Analogous Mechanisms of Resistance to Benzothiazinones and Dinitrobenzamides in Mycobacterium smegmatis

Tuberculosis is still a leading cause of death worldwide. The selection and spread of Mycobacterium tuberculosis multidrug-resistant (MDR-TB) and extensively drug-resistant strains (XDR-TB) is a severe public health problem. Recently, two different classes of chemical series, the benzothiazinones (BTZ) and the dinitrobenzamide (DNB) derivatives have been found to be highly active against M. tuberculosis, including XDR-TB strains. The target of BTZs is DprE1 protein which works in concert with DprE2 to form the heteromeric decaprenylphosphoryl-β-D-ribose 2′-epimerase, involved in Decaprenyl-Phospho-Arabinose (DPA) biosynthesis. Interestingly, it has been shown that the DNBs block the same pathway thus suggesting that both drugs could share the same target. Moreover, in Mycobacterium smegmatis the overexpression of the NfnB nitroreductase led to the inactivation of the BTZs by reduction of a critical nitro-group to an amino-group. In this work several spontaneous M. smegmatis mutants resistant to DNBs were isolated. Sixteen mutants, showing high levels of DNB resistance, exhibited a mutation in the Cys394 of DprE1. Using fluorescence titration and mass spectrometry it has been possible to monitor the binding between DprE1 and DNBs, achieving direct evidence that MSMEG_6382 is the cellular target of DNBs in mycobacteria. Additionally, M. smegmatis mutants having low levels of resistance to DNBs harbor various mutations in MSMEG_6503 gene encoding the transcriptional repressor of the nitroreductase NfnB. By LC/MS2 analysis it has been demonstrated that NfnB is responsible for DNB inactivation. Taken together, our data demonstrate that both DNB and BTZ drugs share common resistance mechanisms in M. smegmatis

Analogous Mechanisms of Resistance to Benzothiazinones and Dinitrobenzamides in Mycobacterium Smegmatis

Tuberculosis is still a leading cause of death worldwide. The selection and spread of Mycobacterium tuberculosis multidrug-resistant (MDR-TB) and extensively drug-resistant strains (XDR-TB) is a severe public health problem. Recently, two different classes of chemical series, the benzothiazinones (BTZ) and the dinitrobenzamide (DNB) derivatives have been found to be highly active against M. tuberculosis, including XDR-TB strains. The target of BTZs is DprE1 protein which works in concert with DprE2 to form the heteromeric decaprenylphosphoryl-β-D-ribose 2\u27-epimerase, involved in Decaprenyl-Phospho-Arabinose (DPA) biosynthesis. Interestingly, it has been shown that the DNBs block the same pathway thus suggesting that both drugs could share the same target. Moreover, in Mycobacterium smegmatis the overexpression of the NfnB nitroreductase led to the inactivation of the BTZs by reduction of a critical nitro-group to an amino-group. In this work several spontaneous M. smegmatis mutants resistant to DNBs were isolated. Sixteen mutants, showing high levels of DNB resistance, exhibited a mutation in the Cys394 of DprE1. Using fluorescence titration and mass spectrometry it has been possible to monitor the binding between DprE1 and DNBs, achieving direct evidence that MSMEG_6382 is the cellular target of DNBs in mycobacteria. Additionally, M. smegmatis mutants having low levels of resistance to DNBs harbor various mutations in MSMEG_6503 gene encoding the transcriptional repressor of the nitroreductase NfnB. By LC/MS analysis it has been demonstrated that NfnB is responsible for DNB inactivation. Taken together, our data demonstrate that both DNB and BTZ drugs share common resistance mechanisms in M. smegmatis

High Content Screening Identifies Decaprenyl-Phosphoribose 2’ Epimerase as a Target for Intracellular Antimycobacterial Inhibitors

