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1.
Cell Death Differ ; 16(7): 976-83, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19444282

ABSTRACT

Autophagy is a cell biological process, enabling cells to autodigest their own cytosol when starved, remove cytoplasmic protein aggregates too large for proteasomal degradation, eliminate aberrant or over-proliferated organelles, and sanitize the cytoplasm by killing intracellular microbes. The role of autophagy has been expanded in recent years to include diverse immunological effector and regulatory functions. In this review, we summarize the multiple immunological roles of autophagy uncovered to date and focus primarily on details of induction of autophagy by pattern recognition receptors, as a newly established Toll-like receptor output. Taken together with other links between autophagy and innate and adaptive immunity processes, this cell-autonomous antimicrobial defense may be evolutionarily positioned at the root of immunity with the multiple innate and adaptive immunity connections uncovered to date reflecting a co-evolution of this ancient cell-defense mechanism and more advanced immunological systems in metazoans.


Subject(s)
Autophagy/immunology , Phagosomes/immunology , Toll-Like Receptors/metabolism , Animals , Cytokines/immunology , Cytokines/metabolism , Humans , Immunity, Active , Immunity, Innate , Microtubule-Associated Proteins/immunology , Microtubule-Associated Proteins/metabolism , Myeloid Differentiation Factor 88/immunology , Myeloid Differentiation Factor 88/metabolism , Protein Kinases/immunology , Protein Kinases/metabolism , Signal Transduction/immunology , Toll-Like Receptors/immunology , Vesicular Transport Proteins/immunology , Vesicular Transport Proteins/metabolism
2.
Microbiology (Reading) ; 148(Pt 10): 3139-3144, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12368447

ABSTRACT

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.


Subject(s)
Heat-Shock Response , Macrophages/microbiology , Mycobacterium tuberculosis/drug effects , Oxidative Stress , Peroxidases/genetics , Peroxynitrous Acid/pharmacology , Cell Line , Drug Resistance, Bacterial , Humans , Microbial Sensitivity Tests , Mycobacterium tuberculosis/growth & development , Mycobacterium tuberculosis/pathogenicity , Peroxiredoxins
3.
Proc Natl Acad Sci U S A ; 98(24): 13972-7, 2001 Nov 20.
Article in English | MEDLINE | ID: mdl-11717455

ABSTRACT

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.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cystic Fibrosis/metabolism , Glycoproteins , Lung/metabolism , Membrane Proteins , Pseudomonas aeruginosa/metabolism , Acids , Bacterial Adhesion , Cell Line , Glycosylation , Green Fluorescent Proteins , Humans , Luminescent Proteins/metabolism , Membrane Glycoproteins/metabolism , Proton-Translocating ATPases/antagonists & inhibitors , Pseudomonas aeruginosa/physiology , Recombinant Fusion Proteins/metabolism , Sialyltransferases/metabolism , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , trans-Golgi Network/metabolism
4.
Proc Natl Acad Sci U S A ; 98(22): 12706-11, 2001 Oct 23.
Article in English | MEDLINE | ID: mdl-11675502

ABSTRACT

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.


Subject(s)
Bacterial Proteins/physiology , Mycobacterium tuberculosis/pathogenicity , Signal Transduction , Animals , Humans , Macrophages/microbiology , Mice , Mice, Inbred C57BL , Mycobacterium tuberculosis/genetics , Organ Specificity , Virulence
5.
Infect Immun ; 69(10): 5967-73, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11553532

ABSTRACT

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.


Subject(s)
Antioxidants/metabolism , Mycobacterium tuberculosis/enzymology , Oxidative Stress , Peroxidases/metabolism , Animals , Female , Gene Expression Profiling , Hydrogen Peroxide/pharmacology , Mice , Mice, Inbred BALB C , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/pathogenicity , Peroxidases/genetics , Peroxiredoxins , Recombination, Genetic , Virulence
6.
J Cell Biol ; 154(3): 631-44, 2001 Aug 06.
Article in English | MEDLINE | ID: mdl-11489920

ABSTRACT

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.


