Advanced Quantification Methods To Improve the 18b Dormancy Model for Assessing the Activity of Tuberculosis Drugs In Vitro.

One of the reasons for the lengthy tuberculosis (TB) treatment is the difficulty to treat the nonmultiplying mycobacterial subpopulation. In order to assess the ability of (new) TB drugs to target this subpopulation, we need to incorporate dormancy models in our preclinical drug development pipeline. In most available dormancy models, it takes a long time to create a dormant state, and it is difficult to identify and quantify this nonmultiplying condition. The Mycobacterium tuberculosis 18b strain might overcome some of these problems, because it is dependent on streptomycin for growth and becomes nonmultiplying after 10 days of streptomycin starvation but still can be cultured on streptomycin-supplemented culture plates. We developed our 18b dormancy time-kill kinetics model to assess the difference in the activity of isoniazid, rifampin, moxifloxacin, and bedaquiline against log-phase growth compared to the nonmultiplying M. tuberculosis subpopulation by CFU counting, including a novel area under the curve (AUC)-based approach as well as time-to-positivity (TTP) measurements. We observed that isoniazid and moxifloxacin were relatively more potent against replicating bacteria, while rifampin and high-dose bedaquiline were equally effective against both subpopulations. Moreover, the TTP data suggest that including a liquid culture-based method could be of additional value, as it identifies a specific mycobacterial subpopulation that is nonculturable on solid media. In conclusion, the results of our study underline that the time-kill kinetics 18b dormancy model in its current form is a useful tool to assess TB drug potency and thus has its place in the TB drug development pipeline.

Antimicrobial resistance in leprosy: results of the first prospective open survey conducted by a WHO surveillance network for the period 2009-15.

OBJECTIVES: Antimicrobial resistance (AMR) is a priority for surveillance in bacterial infections. For leprosy, AMR has not been assessed because Mycobacterium leprae does not grow in vitro. We aim to obtain AMR data using molecular detection of resistance genes and to conduct a prospective open survey of resistance to antileprosy drugs in countries where leprosy is endemic through a WHO surveillance network. METHODS: From 2009 to 2015, multi-bacillary leprosy cases at sentinel sites of 19 countries were studied for resistance to rifampicin, dapsone and ofloxacin by PCR sequencing of the drug-resistance-determining regions of the genes rpoB, folP1 and gyrA. RESULTS: Among 1932 (1143 relapse and 789 new) cases studied, 154 (8.0%) M. leprae strains were found with mutations conferring resistance showing 182 resistance traits (74 for rifampicin, 87 for dapsone and 21 for ofloxacin). Twenty cases showed rifampicin and dapsone resistance, four showed ofloxacin and dapsone resistance, but no cases were resistant to rifampicin and ofloxacin. Rifampicin resistance was observed among relapse (58/1143, 5.1%) and new (16/789, 2.0%) cases in 12 countries. India, Brazil and Colombia reported more than five rifampicin-resistant cases. CONCLUSIONS: This is the first study reporting global data on AMR in leprosy. Rifampicin resistance emerged, stressing the need for expansion of surveillance. This is also a call for vigilance on the global use of antimicrobial agents, because ofloxacin resistance probably developed in relation to the general intake of antibiotics for other infections as it is not part of the multidrug combination used to treat leprosy.

Mechanistic insight into mycobacterial MmpL protein function.

Mycobacterial cell walls are complex structures containing a broad range of unusual lipids, glycolipids and other polymers, some of which act as immunomodulators or virulence determinants. Better understanding of the enzymes involved in export processes would enlighten cell wall biogenesis. Bernut et al. () present the findings of a structural and functional investigation of one of the most important transporter families, the MmpL proteins, members of the resistance-nodulation-cell division (RND) superfamily. A Tyr842His missense mutation in the mmpL4a gene was shown to be responsible for the smooth-to-rough morphotype change of the near untreatable opportunistic pathogen Mycobacterium bolletii due to its failure to export a glycopeptidolipid (GPL). This mutation was pleiotropic and markedly increased virulence in infection models. Tyr842 is well conserved in all actinobacterial MmpL proteins suggesting that it is functionally important and this was confirmed by several approaches including replacing the corresponding residue in MmpL3 of Mycobacterium tuberculosis. Structural modelling combined with experimental results showed Tyr842 to be a critical residue for mediating the proton motive force required for GPL export. This mechanistic insight applies to all MmpL proteins and probably to all RND transporters.

Genome-wide re-sequencing of multidrug-resistant Mycobacterium leprae Airaku-3.

