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Pathogenic mycobacteria have the ability to persist in phagocytic cells and to suppress the immune system. The glycolipid lipoarabinomannan (LAM), in particular its mannose cap, has been shown to inhibit phagolysosome fusion and to induce immunosuppressive IL−10 production via interaction with the mannose receptor or DC-SIGN. Hence, the current paradigm is that the mannose cap of LAM is a crucial factor in mycobacterial virulence. However, the above studies were performed with purified LAM, never with live bacteria. Here we evaluate the biological properties of capless mutants of Mycobacterium marinum and M. bovis BCG, made by inactivating homologues of Rv1635c. We show that its gene product is an undecaprenyl phosphomannose-dependent mannosyltransferase. Compared with parent strain, capless M. marinum induced slightly less uptake by and slightly more phagolysosome fusion in infected macrophages but this did not lead to decreased survival of the bacteria in vitro, nor in vivo in zebra fish. Loss of caps in M. bovis BCG resulted in a sometimes decreased binding to human dendritic cells or DC-SIGN-transfected Raji cells, but no differences in IL-10 induction were observed. In mice, capless M. bovis BCG did not survive less well in lung, spleen or liver and induced a similar cytokine profile. Our data contradict the current paradigm and demonstrate that mannose-capped LAM does not dominate the Mycobacterium–host interaction. [ABSTRACT FROM AUTHOR]

Histidine-requiring auxotrophs of Mycobacterium smegmatis were isolated following W-methyl-W-nitro-N'-nitrosoguanidine treatment. One of these mutants, his5, was transformed with an M. smegmatis shuttle cosmid library, and complementing clones were isolated at a frequency of approximately 1%. A 2.3 kb fragment was subcloned and sequenced, and found to contain the start of an operon including the hisD gene and part of the hisC gene. No hisG gene was detected upstream of hisD, suggesting that the regulation of histidine biosynthesis in mycobacteria may differ from that of Escherichia coli. The strategy used here will allow the molecular genetics of complex mycobacterial-specific biosynthetic pathways involved in the virulence of pathogenic species to be studied. [ABSTRACT FROM AUTHOR]

Despite the great diversity of malonate semialdehyde decarboxylases (MSADs), one of five subgroups of the tautomerase superfamily (TSF) found throughout the biosphere, their distribution among strains within the genus Mycobacterium remains unknown. In this study, we sought to investigate the phylogenetic distribution of MSAD genes of mycobacterial species via genome analysis of 192 different reference Mycobacterium species or subspecies retrieved from NCBI databases. We found that in a total of 87 of 192 strains (45.3%), MSAD-1 and MSAD-2 were distributed in an exclusive manner among Mycobacterium species except for 12 strains, including Mycobacterium chelonae members, with both in their genome. Of note, Mycobacterium strains better adapted to the host and of high virulence potential, such as the Mycobacterium tuberculosis complex, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium ulcerans, and Mycobacterium avium subsp. paratuberculosis, had no orthologs of MSAD in their genome, suggesting MSAD loss during species differentiation in pathogenic slow growing Mycobacterium. To investigate the MSAD distribution among strains of M. avium subspecies, the genome sequences of a total of 255 reference strains from the four subspecies of M. avium (43 of subspecies avium, 162 of subspecies hominissuis, 49 of subspecies paratuberculosis, and 1 of subspecies silvaticum) were further analyzed. We found that only 121 of 255 strains (47.4%) had MSADs in their genome, with none of the 49 M. avium subsp. paratuberculosis strains having MSAD genes. Even in 13 of 121 M. avium strains with the MSAD-1 gene in their genome, deletion mutations in the 98th codon causing premature termination of MSAD were found, further highlighting the occurrence of MSAD pseudogenization during species or subspecies differentiation of M. avium. In conclusion, our data indicated that there are two distinct types of MSADs, MSAD1 and MSAD-2, among strains in the Mycobacterium genus, but more than half of the strains, including pathogenic mycobacteria, M. tuberculosis and M. leprae, have no orthologs in their genome, suggesting MSAD loss during host adaptation of pathogenic mycobacteria. In the future, the role of two distinct MSADs, MSAD-1 and MSAD-2, in mycobacterial pathogenesis or evolution should be investigated. [ABSTRACT FROM AUTHOR]

