scholarly journals A Specific Polymorphism in Mycobacterium tuberculosis H37Rv Causes Differential ESAT-6 Expression and Identifies WhiB6 as a Novel ESX-1 Component

2014 ◽  
Vol 82 (8) ◽  
pp. 3446-3456 ◽  
Author(s):  
Luis Solans ◽  
Nacho Aguiló ◽  
Sofía Samper ◽  
Alexandre Pawlik ◽  
Wafa Frigui ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Genetic Locus ◽  
Laboratory Strain ◽  
Single Nucleotide ◽  
Content Type ◽  
Clinical Strains ◽  
Mycobacterium Tuberculosis H37rv ◽  
H37rv Strain ◽  
Nucleotide Insertion ◽  
Secretory Apparatus

ABSTRACTThe ESX-1 secreted virulence factor ESAT-6 is one of the major and most well-studied virulence factors ofMycobacterium tuberculosis, given that its inactivation severely attenuates virulent mycobacteria. In this work, we show that clinical isolates ofM. tuberculosisproduce and secrete larger amounts of ESAT-6 than the widely usedM. tuberculosisH37Rv laboratory strain. A search for the genetic polymorphisms underlying this observation showed thatwhiB6(rv3862c), a gene upstream of the ESX-1 genetic locus that has not previously been found to be implicated in the regulation of the ESX-1 secretory apparatus, presents a unique single nucleotide insertion in its promoter region in strains H37Rv and H37Ra. This polymorphism is not present in any of the other publicly availableM. tuberculosiscomplex genomes or in any of the 76 clinicalM. tuberculosisisolates analyzed in our laboratory. We demonstrate that in consequence, the virulence master regulator PhoP downregulateswhiB6expression in H37Rv, while it upregulates its expression in clinical strains. Importantly, reintroduction of the wild-type (WT) copy ofwhiB6in H37Rv restored ESAT-6 production and secretion to the level of clinical strains. Hence, we provide clear evidence that inM. tuberculosis—with the exception of the H37Rv strain—ESX-1 expression is regulated by WhiB6 as part of the PhoP regulon, which adds another level of complexity to the regulation of ESAT-6 secretion with a potential role in virulence adaptation.

scholarly journals Mutations in the CCGTTCACA DnaA Box of Mycobacterium tuberculosis oriC That Abolish Replication of oriC Plasmids Are Tolerated on the Chromosome

2002 ◽  
Vol 184 (14) ◽  
pp. 3848-3855 ◽  
Author(s):  
Jaroslaw Dziadek ◽  
Malini Rajagopalan ◽  
Tanya Parish ◽  
Natalia Kurepina ◽  
Rebecca Greendyke ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Hot Spot ◽  
Laboratory Strain ◽  
Functional Requirements ◽  
Clinical Strains ◽  
Replication Assay ◽  
Dnaa Protein ◽  
Mutant Sequence ◽  
Dnaa Box

ABSTRACT The origin of replication (oriC) region in some clinical strains of Mycobacterium tuberculosis is a hot spot for IS6110 elements. To understand how clinical strains with insertions in oriC can replicate their DNA, we characterized the oriC regions of some clinical strains. Using a plasmid-based oriC-dependent replication assay, we showed that IS6110 insertions that disrupted the DnaA box sequence CCGTTCACA abolished oriC activity in M. tuberculosis. Furthermore, by using a surface plasmon resonance technique we showed that purified M. tuberculosis DnaA protein binds native but not mutant DnaA box sequence, suggesting that stable interactions of the DnaA protein with the CCGTTCACA DnaA box are crucial for replication of oriC plasmids in vivo. Replacement by homologous recombination of the CCGTTCACA DnaA box sequence of the laboratory strain M. tuberculosis H37Ra with a mutant sequence did not result in nonviability. Together, these results suggest that M. tuberculosis strains have evolved mechanisms to tolerate mutations in the oriC region and that functional requirements for M. tuberculosis oriC replication are different for chromosomes and plasmids.

scholarly journals Identification of Novel Efflux Proteins Rv0191, Rv3756c, Rv3008, and Rv1667c Involved in Pyrazinamide Resistance in Mycobacterium tuberculosis

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Yumeng Zhang ◽  
Jia Zhang ◽  
Peng Cui ◽  
Ying Zhang ◽  
Wenhong Zhang
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Efflux Pump ◽  
Binding Studies ◽  
Content Type ◽  
Clinical Strains ◽  
Efflux Pump Inhibitors ◽  
Level Resistance ◽  
Pyrazinoic Acid ◽  
Increased Susceptibility ◽  
Efflux Proteins

