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Mycobacterium tuberculosis AND drug use and [keywords]
- Contributions of Unique Active Site Residues of Eukaryotic UDP-Galactopyranose Mutases to Substrate Recognition and Active Site Dynamics. [JOURNAL ARTICLE]
- Biochemistry 2014 Nov 20.
UDP-galactopyranose mutase (UGM) catalyzes the interconversion between UDP-galactopyranose and UDP-galactofuranose. Absent in humans, galactofuranose is found in bacterial and fungal cell walls and is a cell surface virulence factor in protozoan parasites. For these reasons, UGMs are targets for drug discovery. Here, we report a mutagenesis and structural study of the UGMs from Aspergillus fumigatus and Trypanosoma cruzi focused on active site residues that are conserved in eukaryotic UGMs but are absent or different in bacterial UGMs. Kinetic analysis of the variants F66A, Y104A, Q107A, N207A, and Y317A (A. fumigatus numbering) show decreases in kcat/KM values of 200-1000 fold for the mutase reaction. In contrast, none of the mutations strongly affect the kinetics of enzyme activation by NADPH. These results indicate that the targeted residues are important for promoting the transition state conformation for UDP-galactofuranose formation. Crystal structures of the A. fumigatus mutant enzymes were determined in the presence and absence of UDP to understand the structural consequences of the mutations. The structures suggest important roles for Asn207 in stabilizing the closed active site, and Tyr317 in positioning of the uridine ring. Phe66 and the corresponding residue in Mycobacterium tuberculosis UGM (His68) play a role as the backstop, stabilizing the galactopyranose group for nucleophilic attack. Together, these results provide insight into the essentiality of the targeted residues for realizing maximal catalytic activity and a proposal for how conformational changes that close the active site are temporally related and coupled together.
- Metformin as adjunct antituberculosis therapy. [JOURNAL ARTICLE]
- Sci Transl Med 2014 Nov 19; 6(263):263ra159.
The global burden of tuberculosis (TB) morbidity and mortality remains immense. A potential new approach to TB therapy is to augment protective host immune responses. We report that the antidiabetic drug metformin (MET) reduces the intracellular growth of Mycobacterium tuberculosis (Mtb) in an AMPK (adenosine monophosphate-activated protein kinase)-dependent manner. MET controls the growth of drug-resistant Mtb strains, increases production of mitochondrial reactive oxygen species, and facilitates phagosome-lysosome fusion. In Mtb-infected mice, use of MET ameliorated lung pathology, reduced chronic inflammation, and enhanced the specific immune response and the efficacy of conventional TB drugs. Moreover, in two separate human cohorts, MET treatment was associated with improved control of Mtb infection and decreased disease severity. Collectively, these data indicate that MET is a promising candidate host-adjunctive therapy for improving the effective treatment of TB.
- The Host Battles Drug-Resistant Tuberculosis. [JOURNAL ARTICLE]
- Sci Transl Med 2014 Nov 19; 6(263):263fs47.
The repurposed drug metformin reduces unproductive inflammation, refocuses host immune responses on the infectious agent Mycobacterium tuberculosis, and thus may improve treatment outcomes for drug-resistant tuberculosis (Singhal et al., this issue).
- Biochemical and Structural Characterization of Mycobacterial Aspartyl-tRNA Synthetase AspS, a Promising TB Drug Target. [JOURNAL ARTICLE]
- PLoS One 2014; 9(11):e113568.
The human pathogen Mycobacterium tuberculosis is the causative agent of pulmonary tuberculosis (TB), a disease with high worldwide mortality rates. Current treatment programs are under significant threat from multi-drug and extensively-drug resistant strains of M. tuberculosis, and it is essential to identify new inhibitors and their targets. We generated spontaneous resistant mutants in Mycobacterium bovis BCG in the presence of 10× the minimum inhibitory concentration (MIC) of compound 1, a previously identified potent inhibitor of mycobacterial growth in culture. Whole genome sequencing of two resistant mutants revealed in one case a single nucleotide polymorphism in the gene aspS at 535GAC>535AAC (D179N), while in the second mutant a single nucleotide polymorphism was identified upstream of the aspS promoter region. We probed whole cell target engagement by overexpressing either M. bovis BCG aspS or Mycobacterium smegmatis aspS, which resulted in a ten-fold and greater than ten-fold increase, respectively, of the MIC against compound 1. To analyse the impact of inhibitor 1 on M. tuberculosis AspS (Mt-AspS) activity we over-expressed, purified and characterised the kinetics of this enzyme using a robust tRNA-independent assay adapted to a high-throughput screening format. Finally, to aid hit-to-lead optimization, the crystal structure of apo M. smegmatis AspS was determined to a resolution of 2.4 Å.
- In situ characterization of mycobacterial growth inhibition by lytic enzymes expressed in vectorized E. coli. [JOURNAL ARTICLE]
- ACS Synth Biol 2014 Nov 19.
