(Mycobacterium tuberculosis AND drug use and)
- Learning from the past for TB drug discovery in the future. [Review]
- DDDrug Discov Today 2016 Oct 4
- Tuberculosis drug discovery has shifted in recent years from a primarily target-based approach to one that uses phenotypic high-throughput screens. As examples of this, through our EU-funded FP7 coll...
Tuberculosis drug discovery has shifted in recent years from a primarily target-based approach to one that uses phenotypic high-throughput screens. As examples of this, through our EU-funded FP7 collaborations, New Medicines for Tuberculosis was target-based and our more-recent More Medicines for Tuberculosis project predominantly used phenotypic screening. From these projects we have examples of success (DprE1) and failure (PimA) going from drug to target and from target to drug, respectively. It is clear that we still have much to learn about the drug targets and the complex effects of the drugs on Mycobacterium tuberculosis. We propose a more integrated approach that learns from earlier drug discovery efforts that could help to move drug discovery forward.
- Recombinant Leishmania Rab6 (rLdRab6) is recognized by sera from visceral leishmaniasis patients. [Journal Article]
- EPExp Parasitol 2016 Sep 22; 170:135-147
- Rab proteins form the largest branch of the Ras superfamily. Rab proteins are key regulators of intracellular vesicular transport and membrane trafficking. Although RabGTPases are well-recognized tar...
Rab proteins form the largest branch of the Ras superfamily. Rab proteins are key regulators of intracellular vesicular transport and membrane trafficking. Although RabGTPases are well-recognized targets in human diseases but are under-explored therapeutically in the Leishmania parasite. Using a quantitative cytofluorimetric assay, we analyzed the composition and organization of Rab6GTPase protein which was found to be primarily localized on the parasite subpellicular membrane and flagellum due to its association with kinesin motor proteins in the cytoskeletal microtubules. Our aim was to also assess the diagnostic role of recombinant Rab6 protein from Leishmania donovani (rLdRab6) using sera/plasma of Indian visceral leishmaniasis (VL) patients. Receiver-operating characteristic (ROC) curve analysis indicated 100% sensitivity and 100% specificity for rLdRab6-based ELISA which was almost similar in comparison to recombinant K39-based ELISA (95.83% sensitivity and 100% specificity). Sera of patients from another intracellular pathogenic infection, Mycobacterium tuberculosis, did not contain any significant levels of anti-rLdRab6 antibody. Thus rLdRab6 accuracy in visceral leishmaniasis diagnosis makes it a promising antigen for clinical use.
- Whole genome sequencing of Mycobacterium tuberculosis for detection of drug resistance: a systematic review. [Review]
- CMClin Microbiol Infect 2016 Sep 22
- CONCLUSIONS: Whole genome sequencing could be considered a promising alternative to existing phenotypic and molecular drug susceptibility testing methods for rifampicin and isoniazid pending standardization of analytical pipelines. To ensure clinical relevance of WGS for detection of M. tuberculosis complex drug resistance, future studies should include information on clinical outcomes.
- A simplified mycobacterial growth inhibition assay (MGIA) using direct infection of mouse splenocytes and the MGIT system. [Journal Article]
- JMJ Microbiol Methods 2016 Sep 17; 131:7-9
- We describe a simplified Mycobacterial Growth Inhibition Assay (MGIA) for pre-clinical assessment of vaccine-mediated protection in mice. The assay is accomplished by directly infecting splenocytes f...
We describe a simplified Mycobacterial Growth Inhibition Assay (MGIA) for pre-clinical assessment of vaccine-mediated protection in mice. The assay is accomplished by directly infecting splenocytes from vaccinated mice with Mycobacterium tuberculosis and quantifying mycobacteria using Mycobacterial Growth Indicator Tubes (MGIT). Vaccine-mediated immunogenicity detected by this assay correlated with protection.