A critical feature of Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB), is its ability to survive and multiply within macrophages, making these host cells an ideal niche for persisting microbes. Killing the intracellular tubercle bacilli is a key requirement for efficient tuberculosis treatment, yet identifying potent inhibitors has been hampered by labor-intensive techniques and lack of validated targets. Here, we present the development of a phenotypic cell-based assay that uses automated confocal fluorescence microscopy for high throughput screening of chemicals that interfere with the replication of M. tuberculosis within macrophages. Screening a library of 57,000 small molecules led to the identification of 135 active compounds with potent intracellular anti-mycobacterial efficacy and no host cell toxicity. Among these, the dinitrobenzamide derivatives (DNB) showed high activity against M. tuberculosis, including extensively drug resistant (XDR) strains. More importantly, we demonstrate that incubation of M. tuberculosis with DNB inhibited the formation of both lipoarabinomannan and arabinogalactan, attributable to the inhibition of decaprenyl-phospho-arabinose synthesis catalyzed by the decaprenyl-phosphoribose 2\u27 epimerase DprE1/DprE2. Inhibition of this new target will likely contribute to new therapeutic solutions against emerging XDR-TB. Beyond validating the high throughput/content screening approach, our results open new avenues for finding the next generation of antimicrobials

High Content Screening Identifies Decaprenyl-Phosphoribose 2′ Epimerase as a Target for Intracellular Antimycobacterial Inhibitors

A critical feature of Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB), is its ability to survive and multiply within macrophages, making these host cells an ideal niche for persisting microbes. Killing the intracellular tubercle bacilli is a key requirement for efficient tuberculosis treatment, yet identifying potent inhibitors has been hampered by labor-intensive techniques and lack of validated targets. Here, we present the development of a phenotypic cell-based assay that uses automated confocal fluorescence microscopy for high throughput screening of chemicals that interfere with the replication of M. tuberculosis within macrophages. Screening a library of 57,000 small molecules led to the identification of 135 active compounds with potent intracellular anti-mycobacterial efficacy and no host cell toxicity. Among these, the dinitrobenzamide derivatives (DNB) showed high activity against M. tuberculosis, including extensively drug resistant (XDR) strains. More importantly, we demonstrate that incubation of M. tuberculosis with DNB inhibited the formation of both lipoarabinomannan and arabinogalactan, attributable to the inhibition of decaprenyl-phospho-arabinose synthesis catalyzed by the decaprenyl-phosphoribose 2′ epimerase DprE1/DprE2. Inhibition of this new target will likely contribute to new therapeutic solutions against emerging XDR-TB. Beyond validating the high throughput/content screening approach, our results open new avenues for finding the next generation of antimicrobials

High Content Phenotypic Cell-Based Visual Screen Identifies Mycobacterium tuberculosis Acyltrehalose-Containing Glycolipids Involved in Phagosome Remodeling

The ability of the tubercle bacillus to arrest phagosome maturation is considered one major mechanism that allows its survival within host macrophages. To identify mycobacterial genes involved in this process, we developed a high throughput phenotypic cell-based assay enabling individual sub-cellular analysis of over 11,000 Mycobacterium tuberculosis mutants. This very stringent assay makes use of fluorescent staining for intracellular acidic compartments, and automated confocal microscopy to quantitatively determine the intracellular localization of M. tuberculosis. We characterised the ten mutants that traffic most frequently into acidified compartments early after phagocytosis, suggesting that they had lost their ability to arrest phagosomal maturation. Molecular analysis of these mutants revealed mainly disruptions in genes involved in cell envelope biogenesis (fadD28), the ESX-1 secretion system (espL/Rv3880), molybdopterin biosynthesis (moaC1 and moaD1), as well as in genes from a novel locus, Rv1503c-Rv1506c. Most interestingly, the mutants in Rv1503c and Rv1506c were perturbed in the biosynthesis of acyltrehalose-containing glycolipids. Our results suggest that such glycolipids indeed play a critical role in the early intracellular fate of the tubercle bacillus. The unbiased approach developed here can be easily adapted for functional genomics study of intracellular pathogens, together with focused discovery of new anti-microbials

Transient Requirement of the PrrA-PrrB Two-Component System for Early Intracellular Multiplication of Mycobacterium tuberculosis

Adaptive regulation of gene expression in response to environmental changes is a general property of bacterial pathogens. By screening an ordered transposon mutagenesis library of Mycobacterium tuberculosis, we have identified three mutants containing a transposon in the coding sequence or in the 5′ regions of genes coding for two-component signal transduction systems (trcS, regX3, prrA). The intracellular multiplication capacity of the three mutants was investigated in mouse bone marrow-derived macrophages. Only the prrA mutant showed a defect in intracellular growth during the early phase of infection, and this defect was fully reverted when the mutant was complemented with prrA-prrB wild-type copies. The mutant phenotype was transient, as after 1 week this strain recovered full growth capacity to reach levels similar to that of the wild type at day 9. Moreover, a transient induction of prrA promoter activity was observed during the initial phase of macrophage infection, as shown by a prrA promoter-gfp fusion in M. bovis BCG infecting the mouse macrophages. The concordant transience of the prrA mutant phenotype and prrA promoter activity indicates that the PrrA-PrrB two-component system is involved in the environmental adaptation of M. tuberculosis, specifically in an early phase of the intracellular growth, and that, similar to other facultative intracellular parasites, M. tuberculosis can use genes temporarily required at different stages in the course of macrophage infection