Genotyping and molecular characterization of drug resistance mechanisms in Mycobacterium leprae enables disease transmission and drug resistance trends to be monitored. In the present study, we performed genome-wide analysis of Airaku-3, a multidrug-resistant strain with an unknown mechanism of resistance to rifampicin. We identified 12 unique non-synonymous single-nucleotide polymorphisms (SNPs) including two in the transporter-encoding ctpC and ctpI genes. In addition, two SNPs were found that improve the resolution of SNP-based genotyping, particularly for Venezuelan and South East Asian strains of M. leprae.

Transcriptional regulation of lipid homeostasis in mycobacteria.

Mycolic acids are major components of the cell envelope of mycobacteria, such as Mycobacterium tuberculosis, and play an important role in its architecture, impermeability and interaction with the environment. Synthesis of mycolic acids is carried out by two types of fatty acid synthases (FAS) working in concert: type I FAS, a multifunctional enzyme capable of de novo synthesis of medium-chain fatty acids, and type II FAS, responsible for their elongation. In this article we report the identification and characterization of a transcriptional regulator (MabR), whose binding to the FAS-II promoter region was demonstrated in vitro and in vivo. Overexpression and knock-down studies in Mycobacterium smegmatis revealed the repressor nature of MabR, with reduced amounts of FAS-II transcripts and fatty acids in the overproducing strain. Under these conditions, downregulation of fas transcription was also observed, thereby suggesting the existence of cross-talk between the two FAS, mediated by MabR. Finally, the finding that a mabR knock-out mutant could only be obtained in a merodiploid strain of M. smegmatis, confirmed the predicted essentiality, thus implying an essential role for MabR in mycobacterial fatty acid metabolism.

Effect of serial subculturing on the genetic composition and cytotoxic activity of Mycobacterium tuberculosis.

Continuous subculture has been observed to produce changes in the virulence of micro-organisms, e.g. rabies virus, poliovirus and Mycobacterium bovis BCG. The latter has been used as a vaccine for tuberculosis for the last 100 years; however, in some instances its efficacy has been observed to be very low. In order to determine whether similar changes can be produced in Mycobacterium tuberculosis, we selected four isolates, M. tuberculosis H37Rv, a Beijing strain (DR-689), and two more isolates with deletion of the phospholipase C locus (plcA-plcB-plcC ), and subjected them to serial culturing on Middlebrook 7H9 medium, with or without ox bile. After 100 passages, we performed RFLP-IS6110 analysis to determine whether genomic changes were produced. We also checked their genomic composition by microarray analysis. Changes in virulence were studied by measuring the cytotoxic effect of parental and subcultured isolates on a THP-1 macrophage monolayer. The most visible change was the change of position of an IS6110 band of approximately 1400 bp to approximately 1600 bp in the Beijing isolate subcultured in the ox bile medium. Analysis by microarray and PCR confirmation did not reveal any genomic changes. Cytotoxic activity was decreased in the isolates at levels close to that of BCG, and more consistently in those subcultured in the presence of ox bile.

Towards new tuberculosis drugs.

The need for better drugs to treat tuberculosis has never been greater. Despite insufficient funding for discovery research, intensive efforts have been made to find and develop new lead compounds capable of reducing the duration of the present treatment known as DOTS (directly observed therapy short course), from 6 to under 4 months. This minireview describes the progress achieved during the last 5 years and highlights some of the successes without neglecting the problems.

Proteomic identification of M. tuberculosis protein kinase substrates: PknB recruits GarA, a FHA domain-containing protein, through activation loop-mediated interactions.

Genes for functional Ser/Thr protein kinases (STPKs) are ubiquitous in prokaryotic genomes, but little is known about their physiological substrates and their actual involvement in bacterial signal transduction pathways. We report here the identification of GarA (Rv1827), a Forkhead-associated (FHA) domain-containing protein, as a putative physiological substrate of PknB, an essential Ser/Thr protein kinase from Mycobacterium tuberculosis. Using a global proteomic approach, GarA was found to be the best detectable substrate of the PknB catalytic domain in non-denatured whole-cell protein extracts from M. tuberculosis and the saprophyte Mycobacterium smegmatis. Enzymological and binding studies of the recombinant proteins demonstrate that docking interactions between the activation loop of PknB and the C-terminal FHA domain of GarA are required to enable efficient phosphorylation at a single N-terminal threonine residue, Thr22, of the substrate. The predicted amino acid sequence of the garA gene, including both the N-terminal phosphorylation motif and the FHA domain, is strongly conserved in mycobacteria and other related actinomycetes, suggesting a functional role of GarA in putative STPK-mediated signal transduction pathways. The ensuing model of PknB-GarA interactions suggests a substrate recruitment mechanism that might apply to other mycobacterial kinases bearing multiple phosphorylation sites in their activation loops.