ABSTRACT Pyrazinamide (PZA) is a critical drug used for the treatment of tuberculosis (TB). PZA is a prodrug that requires conversion to the active component pyrazinoic acid (POA) by pyrazinamidase (PZase) encoded by the pncA gene. Although resistance to PZA is mostly caused by pncA mutations and less commonly by rpsA, panD, and clpC1 mutations, clinical strains without these mutations are known to exist. While efflux of POA was demonstrated in Mycobacterium tuberculosis previously, the efflux proteins involved have not been identified. Here we performed POA binding studies with an M. tuberculosis proteome microarray and identified four efflux proteins (Rv0191, Rv3756c, Rv3008, and Rv1667c) that bind POA. Overexpression of the four efflux pump genes in M. tuberculosis caused low-level resistance to PZA and POA but not to other drugs. Furthermore, addition of efflux pump inhibitors such as reserpine, piperine, and verapamil caused increased susceptibility to PZA in M. tuberculosis strains overexpressing the efflux proteins Rv0191, Rv3756c, Rv3008, and Rv1667c. Our studies indicate that these four efflux proteins may be responsible for PZA/POA efflux and cause PZA resistance in M. tuberculosis. Future studies are needed to assess their roles in PZA resistance in clinical strains.

scholarly journals Proteomics of Culture Filtrate of Prevalent Mycobacterium tuberculosis Strains: 2D-PAGE Map and MALDI-TOF/MS Analysis

2017 ◽  
Vol 22 (9) ◽  
pp. 1142-1149 ◽  
Author(s):  
Gavish Kumar ◽  
Hari Shankar ◽  
Divakar Sharma ◽  
Prashant Sharma ◽  
Deepa Bisht ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Culture Filtrate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Critical Role ◽  
Laboratory Strain ◽  
Proteomic Profiling ◽  
East African ◽  
Mycobacterium Tuberculosis H37rv ◽  
Flight Mass Spectrometry ◽  
Central Asian

Although diverse efforts have been done to identify biomarkers for control of tuberculosis using laboratory strain Mycobacterium tuberculosis H37Rv, the disease still poses a threat to mankind. There are many emerging M. tuberculosis strains, and proteomic profiling of these strains might be important to find out potential targets for diagnosis and/or prevention of tuberculosis. We evaluated the comparative proteomic profiling of culture filtrate (CF) proteins from prevalent M. tuberculosis strains (Central Asian or Delhi type; CAS1_Del, East African-Indian; EAI-3 and Beijing family) by 2D polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization–time-of-flight mass spectrometry. As a result, we could identify 12 CF proteins (Rv0066c, Rv1310, Rv3375, Rv1415, Rv0567, Rv1886c, Rv3803c, Rv3804c, Rv2031c, Rv1038c, Rv2809, and Rv1911c), which were consistently increased in all prevalent M. tuberculosis strains, and interestingly, two CF proteins (Rv2809, Rv1911c) were identified with unknown functions. Consistent increased intensity of these proteins suggests their critical role for survival of prevalent M. tuberculosis isolates, and some of these proteins may also have potential as diagnostic and vaccine candidates for tuberculosis, which needs to be further explored by immunological analysis.

SYNTHESIS AND ANTIMICROBIAL ACTIVITY OF SOME NEW 1,3,4- OXADIAZOLES, 1,2,4-TRIAZOLES AND 1,3,4-THIADIAZOLES

INDIAN DRUGS ◽  
2016 ◽  
Vol 53 (06) ◽  
pp. 18-23
Author(s):  
U. V. Laddi ◽  
◽  
S. R. Desai
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Activity ◽  
Mycobacterium Tuberculosis ◽  
Antitubercular Activity ◽  
Tuberculosis H37rv ◽  
Research Centre ◽  
Methyl Ethyl ◽  
Rhizoctonia Bataticola ◽  
Mycobacterium Tuberculosis H37rv ◽  
H37rv Strain

Some new 5-[(((α-phenyl/methyl)benzylidene)amino)oxy]methyl/ethyl-2-[4-(substituted aryl)/allyl)] amino-1,3,4-oxadiazoles (4a-p), 3-[(((α-phenyl/methyl)- benzylidene) amino)oxy]methyl/ethyl-4-(4- substitutedaryl)/allyl-5-mercapto-1,2,4-triazoles (5a-p) and 5-[(((α-phenyl/methyl)-benzylidene)amino) oxy]- methyl/ethyl-2-[4-(substituted aryl)/allyl)]amino-1,3,4-thiadiazoles (6a-p) were prepared starting from α/β-[((α-(phenyl/methyl)benzylidene)amino)oxy acetic/propionic acid hydrazides (1a-d). The structures of all the compounds have been established by elemental and spectral (IR, 1HNMR and mass) analysis. All the newly synthesised compounds have been screened for their antimicrobial activity against Escherichia coli, Bacillus cirroflagellosus, Aspergillus niger and Rhizoctonia bataticola. Some of the newly synthesised compounds have been evaluated for antituberculosis activity against Mycobacterium tuberculosis H37Rv strain by BACTEC radiometric system at Southern Research Institute, Birmingham, AL and Frederick Research Centre, Frederick, MD. Significant antimicrobial activity is observed against Escherichia coli and Rhizoctonia bataticola. A few compounds also exhibited interesting antitubercular activity against Mycobacterium tuberculosis H37Rv strain.

