scholarly journals Identification of the Missing trans-Acting Enoyl Reductase Required for Phthiocerol Dimycocerosate and Phenolglycolipid Biosynthesis in Mycobacterium tuberculosis

2007 ◽  
Vol 189 (13) ◽  
pp. 4597-4602 ◽  
Author(s):  
Roxane Siméone ◽  
Patricia Constant ◽  
Christophe Guilhot ◽  
Mamadou Daffé ◽  
Christian Chalut
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Mycobacterium Bovis ◽  
Reductase Activity ◽  
Polyketide Synthases ◽  
Type I ◽  
Wild Type ◽  
Enoyl Reductase ◽  
Phthiocerol Dimycocerosates ◽  
Activity Domain

ABSTRACT Phthiocerol dimycocerosates (DIM) and phenolglycolipids (PGL) are functionally important surface-exposed lipids of Mycobacterium tuberculosis. Their biosynthesis involves the products of several genes clustered in a 70-kb region of the M. tuberculosis chromosome. Among these products is PpsD, one of the modular type I polyketide synthases responsible for the synthesis of the lipid core common to DIM and PGL. Bioinformatic analyses have suggested that this protein lacks a functional enoyl reductase activity domain required for the synthesis of these lipids. We have identified a gene, Rv2953, that putatively encodes an enoyl reductase. Mutation in Rv2953 prevents conventional DIM formation and leads to the accumulation of a novel DIM-like product. This product is unsaturated between C-4 and C-5 of phthiocerol. Consistently, complementation of the mutant with a functional pks15/1 gene from Mycobacterium bovis BCG resulted in the accumulation of an unsaturated PGL-like substance. When an intact Rv2953 gene was reintroduced into the mutant strain, the phenotype reverted to the wild type. These findings indicate that Rv2953 encodes a trans-acting enoyl reductase that acts with PpsD in phthiocerol and phenolphthiocerol biosynthesis.

scholarly journals Mycobacterium bovis Requires P27 (LprG) To Arrest Phagosome Maturation and Replicate within Bovine Macrophages

2016 ◽  
Vol 85 (3) ◽  
Author(s):  
Cristina Lourdes Vázquez ◽  
María Verónica Bianco ◽  
Federico Carlos Blanco ◽  
Marina Andrea Forrellad ◽  
Maximiliano Gabriel Gutierrez ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Host Cell ◽  
Mutant Strain ◽  
Mycobacterium Bovis ◽  
Phagosome Maturation ◽  
Wild Type ◽  
Content Type ◽  
Bovine Macrophage ◽  
Wild Type Phenotype ◽  
First Time

ABSTRACT Mycobacterium bovis causes tuberculosis in a wide variety of mammals, with strong tropism for cattle and eventually humans. P27, also called LprG, is among the proteins involved in the mechanisms of the virulence and persistence of M. bovis and Mycobacterium tuberculosis. Here, we describe a novel function of P27 in the interaction of M. bovis with its natural host cell, the bovine macrophage. We found that a deletion in the p27-p55 operon impairs the replication of M. bovis in bovine macrophages. Importantly, we show for the first time that M. bovis arrests phagosome maturation in a process that depends on P27. This effect is P27 specific since complementation with wild-type p27 but not p55 fully restored the wild-type phenotype of the mutant strain; this indicates that P55 plays no important role during the early events of M. bovis infection. In addition, we also showed that the presence of P27 from M. smegmatis decreases the association of LAMP-3 with bead phagosomes, indicating that P27 itself blocks phagosome-lysosome fusion by modulating the traffic machinery in the cell host.

scholarly journals Establishing Virulence Associated Polyphosphate Kinase 2 as a drug target for Mycobacterium tuberculosis

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Mamta Singh ◽  
Prabhakar Tiwari ◽  
Garima Arora ◽  
Sakshi Agarwal ◽  
Saqib Kidwai ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Guinea Pigs ◽  
High Throughput Screening ◽  
Drug Target ◽  
Wild Type Strain ◽  
Wild Type ◽  
Inorganic Polyphosphate ◽  
Phosphate Donor ◽  
Microbial Stress

