it is an important virulence determinant but at the same time its activity levels determine sensitivity to INH. Thus, it is important to delineate the regulation and expression of katG, as this not only can aid understanding of how M. tuberculosis survives and persists in the host but also may provide information of relevance for better management of INH therapy. Here, we report the first extensive analysis of the katG promoter activity examined both in vitro and in vivo. Using S1 nuclease protection analysis, we mapped the katG mRNA 5' ends and demonstrated that two promoters, P(1)furA and P(1)katG, control transcription of katG. The furA and katG genes are cotranscribed from P(1)furA. Both P(1)furA and P(1)katG promoters show induction upon challenge with hydrogen peroxide and cumene hydroperoxide. Studies carried out using the transcriptional fusions P(1)furA-gfp, P(1)katG-gfp, and P(1)furA-P(1)katG-gfp confirmed the existence of two katG promoters. In addition, we showed that both promoters are expressed in vivo during intracellular growth of virulent M. tuberculosis H37Rv. P(1)furA is induced early upon infection, and P(1)katG becomes active only upon extended growth in macrophages. These studies delineate the transcriptional organization of the furA-katG region and indicate differential regulation in vivo of the two katG promoters. These phenomena most likely reflect the differing demands at sequential stages of the infection cycle and may provide information for improved understanding of host-pathogen interactions in tuberculosis and for further optimization of INH chemotherapy.

Bacterial Proteins↗
PubMed2000

Regulators of membrane trafficking and Mycobacterium tuberculosis phagosome maturation block.

The biogenesis and maturation of phagosomes is an area of study which has been employing aspects of proteomic analyses and variations on that theme by identifying components on isolated organelles and following their dynamic changes and interactions with the endocytic pathway. In the case of Mycobacterium tuberculosis phagosome, the arrest of its maturation in infected macrophages, referred to in classical texts as the inhibition of phagosome-lysosome fusion, represents a phenomenon that is used to functionally dissect the phagosomal maturation pathway. In this review, we summarize the recent studies on regulators of membrane trafficking and other organelle components in the context of phagosomal biogenesis and mycobacterial phagosome maturation arrest.

Amino Acid Sequence↗
PubMed2000

Membrane-to-cytosol redistribution of ECF sigma factor AlgU and conversion to mucoidy in Pseudomonas aeruginosa isolates from cystic fibrosis patients.

The conversion to mucoid phenotype in Pseudomonas aeruginosa during chronic infections in cystic fibrosis (CF) is due to mutations in the algU mucABCD gene cluster. This cluster encodes an extreme stress response system conserved in Gram-negative bacteria. The system includes an ECF sigma factor, AlgU (sigmaE), an inner membrane protein, MucA, which inhibits AlgU activity, and MucB, a periplasmic protein that negatively controls AlgU. In this work, we investigated whether and how these factor interact to transduce signals between different cellular compartments. The mutation mucADeltaG440, which renders a large fraction of P. aeruginosa CF isolates mucoid, did not abrogate AlgU-MucA interactions, although it eliminated MucA-MucB interactions in the yeast two-hybrid system. The mucADeltaG440 truncation of the periplasmic C-terminal tail of MucA destabilized the molecule resulting in low or undetectable steady-state levels in P. aeruginosa. Somewhat reduced levels of MucA were also seen in cells with inactivated mucB or with the mucACF53 allele carrying the missense P184S mutation, which mildly affected interactions with MucB. The events downstream from MucA destabilization were also investigated. AlgU was found to associate with inner membranes in mucA+ cells. In mutants destabilizing MucA, a limited redistribution of AlgU from the membrane to the cytosol was observed. The redistribution was spontaneous in mucADeltaG440 cells, while in mucB and mucACF53 mutants it required additional signals. Despite a large reduction in MucA levels in mucADeltaG440 cells, only a small fraction of AlgU was redistributed to the cytosol and a significant portion of this sigma factor remained membrane bound and behaved as a peripheral inner membrane protein. The fraction of AlgU that depended on MucA for association with the membrane also brought RNA polymerase into this compartment. These results are consistent with a model in which MucB-MucA-AlgU-RNA polymerase interactions at the membrane allow transduction of potentially lethal stress signals with both rapid reaction times of the preassembled complexes and efficient resupply at the membrane from the prebound components.

Alleles↗
PubMed2000

Dual regulation of mucoidy in Pseudomonas aeruginosa and sigma factor antagonism.