Subject(s)
Macrolides , Mycobacterium tuberculosis , Phagosomes/immunology , Phosphatidylinositol 3-Kinases/metabolism , Tuberculosis, Pulmonary/metabolism , Vesicular Transport Proteins , rab5 GTP-Binding Proteins/metabolism , Androstadienes/pharmacology , Animals , Anti-Bacterial Agents/pharmacology , Antibodies/pharmacology , Carrier Proteins/metabolism , Cell Line , Chromones/pharmacology , Enzyme Inhibitors/pharmacology , Glycosylation , Lipopolysaccharides/pharmacology , Lysophospholipids/metabolism , Macrophages/cytology , Macrophages/metabolism , Macrophages/microbiology , Membrane Proteins/immunology , Membrane Proteins/metabolism , Microinjections , Microspheres , Monoglycerides , Morpholines/pharmacology , Phosphatidylinositol 3-Kinases/immunology , Qa-SNARE Proteins , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins , Transport Vesicles/metabolism , Tuberculosis, Pulmonary/immunology , Wortmannin
7.
Front Biosci ; 6: D890-7, 2001 Aug 01.
Article in English | MEDLINE | ID: mdl-11487480

ABSTRACT

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.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis/complications , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/pathogenicity , Respiratory Tract Infections/microbiology , Animals , Bacterial Adhesion , Biofilms/growth & development , Cystic Fibrosis/metabolism , Cystic Fibrosis/microbiology , Disease Models, Animal , Humans , Inflammation/immunology , Mice , Nitric Oxide Synthase/biosynthesis , Nitric Oxide Synthase Type II , Pseudomonas Infections/complications , Pseudomonas Infections/metabolism , Pseudomonas aeruginosa/physiology , Respiratory Mucosa/enzymology , Respiratory Mucosa/microbiology , Respiratory Tract Infections/complications , Respiratory Tract Infections/metabolism , Sialyltransferases/metabolism
8.
Mol Microbiol ; 40(4): 879-89, 2001 May.
Article in English | MEDLINE | ID: mdl-11401695

ABSTRACT

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.


Subject(s)
Antitubercular Agents/pharmacology , Bacterial Proteins/genetics , Isoniazid/pharmacology , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/pathogenicity , Peroxidases/genetics , Repressor Proteins/genetics , Animals , Bacterial Proteins/drug effects , Bacterial Proteins/metabolism , Base Sequence , Drug Resistance, Microbial/genetics , Gene Deletion , Gene Expression Regulation, Bacterial , Genetic Complementation Test , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Mycobacterium tuberculosis/drug effects , Operon , Peroxidases/drug effects , Peroxidases/metabolism , Peroxiredoxins , Promoter Regions, Genetic , Repressor Proteins/drug effects , Repressor Proteins/metabolism , Siderophores/metabolism , Superoxide Dismutase/genetics , Tuberculosis/microbiology , Virulence/genetics
9.
J Bacteriol ; 183(13): 4033-9, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11395468

ABSTRACT

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.


Subject(s)
Macrophages/microbiology , Mycobacterium tuberculosis/genetics , Peroxidases/genetics , Promoter Regions, Genetic , Bacterial Proteins/genetics , Benzene Derivatives/pharmacology , Gene Expression Regulation, Bacterial , Hydrogen Peroxide/pharmacology , Mycobacterium tuberculosis/enzymology , Oxidative Stress/genetics , RNA, Bacterial/biosynthesis , RNA, Messenger/biosynthesis , Repressor Proteins/genetics , Transcription, Genetic
11.
Mol Microbiol ; 39(5): 1174-85, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11251835

ABSTRACT

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.


Subject(s)
Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial , Mycobacterium smegmatis/enzymology , Peroxidases/genetics , Peroxidases/metabolism , Repressor Proteins/genetics , Bacterial Proteins/metabolism , Benzene Derivatives/pharmacology , Hydrogen Peroxide/pharmacology , Iron/metabolism , Isoniazid , Molecular Sequence Data , Mycobacterium smegmatis/drug effects , Mycobacterium smegmatis/genetics , Mycobacterium smegmatis/growth & development , Peroxiredoxins , Repressor Proteins/metabolism
12.
Electrophoresis ; 21(16): 3378-85, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11079558

ABSTRACT

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.


Subject(s)
Mycobacterium tuberculosis/metabolism , Phagosomes/metabolism , Amino Acid Sequence , Biological Transport , Electrophoresis, Gel, Two-Dimensional , GTP Phosphohydrolases/metabolism , Membrane Fusion , Microfilament Proteins/chemistry , Microfilament Proteins/metabolism , Molecular Sequence Data , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/ultrastructure , Proteome , Sequence Homology, Amino Acid
13.
J Bacteriol ; 182(13): 3832-8, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10851001

ABSTRACT

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.


Subject(s)
ATP-Binding Cassette Transporters/physiology , Bacterial Proteins , Mycobacterium tuberculosis/physiology , Signal Transduction , ATP-Binding Cassette Transporters/genetics , Animals , Cell Line , Gene Expression , Humans , Macrophages/microbiology , Mice , Mycobacterium bovis/genetics , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/growth & development , Mycobacterium tuberculosis/metabolism , Promoter Regions, Genetic
14.
Mol Microbiol ; 36(2): 314-27, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10792719

ABSTRACT

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.