Abstract Inorganic polyphosphate (PolyP) plays an essential role in microbial stress adaptation, virulence and drug tolerance. The genome of Mycobacterium tuberculosis encodes for two polyphosphate kinases (PPK-1, Rv2984 and PPK-2, Rv3232c) and polyphosphatases (ppx-1, Rv0496 and ppx-2, Rv1026) for maintenance of intracellular PolyP levels. Microbial polyphosphate kinases constitute a molecular mechanism, whereby microorganisms utilize PolyP as phosphate donor for synthesis of ATP. In the present study we have constructed ppk-2 mutant strain of M. tuberculosis and demonstrate that PPK-2 enzyme contributes to its ability to cause disease in guinea pigs. We observed that ppk-2 mutant strain infected guinea pigs had significantly reduced bacterial loads and tissue pathology in comparison to wild type infected guinea pigs at later stages of infection. We also report that in comparison to the wild type strain, ppk-2 mutant strain was more tolerant to isoniazid and impaired for survival in THP-1 macrophages. In the present study we have standardized a luciferase based assay system to identify chemical scaffolds that are non-cytotoxic and inhibit M. tuberculosis PPK-2 enzyme. To the best of our knowledge this is the first study demonstrating feasibility of high throughput screening to obtain small molecule PPK-2 inhibitors.

scholarly journals Activation of 2,4-Diaminoquinazoline in Mycobacterium tuberculosis by Rv3161c, a Putative Dioxygenase

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Eduard Melief ◽  
Shilah A. Bonnett ◽  
Edison S. Zuniga ◽  
Tanya Parish
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Type A ◽  
Wild Type ◽  
Metabolite Analysis ◽  
Content Type ◽  
Data Support ◽  
In The Wild

ABSTRACT The diaminoquinazoline series has good potency against Mycobacterium tuberculosis. Resistant isolates have mutations in Rv3161c, a putative dioxygenase. We carried out metabolite analysis on a wild-type strain and an Rv3161c mutant strain after exposure to a diaminoquinazoline. The parental compound was found in intracellular extracts from the mutant but not the wild type. A metabolite consistent with a monohydroxylated form was identified in the wild type. These data support the hypothesis that Rv3161c metabolizes diaminoquinazolines in M. tuberculosis.

scholarly journals Analysis of Genomes Changes in Pseudomonas Chlororaphis Subsp. Aurantiaca Strains Producing Phenazines

2021 ◽  
Author(s):  
Anastasia I. Liaudanskaya ◽  
Natalia P. Maximova ◽  
Katsiaryna G. Verameyenka
Keyword(s):  
Mutant Strain ◽  
Phenylalanine Hydroxylase ◽  
Response Regulator ◽  
Trna Genes ◽  
Type I ◽  
Pseudomonas Chlororaphis ◽  
Wild Type ◽  
Mutant Proteins ◽  
Domain Structures ◽  
Genome Annotations

Abstract Genomes of three strains – phenazine producers – Pseudomonas chlororaphis subsp. aurantiaca (B-162 (wild-type), mutant strain B-162/255 and its derivatives B-162/17) were sequenced and compared. All genome annotations revealed 6347 CDS, 5 rRNA clusters (5S, 16S, 23S) and 59 tRNA genes. Comparison analysis of wild-type strain and B-162/255 mutant strain genomes allowed revealing 32 mutations. 19 new mutations were detected upon comparison of genomes strains B-162/255 and B-162/17. Further bioinformatics analysis allowed predicting mutant proteins` functions and secondary structures of five gene products, mutations in which might potentially have influence on phenazine synthesis and secretion in Pseudomonas bacteria. These genes are phenylalanine hydroxylase transcriptional activator PhhR, type I secretion system ATPase, transcriptional regulator MvaT, GacA response regulator and histidine kinase. Amino acid substitutions were located in domain structures of corresponding proteins.

scholarly journals Nicotinamide Limits Replication of Mycobacterium tuberculosis and Bacille Calmette-Guérin Within Macrophages

2019 ◽  
Vol 221 (6) ◽  
pp. 989-999
Author(s):  
Jason D Simmons ◽  
Glenna J Peterson ◽  
Monica Campo ◽  
Jenny Lohmiller ◽  
Shawn J Skerrett ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nicotinic Acid ◽  
Mycobacterium Bovis ◽  
Related Compound ◽  
Antimicrobial Properties ◽  
Broth Culture ◽  
Wild Type ◽  
Bacille Calmette Guerin ◽  
Macrophage Function ◽  
Modest Activity