The conversion to mucoid, exopolysaccharide alginate-overproducing phenotype in Pseudomonas aeruginosa during chronic respiratory infections in cystic fibrosis patients occurs via mutations that activate the alternative sigma factor AlgU (sigmaE). In this study, we demonstrate that conversion to mucoidy can be caused via a second, algU-independent pathway, in which alginate production and transcription of the critical algD promoter depend on another alternative sigma factor, RpoN (sigma54). The algD promoters dependent on sigma54 and sigmaE showed a complete overlap resulting in identical mRNA 5' ends. The two pathways were not independent, as sigma54 also repressed sigmaE-dependent transcription of algD both in vitro and in vivo. The negative regulatory effect of sigma54 on sigmaE-dependent algD expression was based on sigma54 binding to the algD promoter and its interference with sigmaE-dependent transcription. This phenomenon, referred to here as sigma factor antagonism, reflects the unique properties of sigma54, which lacks an intrinsic ability to form open transcription initiation complexes. We propose that this peculiar feature of sigma54 has evolved in part to allow its recruitment as a repressor of certain promoter subsets. The repression of algD by sigma54 also depends on environmental conditions, supporting the notion that sigma factor antagonism plays a physiological role in controlling alginate production in P. aeruginosa during adaptation to different ecological sites (e.g. biofilm development, stress and other growth conditions) and unique environments in the chronically infected host.

Alginates↗
PubMed2000

Innate lung defenses and compromised Pseudomonas aeruginosa clearance in the malnourished mouse model of respiratory infections in cystic fibrosis.

Cystic fibrosis (CF) is characterized by dysfunction of the digestive and respiratory tracts resulting in generalized malnutrition and chronic respiratory infections. Chronic lung infections with Pseudomonas aeruginosa, intense neutrophil-dominated airway inflammation, and progressive lung disease are the major cause of high morbidity and mortality in CF. Here we investigated the effects of malnutrition in CF on innate lung defenses, susceptibility to P. aeruginosa colonization, and associated inflammation, using aerosol models of acute and chronic infections in normal, malnourished, and transgenic mice. CFTR(m1Unc-/-) knockout mice displayed body weight variations and showed variable pulmonary clearance of P. aeruginosa. This variability was not detected in bitransgenic CFTR(m1Unc-/-)(FABP-hCFTR) mice in which the intestinal defect had been corrected. Diet-induced protein calorie malnutrition in C57BL/6J mice resulted in impaired pulmonary clearance of P. aeruginosa. Tumor necrosis factor alpha (TNF-alpha) and nitrite levels detected upon exposure to P. aeruginosa aerosols were lower in the lungs of the malnourished C57BL/6J mice relative than in lungs of mice fed a normal diet. The role of TNF-alpha and reactive nitrogen intermediates in P. aeruginosa clearance was tested in TNF-alpha and inducible nitric oxide synthase (iNOS) knockout mice. P. aeruginosa clearance was diminished in transgenic TNF-alpha- and iNOS-deficient mice. In contrast to the effects of TNF-alpha and iNOS, gamma interferon knockout mice retained a full capacity to eliminate P. aeruginosa from the lung. Malnutrition also contributed to excessive inflammation in C57BL/6J mice upon chronic challenge with P. aeruginosa. The repeatedly infected malnourished host did not produce interleukin-10, a major anti-inflammatory cytokine absent or diminished in the bronchoalveolar fluids of CF patients. These results are consistent with a model in which defective CFTR in the intestinal tract leads to nutritional deficiency which in turn contributes to compromised innate lung defenses, bacterial colonization, and excessive inflammation in the CF respiratory tract.

Administration, Inhalation↗
PubMed2000

An essential two-component signal transduction system in Mycobacterium tuberculosis.

The bacterial two-component signal transduction systems regulate adaptation processes and are likely to play a role in Mycobacterium tuberculosis physiology and pathogenesis. The previous initial characterization of an M. tuberculosis response regulator from one of these systems, mtrA-mtrB, suggested its transcriptional activation during infection of phagocytic cells. In this work, we further characterized the mtrA response regulator from M. tuberculosis H37Rv. Inactivation of mtrA on the chromosome of M. tuberculosis H37Rv was possible only in the presence of plasmid-borne functional mtrA, suggesting that this response regulator is essential for M. tuberculosis viability. In keeping with these findings, expression of mtrA in M. tuberculosis H37Rv was detectable during in vitro growth, as determined by S1 nuclease protection and primer extension analyses of mRNA levels and mapping of transcript 5' ends. The mtrA gene was expressed differently in virulent M. tuberculosis and the vaccine strain M. tuberculosis var. bovis BCG during infection of macrophages, as determined by monitoring of mtrA-gfp fusion activity. In M. bovis BCG, mtrA was induced upon entry into macrophages. In M. tuberculosis H37Rv, its expression was constitutive and unchanged upon infection of murine or human monocyte-derived macrophages. In conclusion, these results identify mtrA as an essential response regulator gene in M. tuberculosis which is differentially expressed in virulent and avirulent strains during growth in macrophages.