Subject(s)
Bacterial Proteins/metabolism , Cell Membrane/metabolism , Cystic Fibrosis/microbiology , Cytosol/metabolism , Pseudomonas aeruginosa/pathogenicity , Sigma Factor/metabolism , Alleles , Bacterial Proteins/genetics , DNA-Directed RNA Polymerases/metabolism , Heat-Shock Response , Mutation , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/isolation & purification , Pseudomonas aeruginosa/physiology , Sigma Factor/genetics , Two-Hybrid System Techniques
15.
Mol Microbiol ; 36(2): 341-51, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10792721

ABSTRACT

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.


Subject(s)
Carbohydrate Dehydrogenases/genetics , Cystic Fibrosis/microbiology , DNA-Binding Proteins , DNA-Directed RNA Polymerases/metabolism , Gene Expression Regulation, Bacterial , Pseudomonas aeruginosa/genetics , Sigma Factor/metabolism , Transcription Factors/metabolism , Alginates/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA-Directed RNA Polymerases/genetics , Glucuronic Acid , Hexuronic Acids , Humans , Nitrogen/pharmacology , Promoter Regions, Genetic , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/pathogenicity , RNA Polymerase Sigma 54 , Sigma Factor/genetics , Transcription Factors/genetics , Transcription, Genetic
16.
Infect Immun ; 68(4): 2142-7, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10722612

ABSTRACT

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.


Subject(s)
Cystic Fibrosis/immunology , Lung/microbiology , Protein-Energy Malnutrition/immunology , Pseudomonas aeruginosa/metabolism , Respiratory Tract Infections/immunology , Administration, Inhalation , Animals , Chemokine CXCL2 , Chemotactic Factors/biosynthesis , Cystic Fibrosis/microbiology , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/physiology , Cytokines/genetics , Cytokines/physiology , Disease Models, Animal , Genotype , Interferon-gamma/genetics , Interferon-gamma/physiology , Interleukin-10/biosynthesis , Intestines/immunology , Intestines/microbiology , Lung/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Monokines/biosynthesis , Neutrophils/cytology , Neutrophils/microbiology , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase/physiology , Nitric Oxide Synthase Type II , Nitrites/metabolism , Peroxidase/biosynthesis , Protein-Energy Malnutrition/microbiology , Respiratory Tract Infections/microbiology , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/physiology
17.
Mol Microbiol ; 31(6): 1603-9, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10209735

ABSTRACT

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.


Subject(s)
Cation Transport Proteins , Mycobacterium tuberculosis/physiology , Phagosomes/metabolism , rab GTP-Binding Proteins , Carrier Proteins/physiology , Cells, Cultured , Cytokines/physiology , GTP-Binding Proteins/metabolism , Membrane Proteins/physiology , Microscopy, Confocal , Models, Biological , Mycobacterium bovis/metabolism , Phagocytosis/physiology , rab5 GTP-Binding Proteins , rab7 GTP-Binding Proteins
18.
Infect Immun ; 67(1): 460-5, 1999 Jan.
Article in English | MEDLINE | ID: mdl-9864257

ABSTRACT

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.


Subject(s)
Crystallins/biosynthesis , Mycobacterium tuberculosis/metabolism , Nitric Oxide Donors/pharmacology , Sequence Homology, Amino Acid , Amino Acid Sequence , Antigens, Bacterial/chemistry , Crystallins/chemistry , Ferritins/chemistry , Gene Expression Regulation, Bacterial/drug effects , Heat-Shock Proteins/chemistry , Molecular Sequence Data , Molecular Weight , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Nitric Oxide/metabolism , Nitric Oxide Donors/metabolism
19.
J Bacteriol ; 180(18): 4856-64, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9733688