The emergence of extremely drug resistant Mycobacterium tuberculosis necessitates new strategies to combat the pathogen. Engineered bacteria may serve as vectors to deliver proteins to human cells, including mycobacteria-infected macrophages. In this work we target Mycobacterium smegmatis, a non-pathogenic tuberculosis model, with E. coli modified to express Trehalose Dimycolate Hydrolase (TDMH), a membrane-lysing serine esterase. We show that TDMH-expressing E. coli are capable of lysing mycobacteria in vitro and at low pH. Vectorized E. coli producing TDMH were found suppress the proliferation of mycobacteria in infected macrophages.
- Unraveling Mycobacterium tuberculosis genomic diversity and evolution in Lisbon, Portugal, a highly drug resistant setting. [JOURNAL ARTICLE]
- BMC Genomics 2014 Nov 18; 15(1):991.
Multidrug- (MDR) and extensively drug resistant (XDR) tuberculosis (TB) presents a challenge to disease control and elimination goals. In Lisbon, Portugal, specific and successful XDR-TB strains have been found in circulation for almost two decades.In the present study we have genotyped and sequenced the genomes of 56 Mycobacterium tuberculosis isolates recovered mostly from Lisbon. The genotyping data revealed three major clusters associated with MDR-TB, two of which are associated with XDR-TB. Whilst the genomic data contributed to elucidate the phylogenetic positioning of circulating MDR-TB strains, showing a high predominance of a single SNP cluster group 5. Furthermore, a genome-wide phylogeny analysis from these strains, together with 19 publicly available genomes of Mycobacterium tuberculosis clinical isolates, revealed two major clades responsible for M/XDR-TB in the region: Lisboa3 and Q1 (LAM).The data presented by this study yielded insights on microevolution and identification of novel compensatory mutations associated with rifampicin resistance in rpoB and rpoC. The screening for other structural variations revealed putative clade-defining variants. One deletion in PPE41, found among Lisboa3 isolates, is proposed to contribute to immune evasion and as a selective advantage. Insertion sequence (IS) mapping has also demonstrated the role of IS6110 as a major driver in mycobacterial evolution by affecting gene integrity and regulation.Globally, this study contributes with novel genome-wide phylogenetic data and has led to the identification of new genomic variants that support the notion of a growing genomic diversity facing both setting and host adaptation.
- Molecular epidemiology of tuberculosis in Sicily, Italy: what has changed after a decade? [JOURNAL ARTICLE]
- BMC Infect Dis 2014 Nov 19; 14(1):602.
BackgroundWe aimed to investigate the molecular epidemiology of Mycobacterium tuberculosis complex (MTBC) isolates in the province of Palermo, Sicily, Italy, by characterizing 183 isolates identified in the years 2004¿2012. A comparison with 104 MTBC strains identified in the same geographic area in the years 1994¿2000 was also carried out.MethodsOne hundred eighty-three MTBC isolates identified in Palermo, Italy, in the years 2004¿2012 were analyzed by spoligotyping and the 24 mycobacterial interspersed repetitive unit (MIRU)-variable-number tandem-repeat (VNTR) method typing. Susceptibility testing to streptomycin, isoniazid, rifampin and ethambutol was also performed. Furthermore, the spoligotyping dataset obtained from 104 MTBC isolates identified from 1994 to 2000 was reanalyzed. Distribution into lineages and clustering of isolates in the two periods was compared.ResultsOne hundred seventy-seven out of the 183 isolates of MTBC submitted to molecular typing were fully characterized. Of these, 108 were from Italian-born and 69 from foreign-born individuals. Eleven different lineages and 35 families-subfamilies were identified with the most represented lineages being Haarlem (26.5%), T (19.2%), LAM (13.6%) and S (8.5%). Except for the Haarlem lineage, where isolates from foreign-born patients were overrepresented, the distribution of isolates in the families belonging to the Euro-American clone reflected the proportions of the two subpopulations. A total of 27 (15.2%) strains were clustered and three clusters were mixed. Approximately 25% of the 183 MTBC isolates under study proved to be resistant to at least one antiTB drug, with only three isolates categorized as multidrug resistant (MDR). When MTBC isolates identified in the years 1994¿2000 were reanalyzed, lineages T (30.8%), LAM (29.8%), Haarlem (16.3%) and S (13.5%) proved to be predominant. No MTBC isolates belonging to CAM, U, CAS, Turkish and Ural lineages were identified.ConclusionsA wide heterogeneity was detected among the MTBC strains isolated in the years 2004¿2012. Six lineages were not present among the isolates of the period 1994¿2000. Comparison between distribution of lineages in the two consecutive periods depicts rapid and deep changes in the TB epidemiology in Palermo, Italy. An universal and continued laboratory-based surveillance of TB in Sicily is required.
- Measuring Glutathione Redox Potential of HIV-1 Infected Macrophages. [JOURNAL ARTICLE]
- J Biol Chem 2014 Nov 18.