- Interactions of linezolid and second-line anti-tuberculosis agents against multidrug-resistant Mycobacterium tuberculosis in vitro and in vivo. [Journal Article]
- IJInt J Infect Dis 2016 Sep 7; 52:23-28
- CONCLUSIONS: These combinations of LZD and second-line anti-TB drugs were all active against MDR-TB with indifference or an additive effect, except LZD + capreomycin, which showed partial synergy.
- Impact of LCP proteins on cell wall biosynthesis in Corynebacterium glutamicum. [Journal Article]
- JBJ Bacteriol 2016 Aug 22
- Proteins of the LCP (LytR, CpsA, Psr)-family were shown to inherit important roles in bacterial cell wall biosynthesis. However, their exact function in the formation of the complex cell wall structu...
Proteins of the LCP (LytR, CpsA, Psr)-family were shown to inherit important roles in bacterial cell wall biosynthesis. However, their exact function in the formation of the complex cell wall structures of the corynebacteriales, including the prominent pathogens Mycobacterium tuberculosis and Corynebacterium diphtheriae, remains unclear. Here we analyzed the role of the LCP proteins LcpA and LcpB of Corynebacterium glutamicum both of which localize at regions of nascent cell wall biosynthesis. A strain lacking lcpB did not show any growth related or morphological phenotype under the tested conditions. In contrast, conditional silencing of the essential lcpA resulted in severe growth defects and drastic morphological changes. Compared to the wild-type, the cell wall of this mutant contained significantly less mycolic acids and a reduced amount of arabinogalactan. In particular rhamnose, a specific sugar component of the linker that connects arabinogalactan and peptidoglycan, was decreased. Complementation studies of the lcpA-silencing strain with several mutated and truncated LcpA variants suggested that both periplasmic domains are essential for function whereas the cytoplasmic N-terminal part is dispensable. Successful complementation experiments with proteins of M. tuberculosis and C. diphtheriae revealed a conserved function of LCP proteins in these species. Finally, pyrophosphatase activity of LcpA was shown in an in vitro assay. Taken together, our results suggest that LCP proteins are responsible for the transfer of arabinogalactan onto peptidoglycan in actinobacterial species and support a crucial function of the so far uncharacterized LytR_C domain, which is frequently found C-terminal of the LCP domain in this prokaryotic phylum.
- Parallel reaction monitoring of clinical Mycobacterium tuberculosis lineages reveals pre-existent markers of rifampicin tolerance in the emerging Beijing lineage. [Journal Article]
- JPJ Proteomics 2016 Aug 26; 150:9-17
- CONCLUSIONS: M. tuberculosis is the leading cause of death by a bacterial infection worldwide. Treatment-regimen to eradicate this pathogen make use of the first-line antibiotic rifampicin, which is considered to be the cornerstone of modern day anti-tuberculosis treatment. Despite the potency of rifampicin, there is an increasing occurrence of rifampicin resistant mutants in a specific cluster of M. tuberculosis, the Beijing genotype. Using both a data dependent acquisition and a targeted proteomic approach we identified markers of rifampicin tolerance to be high abundant in members of the M. tuberculosis Beijing genotype, already prior drug exposure. The identification of this M. tuberculosis Beijing specific trait will contribute to improved diagnostics and treatment of M. tuberculosis.
- Universal stress protein Rv2624c alters abundance of arginine and enhances intracellular survival by ATP binding in mycobacteria. [Journal Article]
- SRSci Rep 2016 Oct 20; 6:35462
- The universal stress protein family is a family of stress-induced proteins. Universal stress proteins affect latency and antibiotic resistance in mycobacteria. Here, we showed that Mycobacterium smeg...
