Microarray analysis of defined Mycobacterium tuberculosis populations using RNA amplification strategies

Here we describe two reproducible methods of bacterial RNA amplification that will allow previously intractable host-pathogen interactions during bacterial infection to be explored at the whole genome level by RNA profilin

Methionine sulfoximine resistance in Mycobacterium tuberculosis is due to a single nucleotide deletion resulting in increased expression of the major glutamine synthetase, GlnA1

We investigated the effect of methionine sulfoximine (MetSox), a potent inhibitor of glutamine synthetase, on Mycobacterium tuberculosis. M. tuberculosis encodes four glutamine synthetases, of which MetSox targets the type I enzyme encoded by glnA1. Trancriptional profiling revealed that glutamate synthetase (gltB) and a type II glutamine synthetase (glnA3) were induced after exposure to MetSox. In addition, we observed a high rate (10−5) of spontaneous resistance to MetSox. All resistant strains had a single-nucleotide deletion in the 5’ region of glnA1, and Western analysis revealed that GlnA1 expression was increased in resistant as compared with sensitive strains. These data show that M. tuberculosis can respond to the effect of MetSox inhibition either by up-regulation of GlnA3 or by GlnA1. The high frequency of resistance suggests that MetSox and other compounds specifically targeting GlnA1 are not likely to become successful anti-mycobacterial agents

cDNA-RNA subtractive hybridization reveals increased expression of mycocerosic acid synthase in intracellular Mycobacterium bovis BCG.

Identifying genes that are differentially expressed by Mycobacterium bovis BCG after phagocytosis by macrophages will facilitate the understanding of the molecular mechanisms of host cell-intracellular pathogen interactions. To identify such genes a cDNA-total RNA subtractive hybridization strategy has been used that circumvents the problems both of limited availability of bacterial RNA from models of infection and the high rRNA backgrounds in total bacterial RNA. The subtraction products were used to screen a high-density gridded Mycobacterium tuberculosis genomic library. Sequence data were obtained from 19 differential clones, five of which contained overlapping sequences for the gene encoding mycocerosic acid synthase (mas). Mas is an enzyme involved in the synthesis of multi-methylated long-chain fatty acids that are part of phthiocerol dimycocerosate, a major component of the complex mycobacterial cell wall. Northern blotting and primer extension data confirmed up-regulation of mas in intracellular mycobacteria and also revealed a putative extended -10 promoter structure and a long untranslated upstream region 5' of the mas transcripts, containing predicted double-stranded structures. Furthermore, clones containing overlapping sequences for furB, groEL-2, rplE and fadD28 were identified and the up-regulation of these genes was confirmed by Northern blot analysis. The cDNA-RNA subtractive hybridization enrichment and high density gridded library screening, combined with selective extraction of bacterial mRNA represents a valuable approach to the identification of genes expressed during intra-macrophage residence for bacteria such as M. bovis BCG and the pathogenic mycobacterium, M. tuberculosis

Mycobacterium tuberculosis Expresses a Novel Ph-Dependent Divalent Cation Transporter Belonging to the Nramp Family

Mammalian natural resistance–associated macrophage protein (Nramp) homologues are important determinants of susceptibility to infection by diverse intracellular pathogens including mycobacteria. Eukaryotic Nramp homologues transport divalent cations such as Fe2+, Mn2+, Zn2+, and Cu2+. Mycobacterium tuberculosis and Mycobacterium bovis (bacillus Calmette-Guérin [BCG]) also encode an Nramp homologue (Mramp)

Examining the basis of isoniazid tolerance in nonreplicating Mycobacterium tuberculosis using transcriptional profiling

Background: Understanding how growth state influences Mycobacterium tuberculosis responses to antibiotic exposure provides a window into drug action during patient chemotherapy. In this article, we describe the transcriptional programs mediated by isoniazid (INH) during the transition from log-phase to nonreplicating bacilli, from INH-sensitive to INH-tolerant bacilli respectively, using the Wayne model. Results: INH treatment did not elicit a transcriptional response from nonreplicating bacteria under microarophilic conditions (NRP2), unlike the induction of a robust and well-characterized INH signature in log-phase bacilli. Conclusion: The differential regulation (between drug-free NRP2 and log-phase bacilli) of genes directly implicated in INH resistance could not account for the abrogation of INH killing in nongrowing bacilli. Thus, factors affecting the requirement for mycolic acids and the redox status of bacilli are likely responsible for the reduction in INH efficacy. We speculate on additional mechanisms revealed by transcriptome analysis that might account for INH tolerance

Antimicrobial treatment improves mycobacterial survival in nonpermissive growth conditions