Redox signaling plays a crucial role in the pathogenesis of Human Immunodeficiency Virus type-1 (HIV-1). Majority of HIV redox research relies on measuring redox stress using invasive technologies, which are unreliable and do not inform the contributions of sub-cellular compartments. A major technological leap emerges from the development of genetically encoded redox sensitive green fluorescent proteins (roGFPs), which provide sensitive and compartment-specific insights into redox homeostasis. Here, we exploited a roGFP based specific bioprobe of glutathione redox potential (EGSH; Grx1-roGFP2) and measured sub-cellular changes in EGSH during various phases of HIV-1 infection using U1 monocytic cells (latently infected U937 cells with HIV-1). We show that while U937 and U1 cells demonstrate significantly reduced cytosolic and mitochondrial EGSH (≈ -310 mV), active viral replication induces substantial oxidative stress (EGSH > -240 mV). Furthermore, exposure to a physiologically-relevant oxidant, hydrogen peroxide (H2O2) induces significant deviations in sub-cellular EGSH between U937 and U1, which distinctly modulates susceptibility to apoptosis. Using Grx1-roGFP2, we demonstrate that a marginal increase of about ~25 mV in EGSH is sufficient to switch HIV-1 from latency to reactivation, raising the possibility of purging HIV-1 by redox modulators without triggering detrimental changes in cellular physiology. Importantly, we show that bioactive lipids synthesized by clinical drug resistant isolates of Mycobacterium tuberculosis (Mtb) reactivate HIV-1 through modulation of intracellular EGSH. Finally, the expression analysis of U1 and patient Peripheral Blood Mononuclear Cells (PBMCs) demonstrated a major recalibration of cellular redox homeostatic pathways during persistence and active replication of HIV.
- Delamanid: A Review of Its Use in Patients with Multidrug-Resistant Tuberculosis. [JOURNAL ARTICLE]
- Drugs 2014 Nov 18.
Delamanid (Deltyba(®)), a nitroimidazo-oxazole derivative, is a new anti-tuberculosis (TB) drug which exhibits potent in vitro and in vivo antitubercular activity against drug-susceptible and -resistant strains of Mycobacterium tuberculosis. It is approved in several countries, including Japan and those of the EU, for use as part of an appropriate combination regimen in adults with multidrug-resistant tuberculosis (MDR-TB) when an effective treatment regimen cannot otherwise be composed due to resistance or tolerability. In a robust phase II trial in adult patients with MDR-TB, oral delamanid 100 mg twice daily for 2 months plus an optimized background regimen improved sputum culture conversion rates to a significantly greater extent than placebo. In a 6-month extension study, long-term (≤8 months) treatment with delamanid was associated with a higher incidence of favourable outcomes (i.e. cured or completed all treatment) than short-term (≤2 months) treatment, with an accompanying reduction inunfavourable outcomes as defined by the WHO (i.e. pre-specified proportion of TB-positive sputum cultures, death or treatment discontinuation for ≥2 months without medical approval). Delamanid was not associated with clinically relevant drug-drug interactions, including with antiretroviral drugs and those commonly used in treating TB. Delamanid was generally well tolerated in patients with MDR-TB, with gastrointestinal adverse events and insomnia reported most commonly. Although the incidence of QT interval prolongation was higher with delamanid-based therapy, it was not associated with clinical symptoms such as syncope and arrhythmia. In conclusion, delamanid is a useful addition to the treatment options currently available for patients with MDR-TB.
- Radioiodo-DPA-713 Imaging Correlates with Bactericidal Activity of Tuberculosis Treatments in Mice. [JOURNAL ARTICLE]
- Antimicrob Agents Chemother 2014 Nov 17.
Current tools for monitoring response to tuberculosis treatments have several limitations. Noninvasive biomarkers could accelerate tuberculosis drug development and clinical studies, but to date little progress has been made in developing new imaging technologies for this application. In this study, we developed pulmonary iodo-DPA-713 single-photon emission computed tomography (SPECT) to serially monitor the activity of tuberculosis treatments in live mice, which develop necrotic granulomas and cavitary lesions. C3HeB/FeJ mice were aerosol infected with Mycobacterium tuberculosis and administered either a standard or a highly active bedaquiline containing drug regimen. Serial [(125)I]DPA-713 SPECT imaging was compared with [(18)F]fluorodeoxyglucose (FDG) positron emission tomography (PET) and standard microbiology. Ex vivo studies were performed to characterize and correlate DPA-713 imaging with cellular and cytokine responses. Pulmonary [(125)I]DPA-713 SPECT, but not [(18)F]FDG PET, was able to correctly identify the bactericidal activities of the two tuberculosis treatments as early as four weeks after starting treatment (P < 0.03). DPA-713 readily penetrated the fibrotic rims of necrotic and cavitary lesions. A time dependent decrease in both TNF-α and IFN-γ levels was observed with treatments, with [(125)I]DPA-713 SPECT correlating best with tissue TNF-α levels (ρ = 0.94; P < 0.01). [(124)I]DPA-713 was also evaluated as a PET probe and demonstrated a 4.0-fold higher signal intensity in the infected tuberculous lesions compared to uninfected controls (P = 0.03). These studies provide proof-of-concept for application of a novel noninvasive imaging biomarker to monitor tuberculosis treatments, with potential for translation to humans.