scholarly journals Using Mycobacterium tuberculosis Single-Nucleotide Polymorphisms To Predict Fluoroquinolone Treatment Response

2019 ◽  
Vol 63 (7) ◽  
Author(s):  
Marva Seifert ◽  
Edmund Capparelli ◽  
Donald G. Catanzaro ◽  
Timothy C. Rodwell
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Single Nucleotide Polymorphisms ◽  
Specific Resistance ◽  
Therapeutic Targets ◽  
World Health ◽  
Population Pharmacokinetic ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Content Type ◽  
Level Increase

ABSTRACT Clinical phenotypic fluoroquinolone susceptibility testing of Mycobacterium tuberculosis is currently based on M. tuberculosis growth at a single critical concentration, which provides limited information for a nuanced clinical response. We propose using specific resistance-conferring M. tuberculosis mutations in gyrA together with population pharmacokinetic and pharmacodynamic modeling as a novel tool to better inform fluoroquinolone treatment decisions. We sequenced the gyrA resistance-determining region of 138 clinical M. tuberculosis isolates collected from India, Moldova, Philippines, and South Africa and then determined each strain’s MIC against ofloxacin, moxifloxacin, levofloxacin, and gatifloxacin. Strains with specific gyrA single-nucleotide polymorphisms (SNPs) were grouped into high or low drug-specific resistance categories based on their empirically measured MICs. Published population pharmacokinetic models were then used to explore the pharmacokinetics and pharmacodynamics of each fluoroquinolone relative to the empirical MIC distribution for each resistance category to make predictions about the likelihood of patients achieving defined therapeutic targets. In patients infected with M. tuberculosis isolates containing SNPs associated with a fluoroquinolone-specific low-level increase in MIC, models suggest increased fluoroquinolone dosing improved the probability of achieving therapeutic targets for gatifloxacin and moxifloxacin but not for levofloxacin and ofloxacin. In contrast, among patients with isolates harboring SNPs associated with a high-level increase in MIC, increased dosing of levofloxacin, moxifloxacin, gatifloxacin, or ofloxacin did not meaningfully improve the probability of therapeutic target attainment. We demonstrated that quantifiable fluoroquinolone drug resistance phenotypes could be predicted from rapidly detectable gyrA SNPs and used to support dosing decisions based on the likelihood of patients reaching therapeutic targets. Our findings provide further supporting evidence for the moxifloxacin clinical breakpoint recently established by the World Health Organization.

scholarly journals Correlates between Models of Virulence for Mycobacterium tuberculosis among Isolates of the Central Asian Lineage: a Case for Lysozyme Resistance Testing?

2015 ◽  
Vol 83 (6) ◽  
pp. 2213-2223 ◽  
Author(s):  
Claire Pardieu ◽  
Nicola Casali ◽  
Simon O. Clark ◽  
Richard Hooper ◽  
Ann Williams ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Ex Vivo ◽  
Resistance Testing ◽  
Snp Analysis ◽  
Single Nucleotide ◽  
Content Type ◽  
Differential Outcomes ◽  
Central Asian ◽  
Strain H37rv

Virulence factors (VFs) contribute to the emergence of new humanMycobacterium tuberculosisstrains, are lineage dependent, and are relevant to the development ofM. tuberculosisdrugs/vaccines. VFs were sought withinM. tuberculosislineage 3, which has the Central Asian (CAS) spoligotype. Three isolates were selected from clusters previously identified as dominant in London, United Kingdom. Strain-associated virulence was studied in guinea pig, monocyte-derived macrophage, and lysozyme resistance assays. Whole-genome sequencing, single nucleotide polymorphism (SNP) analysis, and a literature review contributed to the identification of SNPs of interest. The animal model revealed borderline differences in strain-associated pathogenicity.Ex vivo, isolate C72 exhibited statistically significant differences in intracellular growth relative to C6 and C14. SNP candidates inducing lower fitness levels included 123 unique nonsynonymous SNPs, including three located in genes (lysX,caeA, andponA2) previously identified as VFs in the laboratory-adapted reference strain H37Rv and shown to confer lysozyme resistance. C72 growth was most affected by lysozymein vitro. A BLAST search revealed that all three SNPs of interest (C35F, P76Q, and P780R) also occurred in Tiruvallur, India, and in Uganda. Unlike C72, however, no single isolate identified through BLAST carried all three SNPs simultaneously. CAS isolates representative of three medium-sized human clusters demonstrated differential outcomes in models commonly used to estimate strain-associated virulence, supporting the idea that virulence varies within, not just across,M. tuberculosislineages. Three VF SNPs of interest were identified in two additional locations worldwide, which suggested independent selection and supported a role for these SNPs in virulence. The relevance of lysozyme resistance to strain virulence remains to be established.