Abstract Novel antimicrobials for treatment of Mycobacterium tuberculosis are needed. We hypothesized that nicotinamide (NAM) and nicotinic acid (NA) modulate macrophage function to restrict M. tuberculosis replication in addition to their direct antimicrobial properties. Both compounds had modest activity in 7H9 broth, but only NAM inhibited replication in macrophages. Surprisingly, in macrophages NAM and the related compound pyrazinamide restricted growth of bacille Calmette-Guérin but not wild-type Mycobacterium bovis, which both lack a functional nicotinamidase/pyrazinamidase (PncA) rendering each strain resistant to these drugs in broth culture. Interestingly, NAM was not active in macrophages infected with a virulent M. tuberculosis mutant encoding a deletion in pncA. We conclude that the differential activity of NAM and nicotinic acid on infected macrophages suggests host-specific NAM targets rather than PncA-dependent direct antimicrobial properties. These activities are sufficient to restrict attenuated BCG, but not virulent wild-type M. bovis or M. tuberculosis.

scholarly journals A Peptide Permease Mutant of Mycobacterium bovis BCG Resistant to the Toxic Peptides Glutathione andS-Nitrosoglutathione

2000 ◽  
Vol 68 (2) ◽  
pp. 429-436 ◽  
Author(s):  
Renee M. Green ◽  
Anjali Seth ◽  
Nancy D. Connell
Keyword(s):  
Mutant Strain ◽  
Mycobacterium Bovis ◽  
Mammalian Cells ◽  
Sole Source ◽  
Cosmid Library ◽  
Peptide Transporter ◽  
Wild Type ◽  
Genome Database ◽  
Mutant Strains ◽  
Oligopeptide Permease

ABSTRACT Oligopeptides play important roles in bacterial nutrition and signaling. Using sequences from the available genome database forMycobacterium tuberculosis H37Rv, the oligopeptide permease operon (oppBCDA) of Mycobacterium bovis BCG was cloned from a cosmid library. An opp mutant strain was constructed by homologous recombination with an allele ofoppD interrupted by kanamycin and streptomycin resistance markers. The deletion was complemented with a wild-type copy of theopp operon. Two approaches were taken to characterize the peptide transporter defect in this mutant strain. First, growth of wild-type and mutant strains was monitored in media containing a wide variety of peptides as sole source of carbon and/or nitrogen. Among 25 peptides ranging from two to six amino acids in length, none was capable of supporting measurable growth as the sole carbon source in either wild-type or mutant strains. The second approach exploited the resistance of permease mutants to toxic substrates. The tripeptide glutathione (γ-glutamyl-l-cyteinylglycine [GSH]) is toxic to wild-type BCG and was used successfully to characterize peptide uptake in the opp mutant. In 2 mM GSH, growth of the wild-type strain is inhibited, whereas the opp mutant is resistant to concentrations as high as 10 mM. Similar results were found with the tripeptide S-nitrosoglutathione (GSNO), thought to be a donor of NO in mammalian cells. Using incorporation of [3H]uracil to monitor the effects of GSH and GSNO on macromolecular synthesis in growing cells, it was demonstrated that theopp mutant is resistant, whereas the wild type and the mutant complemented with a wild-type copy of the operon are sensitive to both tripeptides. In uptake measurements, incorporation of [3H]GSH is reduced in the mutant compared with wild type and the complemented mutant. Finally, growth of the three strains in the tripeptides suggests that GSH is bacteriostatic, whereas GSNO is bacteriocidal.

scholarly journals A Thiolase of Mycobacterium tuberculosis Is Required for Virulence and Production of Androstenedione and Androstadienedione from Cholesterol

2009 ◽  
Vol 78 (1) ◽  
pp. 275-282 ◽  
Author(s):  
Natasha M. Nesbitt ◽  
Xinxin Yang ◽  
Patricia Fontán ◽  
Irina Kolesnikova ◽  
Issar Smith ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Wild Type Strain ◽  
Cholesterol Metabolism ◽  
Transcriptional Profiling ◽  
Mouse Lung ◽  
Intracellular Pathogen ◽  
Wild Type ◽  
Strain H37rv