ATP-Binding Cassette Transporters↗
PubMed1999

Mycobacterium tuberculosis phagosome.

The arrest of Mycobacterium tuberculosis phagosome maturation in infected macrophages is a phenomenon of dual significance both for the pathogenesis of tuberculosis and as a model system to study interference of microbes with membrane trafficking and organelle biogenesis in host cells. Among other factors, compartment-specialized regulators of vesicular trafficking and other parts of membrane fusion machinery are likely to play a role in these processes. Here we summarize the emerging view of mycobacterial phagosome maturation arrest in the context of the dynamic processes of intracellular membrane trafficking.

Carrier Proteins↗
PubMed1999

Response to reactive nitrogen intermediates in Mycobacterium tuberculosis: induction of the 16-kilodalton alpha-crystallin homolog by exposure to nitric oxide donors.

In contrast to the apparent paucity of Mycobacterium tuberculosis response to reactive oxygen intermediates, this organism has evolved a specific response to nitric oxide challenge. Exposure of M. tuberculosis to NO donors induces the synthesis of a set of polypeptides that have been collectively termed Nox. In this work, the most prominent Nox polypeptide, Nox16, was identified by immunoblotting and by N-terminal sequencing as the alpha-crystallin-related, 16-kDa small heat shock protein, sHsp16. A panel of chemically diverse donors of nitric oxide, with the exception of nitroprusside, induced sHsp16 (Nox16). Nitroprusside, a coordination complex of Fe2+ with a nitrosonium (NO+) ion, induced a 19-kDa polypeptide (Nox19) homologous to the nonheme bacterial ferritins. We conclude that the NO response in M. tuberculosis is dominated by increased synthesis of the alpha-crystallin homolog sHsp16, previously implicated in stationary-phase processes and found in this study to be a major M. tuberculosis protein induced upon exposure to reactive nitrogen intermediates.

Amino Acid Sequence↗
PubMed1998

Microbial pathogenesis in cystic fibrosis: pulmonary clearance of mucoid Pseudomonas aeruginosa and inflammation in a mouse model of repeated respiratory challenge.

Chronic endobronchiolitis compounded by recurring Pseudomonas aeruginosa infections is the major cause of morbidity and mortality in patients with cystic fibrosis (CF). In this study, a mouse model of repeated respiratory exposure to P. aeruginosa was established to facilitate investigations of factors contributing to P. aeruginosa persistence and associated inflammatory processes in the lung. While a single exposure to P. aeruginosa aerosols resulted in only mild histopathological changes, repeated exposure caused significant lung pathology in C57BL/6J mice. The peak of histopathological changes and inflammation in C57BL/6J mice was characterized by subacute lymphohistiocytic bronchopneumonia and persistent elevation of tumor necrosis factor alpha and macrophage inflammatory protein 2 in the lung but not in the serum. When isogenic nonmucoid (mucA+) and mucoid (mucA22) P. aeruginosa strains were compared, the mucoid cells were cleared several-fold less efficiently than the parental nonmucoid strain during the initial stages of the aerosol exposure regimen. However, the microscopic pathology findings and proinflammatory cytokine levels were similar in mice exposed to nonmucoid and mucoid P. aeruginosa throughout the infection. We also tested lung histopathology and proinflammatory cytokines in interleukin 10 (IL-10)-deficient transgenic (IL-10T) mice. Significant mortality was seen in IL-10T mice on initial challenge with P. aeruginosa, although no histopathological differences could be observed in the lungs of C57BL/6J and surviving IL-10T mice after a single exposure. However, increased pathology was detected in IL-10T mice relative to C57BL/6J after repeated challenge with P. aeruginosa. This observation supports the proposals that anti-inflammatory cytokines may play a role in suppressing P. aeruginosa-induced tissue damage during chronic infection.

Animals↗