Comparative mycobacterial genomics as a tool for drug target and antigen discovery.

Genomics and the associated downstream technologies are generating vast data sets that provide new opportunities for understanding and combating both infectious and genetic diseases in humans. The genomic approach has been applied to tuberculosis, a major cause of transmissible morbidity and mortality, with notable success. Complete genome sequences are now available for three members of the Mycobacterium tuberculosis complex and the related intracellular pathogen M. leprae. Many of the predictions generated in silico by genomics have been validated through functional analysis, including studies of the transcriptome and proteome, and led to the identification of essential genes. Knowledge of the latter defines potential targets for new and existing drugs and their specificity can be assessed by comparative genomics with the host or other pathogens. Genomics is also furthering tuberculosis vaccine development by pinpointing potentially antigenic proteins as well as providing better diagnostic tools to detect infection.

Inactivation of the Mycobacterium tuberculosis Nramp orthologue (mntH) does not affect virulence in a mouse model of tuberculosis.

Mycobacterium tuberculosis is an intracellular pathogen which can survive and multiply within the phagosomal compartment of the macrophage, and in doing so has to withstand the various macrophage defense mechanisms, which include limitation of iron and other metals. Analysis of the complete genome sequence of M. tuberculosis revealed an extensive array of cation transporters, including mntH, an orthologue of the eukaryotic Nramp (natural resistance-associated macrophage protein) gene, that encodes a proton-dependent divalent metal transporter. To assess the effect of this transporter on intracellular survival and pathogenesis, an mntH knock-out mutant of M. tuberculosis H37Rv was created and assayed in bone marrow-derived macrophages and in a murine model of tuberculosis. In neither of these systems was any loss of fitness associated with inactivation of mntH, demonstrating that Nramp orthologues are not important determinants of mycobacterial virulence.

A new evolutionary scenario for the Mycobacterium tuberculosis complex.

The distribution of 20 variable regions resulting from insertion-deletion events in the genomes of the tubercle bacilli has been evaluated in a total of 100 strains of Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium canettii, Mycobacterium microti, and Mycobacterium bovis. This approach showed that the majority of these polymorphisms did not occur independently in the different strains of the M. tuberculosis complex but, rather, resulted from ancient, irreversible genetic events in common progenitor strains. Based on the presence or absence of an M. tuberculosis specific deletion (TbD1), M. tuberculosis strains can be divided into ancestral and "modern" strains, the latter comprising representatives of major epidemics like the Beijing, Haarlem, and African M. tuberculosis clusters. Furthermore, successive loss of DNA, reflected by region of difference 9 and other subsequent deletions, was identified for an evolutionary lineage represented by M. africanum, M. microti, and M. bovis that diverged from the progenitor of the present M. tuberculosis strains before TbD1 occurred. These findings contradict the often-presented hypothesis that M. tuberculosis, the etiological agent of human tuberculosis evolved from M. bovis, the agent of bovine disease. M. canettii and ancestral M. tuberculosis strains lack none of these deleted regions, and, therefore, seem to be direct descendants of tubercle bacilli that existed before the M. africanum-->M. bovis lineage separated from the M. tuberculosis lineage. This observation suggests that the common ancestor of the tubercle bacilli resembled M. tuberculosis or M. canettii and could well have been a human pathogen already.

The evolution of mycobacterial pathogenicity: clues from comparative genomics.

Comparative genomics, and related technologies, are helping to unravel the molecular basis of the pathogenesis, host range, evolution and phenotypic differences of the slow-growing mycobacteria. In the highly conserved Mycobacterium tuberculosis complex, where single-nucleotide polymorphisms are rare, insertion and deletion events (InDels) are the principal source of genome plasticity. InDels result from recombinational or insertion sequence (IS)-mediated events, expansion of repetitive DNA sequences, or replication errors based on repetitive motifs that remove blocks of genes or contract coding sequences. Comparative genomic analyses also suggest that loss of genes is part of the ongoing evolution of the slow-growing mycobacterial pathogens and might also explain how the vaccine strain BCG became attenuated.

Role of OxyS of Mycobacterium tuberculosis in oxidative stress: overexpression confers increased sensitivity to organic hydroperoxides.