ABSTRACT Mycobacterium tuberculosis, the causative agent of tuberculosis, is an intracellular pathogen that shifts to a lipid-based metabolism in the host. Moreover, metabolism of the host lipid cholesterol plays an important role in M. tuberculosis infection. We used transcriptional profiling to identify genes transcriptionally regulated by cholesterol and KstR (Rv3574), a TetR-like repressor. The fadA5 (Rv3546) gene, annotated as a lipid-metabolizing thiolase, the expression of which is upregulated by cholesterol and repressed by KstR, was deleted in M. tuberculosis H37Rv. We demonstrated that fadA5 is required for utilization of cholesterol as a sole carbon source in vitro and for full virulence of M. tuberculosis in the chronic stage of mouse lung infection. Cholesterol is not toxic to the fadA5 mutant strain, and, therefore, toxicity does not account for its attenuation. We show that the wild-type strain, H37Rv, metabolizes cholesterol to androst-4-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD) and exports these metabolites into the medium, whereas the fadA5 mutant strain is defective for this activity. We demonstrate that FadA5 catalyzes the thiolysis of acetoacetyl-coenzyme A (CoA). This catalytic activity is consistent with a β-ketoacyl-CoA thiolase function in cholesterol β-oxidation that is required for the production of androsterones. We conclude that the attenuated phenotype of the fadA5 mutant is a consequence of disrupted cholesterol metabolism that is essential only in the persistent stage of M. tuberculosis infection and may be caused by the inability to produce AD/ADD from cholesterol.

scholarly journals The Rhizobium etli argC Gene Is Essential for Arginine Biosynthesis and Nodulation of Phaseolus vulgaris

2001 ◽  
Vol 14 (2) ◽  
pp. 250-254 ◽  
Author(s):  
Simona Ferraioli ◽  
Rosarita Taté ◽  
Emilia Caputo ◽  
Alessandro Lamberti ◽  
Anna Riccio ◽  
...  
Keyword(s):  
Phaseolus Vulgaris ◽  
Mutant Strain ◽  
Wild Type Strain ◽  
Reductase Activity ◽  
Homologous Gene ◽  
Rhizobium Etli ◽  
Wild Type ◽  
Arginine Biosynthesis ◽  
Nodulation Factors ◽  
Wild Type Parent

A Tn5-induced mutant strain (CTNUX5) of Rhizobium etli unable to grow with ammonium as the sole nitrogen source was isolated and characterized. Sequence analysis showed that Tn5 is inserted into an argC-homologous gene. Unlike its wild-type parent (strain CE3), the mutant strain CTNUX5 had an absolute dependency on arginine to grow. The argC gene was cloned from the wild-type strain CE3, and the resulting plasmid, pAR207, after transformation was shown to relieve the arginine auxotrophy of strain CTNUX5. Unlike strain CE3 or CTNUX5-pAR207, strain CTNUX5 showed undetectable levels of N-acetyl-γ-glutamylphosphate reductase activity. Unless ar-ginine was added to the growth medium, strain CTNUX5 was unable to produce flavonoid-inducible lipo-chitin oli-gosaccharides (nodulation factors) and to induce nodules or nodulelike structures on the roots of Phaseolus vulgaris.

scholarly journals Construction and Phenotypic Characterization of an Auxotrophic Mutant of Mycobacterium tuberculosis Defective in l-Arginine Biosynthesis

2002 ◽  
Vol 70 (6) ◽  
pp. 3080-3084 ◽  
Author(s):  
Bhavna G. Gordhan ◽  
Debbie A. Smith ◽  
Heidi Alderton ◽  
Ruth A. McAdam ◽  
Gregory J. Bancroft ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Auxotrophic Mutant ◽  
Phenotypic Characterization ◽  
Wild Type ◽  
Arginine Biosynthesis ◽  
High Concentrations

ABSTRACT A mutant of Mycobacterium tuberculosis defective in the metabolism of l-arginine was constructed by allelic exchange mutagenesis. The argF mutant strain required exogenous l-arginine for growth in vitro, and in the presence of 0.96 mM l-arginine, it achieved a growth rate and cell density in stationary phase comparable to those of the wild type. The mutant strain was also able to grow in the presence of high concentrations of argininosuccinate, but its auxotrophic phenotype could not be rescued by l-citrulline, suggesting that the ΔargF::hyg mutation exerted a polar effect on the downstream argG gene but not on argH. The mutant strain displayed reduced virulence in immunodeficient SCID mice and was highly attenuated in immunocompetent DBA/2 mice, suggesting that l-arginine availability is restricted in vivo.

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