Antimicrobials targeting cell wall biosynthesis are generally considered inactive against nonreplicating bacteria. Paradoxically, we found that under nonpermissive growth conditions, exposure of Mycobacterium bovis BCG bacilli to such antimicrobials enhanced their survival. We identified a transcriptional regulator, RaaS (for regulator of antimicrobial-assisted survival), encoded by bcg1279 (rv1219c) as being responsible for the observed phenomenon. Induction of this transcriptional regulator resulted in reduced expression of specific ATP-dependent efflux pumps and promoted long-term survival of mycobacteria, while its deletion accelerated bacterial death under nonpermissive growth conditions in vitro and during macrophage or mouse infection. These findings have implications for the design of antimicrobial drug combination therapies for persistent infectious diseases, such as tuberculosis

The KRESCENT Program (2005-2015) : an evaluation of the state of Kidney Research Training in Canada

Background: The Kidney Research Scientist Core Education and National Training (KRESCENT) Program was launched in 2005 to enhance kidney research capacity in Canada and foster knowledge translation across the 4 themes of health research. Objective: To evaluate the impact of KRESCENT on its major objectives and on the careers of trainees after its first 10 years. Methods: An online survey of trainees (n = 53) who had completed or were enrolled in KRESCENT was conducted in 2015. Information was also obtained from curriculum vitae (CVs). A bibliometric analysis assessed scientific productivity, collaboration, and impact in comparison with unsuccessful applicants to KRESCENT over the same period. The analysis included a comparison of Canadian with international kidney research metrics from 2000 to 2014. Results: Thirty-nine KRESCENT trainees completed the survey (74%), and 44 trainees (83%) submitted CVs. KRESCENT trainees had a high success rate at obtaining grant funding from the Canadian Institutes of Health Research (CIHR; 79%), and 76% of Post-Doctoral Fellows received academic appointments at the Assistant Professor level within 8 months of completing training. The majority of trainees reported that KRESCENT had contributed significantly to their success in securing CIHR funding (90%), and to the creation of knowledge (93%) and development of new methodologies (50%). Bibliometric analysis revealed a small but steady decline in total international kidney research output from 2000 to 2014, as a percentage of all health research, although overall impact of kidney research in Canada increased from 2000-2005 to 2009- 2014 compared with other countries. KRESCENT trainees demonstrated increased productivity, multiauthored papers, impact, and international collaborations after their training, compared with nonfunded applicants. Conclusions: The KRESCENT Program has fostered kidney research career development and contributed to increased capacity, productivity, and collaboration. To further enhance knowledge creation and translation in kidney research in Canada, programs such as KRESCENT should be sustained via long-term funding partnerships.Mise en contexte: Le programme KRESCENT (Kidney Research Scientist Core Education and National Training) a été lancé en 2005 pour augmenter la capacité de la recherche sur les maladies du rein à travers le Canada, et pour encourager la transmission des connaissances au sein des quatre axes de recherche en santé. Objectifs de l’étude: Cette étude avait pour but d’évaluer les répercussions du programme KRESCENT sur ses principaux objectifs ainsi que des retombées sur la carrière des stagiaires participants, dix ans après sa création. Méthodologie: Un sondage en ligne a été mené en 2015 auprès des stagiaires (n = 53) ayant été admis ou ayant complété le programme KRESCENT. Des renseignements ont également été obtenus par la consultation de curriculum vitae (CV). Une analyse bibliométrique a évalué la productivité scientifique et la collaboration des participants ainsi que les répercussions de leur participation à KRESCENT sur leur carrière. Les données de cette analyse ont été comparées à celles des candidats n’ayant pas été retenus au cours de la même période. L’analyse comprenait également une comparaison des données canadiennes avec celles obtenues en recherche sur les maladies du rein ailleurs dans le monde

A Novel Microfluidic Dielectrophoresis Technology to Enable Rapid Diagnosis of Mycobacteria tuberculosis in Clinical Samples

To achieve the global efforts to end tuberculosis, affordable diagnostics suitable for true point-of-care implementation are required to reach the missing millions. In addition, diagnostics with increased sensitivity and expanded drug susceptibility testing are needed to address drug resistance and to diagnose low-bacterial burden cases. The laboratory-on-a-chip technology described herein used dielectrophoresis to selectively isolate Mycobacterium tuberculosis from sputum samples, purifying the bacterial population ahead of molecular confirmation by multiplex real-time quantitative PCR. After optimization using a panel of 50 characterized sputum samples, the performance of the prototype was assessed against the current gold standards, screening 100 blinded sputum samples using characterized and biobanked sputum provided by Foundation for Innovative New Diagnostics. Concordance with culture diagnosis was 100% for smear-negative samples and 87% for smear-positive samples. Of the smear-positive samples, the high burden sample concordance was 100%. Samples were diagnosed on the basis of visual assessment of the dielectrophoresis array and by multiplex real-time quantitative PCR assay. The results described herein demonstrate the potential of the CAPTURE-XT technology to provide a powerful sample preparation tool that could function as a front-end platform for molecular detection. This versatile tool could equally be applied as a visual detection diagnostic, potentially associated with bacterial identification for low-cost screening or coupled with an expanded PCR assay for genotypic drug susceptibility testing