Mycobacterial genomics has uncovered a novel regulatory gene, oxyS, belonging to the LysR family. There is extensive similarity in the DNA-binding domain of OxyS with that of OxyR, the oxidative stress response protein of many bacteria. Since the oxyR gene of Mycobacterium tuberculosis has been multiply inactivated during evolution it was conceivable that some of its functions could be effected by OxyS. It is shown here that OxyS is produced at low levels and that there are at least three different oxyS alleles present in clinical isolates of M. tuberculosis that are susceptible or resistant to isoniazid. Overproduction or depletion of OxyS did not affect susceptibility to isoniazid but increasing the concentration of the regulator lowered levels of the alkyl hydroperoxide reductase, AhpC, and rendered the tubercle bacillus more susceptible to organic hydroperoxides.

Genomics of Mycobacterium bovis.

The imminent completion of the genome sequence of Mycobacterium bovis will reveal the genetic blueprint for this most successful pathogen. Comparative analysis with the genome sequences of M. tuberculosis and M. bovis BCG promises to expose the genetic basis for the phenotypic differences between the tubercle bacilli, offering unparalleled insight into the virulence factors of the M. tuberculosis complex. Initial analysis of the sequence data has already revealed a novel deletion from M. bovis, as well as identifying variation in members of the PPE family of proteins. As the study of bacterial pathogenicity enters the postgenomic phase, the genome sequence of M. bovis promises to serve as a cornerstone of mycobacterial genetics.

Regulation of catalase-peroxidase (KatG) expression, isoniazid sensitivity and virulence by furA of Mycobacterium tuberculosis.

Mycobacterium tuberculosis has two genes for ferric uptake regulator orthologues, one of which, furA, is situated immediately upstream of katG encoding catalase-peroxidase, a major virulence factor that also activates the prodrug isoniazid. This association suggested that furA might regulate katG and other genes involved in pathogenesis. Transcript mapping showed katG to be expressed from a strong promoter, with consensus -10 and -35 elements, preceding furA. No promoter activity was demonstrated downstream of the furA start codon, using different gene reporter systems, indicating that furA and katG are co-transcribed from a common regulatory region. The respective roles of these two genes in the isoniazid susceptibility and virulence of M. tuberculosis were assessed by combinatorial complementation of a Delta(furA-katG) strain that is heavily attenuated in a mouse model of tuberculosis. In the absence of furA, katG was upregulated, cells became hypersensitive to isoniazid, and full virulence was restored, indicating that furA regulates the transcription of both genes. When furA alone was introduced into the Delta(furA-katG) mutant, survival in mouse lungs was moderately increased, suggesting that FurA could regulate genes, other than katG, that are involved in pathogenesis. These do not include the oxidative stress genes ahpC and sodA, or those for siderophore production.

Familial outbreak of disseminated multidrug-resistant tuberculosis and meningitis.

Rapidly progressive multidrug-resistant tuberculosis (MDR-TB) is well documented in human immunodeficiency virus (HIV) positive subjects, but it is not fully recognised in HIV-negative subjects in the familial environment. We report three cases of MDR-TB in three young HIV-negative subjects from the same family. All the patients showed signs of meningitis during the course of their disease, and in two cases a resistant strain of Mycobacterium tuberculosis was isolated in cerebrospinal fluid. Two of the three subjects died from neurological complications; the other was successful treated utilising both systemic and intrathecal therapy for tuberculous meningitis. By a retrospective analysis of DNA obtained from Lowenstein-Jensen cultures, the strains were confirmed as M. tuberculosis resistant to rifampicin and isoniazid, and were closely related in the two cases where specimens were available for analysis. The resistance was acquired in two patients initially infected with a susceptible strain; in the other patient, the resistance was present on the first sensitivity test for which results were available. This report demonstrates the high risk of fatality from MDR-TB for HIV-negative subjects in the absence of reliable early diagnostic and preventive tools. It also reinforces the concept that genetic susceptibility to M. tuberculosis may be an important factor in the clinical presentation and outcome of MDR-TB.

Massive gene decay in the leprosy bacillus.

Leprosy, a chronic human neurological disease, results from infection with the obligate intracellular pathogen Mycobacterium leprae, a close relative of the tubercle bacillus. Mycobacterium leprae has the longest doubling time of all known bacteria and has thwarted every effort at culture in the laboratory. Comparing the 3.27-megabase (Mb) genome sequence of an armadillo-derived Indian isolate of the leprosy bacillus with that of Mycobacterium tuberculosis (4.41 Mb) provides clear explanations for these properties and reveals an extreme case of reductive evolution. Less than half of the genome contains functional genes but pseudogenes, with intact counterparts in M. tuberculosis, abound. Genome downsizing and the current mosaic arrangement appear to have resulted from extensive recombination events between dispersed repetitive sequences. Gene deletion and decay have eliminated many important metabolic activities including siderophore production, part of the oxidative and most of the microaerophilic and anaerobic respiratory chains, and numerous catabolic systems and their regulatory circuits.