High-Content Screening Technology Combined with a Human Granuloma Model as a New Approach To Evaluate the Activities of Drugs against Mycobacterium tuberculosis

International audienceTuberculosis remains a major health problem due to the emergence of drug-resistant strains of Mycobacterium tuberculosis. Some models have provided valuable information about drug resistance and efficacy; however, the translation of these results into effective human treatments has mostly proven unsuccessful. In this study, we adapted high-content screening (HCS) technology to investigate the activities of antitubercular compounds in the context of an in vitro granuloma model. We observed significant shifts in the MIC 50 s between the activities of the compounds under extracellular and granuloma conditions. T uberculosis (TB) is a respiratory disease that is one of the major causes of mortality and morbidity worldwide. Almost 20 people develop TB and four patients die from the disease every minute somewhere in the world (1). The disease can be cured by drug treatment involving a regimen of several drugs. The World Health Organization (WHO) estimates that there were around 0.5 million new cases of multidrug-resistant TB (MDR-TB) in 2012 (2, 3). A recent report confirmed that MDR-TB is a continuing worldwide health problem, even in high-income countries with a low incidence of TB (4). The immune system undoubtedly plays a major role in TB control (5). Signaling events of the immune system lead to the formation of a granuloma, the hallmark of TB. Granulomas are an immune microenvironment in which the infection can be controlled but also a niche in which bacilli can thrive and modulate immune responses to ensure their survival for long periods without causing damage (6, 7). Granulomas are cell aggregates that form tridimensional and heterogeneous structures (8, 9). We previously described an in vitro granuloma model involving human blood cells either infected with mycobacteria or incubated with mycobacterial antigens (8, 10). Either of these models resulted in the formation of a typical epithelioid granuloma with morphological characteristics and properties of cellular differentiation similar to those of natural granulomas (8, 10, 11). This model may help bridge the gap between the in vitro evaluation of MICs and costly in vivo efficacy studies in guinea pigs. In particular, compounds that are effective in the in vitro granuloma model could be prioritized for guinea pig studies. High-content screening (HCS) technology has been used to identify anti-TB compounds (12–14). This technology has several advantages over traditional phenotypic screening approaches. This technology has mostly been used in single-cell experiments, because it is adapted to identify and analyze images (12, 13); however , in this paper, we focus on the use of granulomas instead of single cells. In this report, we describe the development of a novel HCS method to evaluate the activities of reference compounds against a green fluorescent protein (GFP)-expressing H37Rv strain of Mycobacterium tuberculosis (MTB-GFP) within 5 days, and we compared the MIC 50 values with those of classical liquid cultures (extracellular), intracellular growth (single-cell assay), and within granulomas, all using the same bacterial strain. MTB-GFP strains (13) were grown for 3 weeks at 37°C until the log phase was reached. The test inoculum was prepared by diluting the culture with phosphate-buffered saline (PBS) medium or complete RPMI (RPMIc) medium (containing RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum [FBS], with 1% glutamine, 1% HEPES, 1% nonessential amino acids) to a final bacterial concentration of 1 ϫ 10 6 CFU/ml. In vitro human granuloma formation was performed by incu-bating peripheral blood mononuclear cells (PBMC) of healthy volunteers; the cells were purified by gradient density sedimenta-tion (15) with MTB-GFP using an appropriate multiplicity of infection in RPMIc medium at 37°C with low shaking (125 rpm) for 1 h; they were then washed with PBS containing 2% FBS and suspended in RPMIc medium at a concentration of 3.5 ϫ 10 5 cells per well in 384-well plates (Greiner). The cells were then incu-bated for 3 days at 37°C in a 5% CO 2 atmosphere to allow the formation of granulomas. Intragranuloma bacterial quantification was performed by fixing granulomas with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS and staining with Hoechst (Sigma); each step consisted of a 30-min incubation period at room temperature