ABSTRACT

Oxidative stress response in pathogenic mycobacteria is believed to be of significance for host-pathogen interactions at various stages of infection. It also plays a role in determining the intrinsic susceptibility to isoniazid in mycobacterial species. In this work, we characterized the oxyR-ahpC and furA-katG loci in the nontuberculous pathogen Mycobacterium marinum. In contrast to Mycobacterium smegmatis and like Mycobacterium tuberculosis and Mycobacterium leprae, M. marinum was shown to possess a closely linked and divergently oriented equivalents of the regulator of peroxide stress response oxyR and its subordinate gene ahpC, encoding a homolog of alkyl hydroperoxide reductase. Purified mycobacterial OxyR was found to bind to the oxyR-ahpC promoter region from M. marinum and additional mycobacterial species. Mobility shift DNA binding analyses using OxyR binding sites from several mycobacteria and a panel of in vitro-generated mutants validated the proposed consensus mycobacterial recognition sequence. M. marinum AhpC levels detected by immunoblotting, were increased upon treatment with H2O2, in keeping with the presence of a functional OxyR and its binding site within the promoter region of ahpC. In contrast, OxyR did not bind to the sequences upstream of the katG structural gene, and katG expression did not follow the pattern seen with ahpC. Instead, a new open reading frame encoding a homolog of the ferric uptake regulator Fur was identified immediately upstream of katG in M. marinum. The furA-katG linkage and arrangement are ubiquitous in mycobacteria, suggesting the presence of additional regulators of oxidative stress response and potentially explaining the observed differences in ahpC and katG expression. Collectively, these findings broaden our understanding of oxidative stress response in mycobacteria. They also suggest that M. marinum will be useful as a model system for studying the role of oxidative stress response in mycobacterial physiology, intracellular survival, and other host-pathogen interactions associated with mycobacterial diseases.


Subject(s)
Bacterial Proteins/genetics , DNA-Binding Proteins , Genes, Bacterial , Mycobacterium marinum/genetics , Oxidative Stress , Oxidoreductases/genetics , Peroxidases/genetics , Repressor Proteins/genetics , Transcription Factors/genetics , Amino Acid Sequence , Animals , Cell Line , Chromosome Mapping , Mice , Molecular Sequence Data , Mycobacterium marinum/metabolism , Peroxiredoxins , Rabbits
20.
J Cell Sci ; 111 ( Pt 7): 897-905, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9490634

ABSTRACT

One of the major mechanisms permitting intracellular pathogens to parasitize macrophages is their ability to alter maturation of the phagosome or affect its physical integrity. These processes are opposed by the host innate and adaptive immune defenses, and in many instances mononuclear phagocytes can be stimulated with appropriate cytokines to restrict the growth of the microorganisms within the phagosomal compartment. Very little is known about the effects that cytokines have on phagosome maturation. Here we have used green fluorescent protein (GFP)-labeled mycobacteria and a fixable acidotropic probe, LysoTracker Red DND-99, to monitor maturation of the mycobacterial phagosome. The macrophage compartments that stained with the LysoTracker probe were examined first. This dye was found to colocalize preferentially with the late endosomal and lysosomal markers rab7 and Lamp1, and with a fluid phase marker chased into the late endosomal compartments. In contrast, LysoTracker showed only a minor overlap with the early endosomal marker rab5. Pathogenic mycobacteria are believed to reside in nonacidified vacuoles sequestered away from late endosomal compartments as a part of their intracellular survival strategy. We examined the status of mycobacterial phagosomes in macrophages from IL-10 knockout mice, in quiescent cells, and in mononuclear phagocytes stimulated with the macrophage-activating cytokine IFN-(gamma). When macrophages were derived from the bone marrow of transgenic IL-10 mice lacking this major deactivating cytokine, colocalization of GFP-fluorescing mycobacteria with the LysoTracker staining appeared enhanced, suggestive of increased acidification of the mycobacterial phagosome relative to macrophages from normal mice. When bone marrow-derived macrophages from normal mice or a J774 murine macrophage cell line were stimulated with IFN-(gamma) and LPS, this resulted in increased colocalization of mycobacteria and LysoTracker, but no statistically significant enhancement was observed in IL-10 transgenic animals. These studies are consistent with the interpretation that proinflammatory and anti-inflammatory cytokines affect maturation of mycobacterial phagosomes. Although multiple mechanisms are likely to be at work, we propose the existence of a direct link between cytokine effects on the host cell and phagosome maturation in the macrophage.


Subject(s)
Cytokines/pharmacology , Mycobacterium bovis/physiology , Phagosomes/physiology , Animals , Bone Marrow Cells/metabolism , Bone Marrow Cells/microbiology , Cell Compartmentation , Cell Line , Endosomes/metabolism , Fluorescent Dyes/metabolism , Green Fluorescent Proteins , Hydrogen-Ion Concentration , Interferon-gamma/pharmacology , Interleukin-10/genetics , Luminescent Proteins/metabolism , Lysosomes/metabolism , Macrophages/metabolism , Macrophages/microbiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mycobacterium bovis/drug effects , Mycobacterium bovis/metabolism , Phagosomes/drug effects , Phagosomes/metabolism
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