The universal stress protein family is a family of stress-induced proteins. Universal stress proteins affect latency and antibiotic resistance in mycobacteria. Here, we showed that Mycobacterium smegmatis overexpressing M. tuberculosis universal stress protein Rv2624c exhibits increased survival in human monocyte THP-1 cells. Transcriptome analysis suggested that Rv2624c affects histidine metabolism, and arginine and proline metabolism. LC-MS/MS analysis showed that Rv2624c affects the abundance of arginine, a modulator of both mycobacteria and infected THP-1 cells. Biochemical analysis showed that Rv2624c is a nucleotide-binding universal stress protein, and an Rv2624c mutant incapable of binding ATP abrogated the growth advantage in THP-1 cells. Rv2624c may therefore modulate metabolic pathways in an ATP-dependent manner, changing the abundance of arginine and thus increasing survival in THP-1 cells.
- Identifying and Tackling Emergent Vulnerability in Drug-Resistant Mycobacteria. [Journal Article]
- AIACS Infect Dis 2016 Sep 9; 2(9):592-607
- The global mechanisms and associated molecular alterations that occur in drug-resistant mycobacteria are poorly understood. To address this, we obtain genomics data and then construct a genome-scale ...
The global mechanisms and associated molecular alterations that occur in drug-resistant mycobacteria are poorly understood. To address this, we obtain genomics data and then construct a genome-scale response network in isoniazid-resistant Mycobacterium smegmatis and apply a network-mining algorithm. Through this, we decipher global alterations in an unbiased manner and identify emergent vulnerabilities in resistant bacilli, of which redox response was prominent. Using phenotypic profiling, we find that resistant bacilli exhibit collateral sensitivity to several compounds that block antioxidant responses. We find that nanogram/milliliter concentrations of ebselen, vancomycin, and phenylarsine oxide, in combination with isoniazid, are highly effective against Mycobacterium tuberculosis H37Rv and three clinical drug-resistant strains. Dynamic measurements of cytoplasmic redox potential revealed a surprisingly diminished capacity of clinical drug-resistant strains to counteract oxidative stress, providing a mechanistic basis for efficient and synergistic mycobactericidal activity of the drug combinations. Ebselen and vancomycin appear to be promising repurposable drugs.
New Search Next
- Pyrazinamide Resistance Is Caused by Two Distinct Mechanisms: Prevention of Coenzyme A Depletion and Loss of Virulence Factor Synthesis. [Journal Article]
- AIACS Infect Dis 2016 Sep 9; 2(9):616-626
- Pyrazinamide (PZA) is a critical component of first- and second-line treatments of tuberculosis (TB), yet its mechanism of action largely remains an enigma. We carried out a genetic screen to isolate...
Pyrazinamide (PZA) is a critical component of first- and second-line treatments of tuberculosis (TB), yet its mechanism of action largely remains an enigma. We carried out a genetic screen to isolate Mycobacterium bovis BCG mutants resistant to pyrazinoic acid (POA), the bioactive derivative of PZA, followed by whole genome sequencing of 26 POA resistant strains. Rather than finding mutations in the proposed candidate targets fatty acid synthase I and ribosomal protein S1, we found resistance conferring mutations in two pathways: missense mutations in aspartate decarboxylase panD, involved in the synthesis of the essential acyl carrier coenzyme A (CoA), and frameshift mutations in the vitro nonessential polyketide synthase genes mas and ppsA-E, involved in the synthesis of the virulence factor phthiocerol dimycocerosate (PDIM). Probing for cross resistance to two structural analogs of POA, nicotinic acid and benzoic acid, showed that the analogs share the PDIM- but not the CoA-related mechanism of action with POA. We demonstrated that POA depletes CoA in wild-type bacteria, which is prevented by mutations in panD. Sequencing 10 POA-resistant Mycobacterium tuberculosis H37Rv isolates confirmed the presence of at least 2 distinct mechanisms of resistance to the drug. The emergence of resistance through the loss of a virulence factor in vitro may explain the lack of clear molecular patterns in PZA-resistant clinical isolates, other than mutations in the prodrug-converting enzyme. The apparent interference of POA with virulence pathways may contribute to the drug's excellent in vivo efficacy compared to its modest in vitro potency.