Effects of low incubation temperatures on the bactericidal activity of anti-tuberculosis drugs

OBJECTIVES: to explore the effect of low incubation temperatures and the consequent slowing of bacterial metabolism on the bactericidal action of anti-tuberculosis drugs against Mycobacterium tuberculosis. METHODS: counting of surviving bacteria during exposure of static cultures to 1 mg/L isoniazid, 2 mg/L rifampicin, 0.5 or 2 mg/L TMC207 and 40 or 160 mg/L pyrazinamide, usually for periods of 21 days at temperatures of 37, 25, 22, 19, 16 or 8°C. RESULTS: the bactericidal activities of isoniazid and rifampicin were progressively reduced at 25 and 22°C, and were minimal at lower temperatures. TMC207 was immediately bactericidal at 37°C, in contrast to the early static phase reported with log phase cultures, and showed less change in activity as incubation temperatures were reduced than did rifampicin or isoniazid. Pyrazinamide was more bactericidal when incubation temperatures were reduced below 37°C and when the static seed cultures were most dormant. CONCLUSIONS: these results can be explained by the surmise that at low temperatures bacterial energy is at a low level with only just sufficient ATP to maintain homeostasis, making the bacteria more susceptible to the blocking of ATP synthesis by TMC207. Insufficient ATP at low temperature would also hinder the export of pyrazinoic acid, the toxic product of the pro-drug pyrazinamide, from the mycobacterial cell by an inefficient efflux pump that requires energ

Adjusting to a new home: Mycobacterium tuberculosis gene expression in response to an intracellular lifestyle.

Mycobacterium tuberculosis remains the most significant single species of bacteria causing disease in mankind. The ability of M. tuberculosis to survive and replicate within host macrophages is a pivotal step in its pathogenesis. Understanding the microenvironments that M. tuberculosis encounters within the macrophage and the adaptations that the bacterium undergoes to facilitate its survival will lead to insights into possible therapeutic targets for improved treatment of tuberculosis. This is urgently needed with the emergence of multi- and extensively drug resistant strains of M. tuberculosis. Significant advances have been made in understanding the macrophage response on encountering M. tuberculosis. Complementary information is also accumulating regarding the counter responses of M. tuberculosis during the various stages of its interactions with the host. As such, a picture is emerging delineating the gene expression of intracellular M. tuberculosis at different stages of the interaction with macrophages

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

Dissecting interferon-induced transcriptional programs in human peripheral blood cells

Interferons are key modulators of the immune system, and are central to the control of many diseases. The response of immune cells to stimuli in complex populations is the product of direct and indirect effects, and of homotypic and heterotypic cell interactions. Dissecting the global transcriptional profiles of immune cell populations may provide insights into this regulatory interplay. The host transcriptional response may also be useful in discriminating between disease states, and in understanding pathophysiology. The transcriptional programs of cell populations in health therefore provide a paradigm for deconvoluting disease-associated gene expression profiles.We used human cDNA microarrays to (1) compare the gene expression programs in human peripheral blood mononuclear cells (PBMCs) elicited by 6 major mediators of the immune response: interferons alpha, beta, omega and gamma, IL12 and TNFalpha; and (2) characterize the transcriptional responses of purified immune cell populations (CD4+ and CD8+ T cells, B cells, NK cells and monocytes) to IFNgamma stimulation. We defined a highly stereotyped response to type I interferons, while responses to IFNgamma and IL12 were largely restricted to a subset of type I interferon-inducible genes. TNFalpha stimulation resulted in a distinct pattern of gene expression. Cell type-specific transcriptional programs were identified, highlighting the pronounced response of monocytes to IFNgamma, and emergent properties associated with IFN-mediated activation of mixed cell populations. This information provides a detailed view of cellular activation by immune mediators, and contributes an interpretive framework for the definition of host immune responses in a variety of disease settings

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

Multi-omics technologies applied to tuberculosis drug discovery

Multi-omics strategies are indispensable tools in the search for new anti-tuberculosis drugs. Omics methodologies, where the ensemble of a class of biological molecules are measured and evaluated together, enable drug discovery programs to answer two fundamental questions. Firstly, in a discovery biology approach, to find new targets in druggable pathways for target-based investigation, advancing from target to lead compound. Secondly, in a discovery chemistry approach, to identify the mode of action of lead compounds derived from high-throughput screens, progressing from compound to target. The advantage of multi-omics methodologies in both of these settings is that omics approaches are unsupervised and unbiased to a priori hypotheses, making omics useful tools to confirm drug action, reveal new insights into compound activity, and discover new avenues for inquiry. This review summarizes the application of Mycobacterium tuberculosis omics technologies to the early stages of tuberculosis antimicrobial drug discovery

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

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

Design, synthesis and biological evaluation of a new series of carvedilol derivatives that protect sensory hair cells from aminoglycoside-induced damage by blocking the mechanoelectrical transducer channel

Aminoglycosides (AGs) are broad-spectrum antibiotics used for the treatment of serious bacterial infections but have use-limiting side effects including irreversible hearing loss. Here, we assessed the otoprotective profile of carvedilol in mouse cochlear cultures and in vivo zebrafish assays and investigated its mechanism of protection which, we found, may be mediated by a block of the hair cell’s mechanoelectrical transducer (MET) channel, the major entry route for the AGs. To understand the full otoprotective potential of carvedilol, a series of 18 analogues were prepared and evaluated for their effect against AG-induced damage as well as their affinity for the MET channel. One derivative was found to confer greater protection than carvedilol itself in cochlear cultures and also to bind more tightly to the MET channel. At higher concentrations, both carvedilol and this derivative were toxic in cochlear cultures but not in zebrafish, suggesting a good therapeutic window under in vivo conditions