scholarly journals Rufomycin Targets ClpC1 Proteolysis in Mycobacterium tuberculosis and M. abscessus

2019 ◽  
Vol 63 (3) ◽  
Author(s):  
Mary P. Choules ◽  
Nina M. Wolf ◽  
Hyun Lee ◽  
Jeffrey R. Anderson ◽  
Edyta M. Grzelak ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Atpase Activity ◽  
Cyclic Peptides ◽  
Mycobacterium Abscessus ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Content Type ◽  
Downstream Effects ◽  
Intracellular Proteins ◽  
Cyclomarin A

ABSTRACT ClpC1 is an emerging new target for the treatment of Mycobacterium tuberculosis infections, and several cyclic peptides (ecumicin, cyclomarin A, and lassomycin) are known to act on this target. This study identified another group of peptides, the rufomycins (RUFs), as bactericidal to M. tuberculosis through the inhibition of ClpC1 and subsequent modulation of protein degradation of intracellular proteins. Rufomycin I (RUFI) was found to be a potent and selective lead compound for both M. tuberculosis (MIC, 0.02 μM) and Mycobacterium abscessus (MIC, 0.4 μM). Spontaneously generated mutants resistant to RUFI involved seven unique single nucleotide polymorphism (SNP) mutations at three distinct codons within the N-terminal domain of clpC1 (V13, H77, and F80). RUFI also significantly decreased the proteolytic capabilities of the ClpC1/P1/P2 complex to degrade casein, while having no significant effect on the ATPase activity of ClpC1. This represents a marked difference from ecumicin, which inhibits ClpC1 proteolysis but stimulates the ATPase activity, thereby providing evidence that although these peptides share ClpC1 as a macromolecular target, their downstream effects are distinct, likely due to differences in binding.

scholarly journals Draft Genome Sequence of an Isolate of Extensively Drug-Resistant Mycobacterium tuberculosis from Nepal

2020 ◽  
Vol 9 (4) ◽  
Author(s):  
Sanjay S. Gautam ◽  
Kelvin W. C. Leong ◽  
Manoj Pradhan ◽  
Y. Ibotomba Singh ◽  
Sagar K. Rajbhandari ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Genome Sequence ◽  
Draft Genome ◽  
Draft Genome Sequence ◽  
Drug Resistant ◽  
Single Nucleotide ◽  
Content Type ◽  
Extensively Drug Resistant ◽  
Drug Resistant Phenotype ◽  
First Time

Extensively drug-resistant (XDR) Mycobacterium tuberculosis has become a challenge to the treatment of tuberculosis (TB) in several countries, including Nepal. Here, we report for the first time the draft genome sequence of an isolate of XDR-TB collected in Nepal and describe single-nucleotide variations associated with its extensively drug-resistant phenotype.

scholarly journals Mutation in an Unannotated Protein Confers Carbapenem Resistance in Mycobacterium tuberculosis

2017 ◽  
Vol 61 (3) ◽  
Author(s):  
Pankaj Kumar ◽  
Amit Kaushik ◽  
Drew T. Bell ◽  
Varsha Chauhan ◽  
Fangfang Xia ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Genetic Basis ◽  
Carbapenem Resistance ◽  
Drug Resistant ◽  
Binding Affinities ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Content Type ◽  
Resistant Tuberculosis ◽  
Recent Developments

ABSTRACT β-Lactams are the most widely used antibacterials. Among β-lactams, carbapenems are considered the last line of defense against recalcitrant infections. As recent developments have prompted consideration of carbapenems for treatment of drug-resistant tuberculosis, it is only a matter of time before Mycobacterium tuberculosis strains resistant to these drugs will emerge. In the present study, we investigated the genetic basis that confers such resistance. To our surprise, instead of mutations in the known β-lactam targets, a single nucleotide polymorphism in the Rv2421c-Rv2422 intergenic region was common among M. tuberculosis mutants selected with meropenem or biapenem. We present data supporting the hypothesis that this locus harbors a previously unidentified gene that encodes a protein. This protein binds to β-lactams, slowly hydrolyzes the chromogenic β-lactam nitrocefin, and is inhibited by select penicillins and carbapenems and the β-lactamase inhibitor clavulanate. The mutation results in a W62R substitution that reduces the protein's nitrocefin-hydrolyzing activity and binding affinities for carbapenems.

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