The specific features of the reactivation of "non-culturable" (NC) Mycobacterium tuberculosis (MTB) cells for the following multiplication in liquid medium were defined, as well as the approach for the quantification of these cells by culture methods was suggested. When NC cells obtained in vitro were inoculated into the standard Sauton's liquid medium, a prolonged (up to 20-day) lag phase was observed, in which no cell multiplication was occurred. During the lag phase, dormant cells secreted substances of an unknown nature that inhibited or completely arrested the reactivation and growth of cultures when the initial dormant cell concentration was above 107–108 cells/mL. Addition of meat-peptone broth (3.25 g/L) into a standard Sauton's medium with a 10-fold reduced concentration of glycerol significantly stimulated the reactivation and multiplication of the initially dormant cells inoculated at a concentration above the threshold. This modification of the medium made it possible to detect up to 103 times more cells in the MTB population by the method of the Most Probable Number (MPN) of cells. An increased number of the detected dormant cells in the population (up to an average of 2.5 orders of magnitude) was also achieved by adding RpfB recombinant protein (5 ng/mL) (a protein of the Rpf family, resuscitation promoting factor of dormant bacteria) to the reactivation medium. Perhaps, the action of the Rpf enzyme is related to the products of its enzymatic activity, since an increased MPN value in the dormant cells population was also observed when the products of mycobacteria peptidoglycan hydrolysis obtained by the coaction of RpfB and endopeptidase RipA were added. The addition of sonicated peptidoglycan fragments at a concentration up to 1 μg/mL had a similar effect. These results may be used in approaches to optimize the liquid media composition and culture conditions to identify the pathogen of tuberculosis in clinical samples in a nonculturable state. [ABSTRACT FROM AUTHOR]

Mycobacteria are members of the Actinomycetales order, and they are classified into one family, Mycobacteriaceae. More than 20 mycobacterial species cause disease in humans. The Mycobacterium group, called the Mycobacterium tuberculosis complex (MTBC), has nine closely related species that cause tuberculosis in animals and humans. TB can be detected worldwide and one-fourth of the world's population is contaminated with tuberculosis. According to the WHO, about two million dies from it, and more than nine million people are newly infected with TB each year. Mycobacterium tuberculosis (M. tuberculosis) is the most potential causative agent of tuberculosis and prompts enormous mortality and morbidity worldwide due to the incompletely understood pathogenesis of human tuberculosis. Moreover, modern diagnostic approaches for human tuberculosis are inefficient and have many lacks, while MTBC species can modulate host immune response and escape host immune attacks to sustain in the human body. "Multi-omics" strategies such as genomics, transcriptomics, proteomics, metabolomics, and deep sequencing technologies could be a comprehensive strategy to investigate the pathogenesis of mycobacterial species in humans and offer significant discovery to find out biomarkers at the early stage of disease in the host. Thus, in this review, we attempt to understand an overview of the mission of "omics" approaches in mycobacterial pathogenesis, including tuberculosis, leprosy, and other mycobacterial diseases. [ABSTRACT FROM AUTHOR]

Non-tuberculosis mycobacteria (NTMs) comprise a large group of organisms that are phenotypically diverse. Analysis of the growing number of completed NTM genomes has revealed both significant intra-genus genetic diversity, and a high percentage of predicted genes that appear to be unique to this group. Most NTMs have not been studied, however, the rise in NTM infections in several countries has prompted increasing interest in these organisms. Mycobacterial research has recently benefitted from the development of new genetic tools and a growing number of studies describing the genetic manipulation of NTMs have now been reported. In this review, we discuss the use of both site-specific and random mutagenesis tools in NTMs, highlighting the challenges that exist in applying these techniques to this diverse group of organisms. [ABSTRACT FROM AUTHOR]

A greater understanding of the genes involved in antibiotic resistance in Mycobacterium tuberculosis (Mtb) is necessary for the design of improved therapies. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been previously utilized in mycobacteria to identify novel drug targets by the demonstration of gene essentiality. The work presented here shows that it can also be usefully applied to the study of non-essential genes involved in antibiotic resistance. The expression of an ADP-ribosyltransferase (Arr) involved in rifampicin resistance in Mycobacterium smegmatis was silenced using CRISPRi and the impact on rifampicin susceptibility was measured. Gene silencing resulted in a decrease in the minimum inhibitory concentration (MIC) similar to that previously reported in an arr deletion mutant. There is contradictory evidence for the toxicity of Streptococcus pyogenes dCas9 (dCas9Spy) in the literature. In this study the expression of dCas9Spy in M. smegmatis showed no impact on viability. Silencing was achieved with concentrations of the aTc inducer lower than previously described and with shorter induction times. Finally, designing small guide RNAs (sgRNAs) that target transcription initiation, or the early stages of elongation had the most impact on rifampicin susceptibility. This study demonstrates that CRISPRi based gene silencing can be as impactful as gene deletion for the study of non-essential genes and further contributes to the knowledge on the design and induction of sgRNAs for CRISPRi. This approach can be applied to other non-essential antimicrobial resistance genes such as drug efflux pumps. [ABSTRACT FROM AUTHOR]

Multi-drug resistant tuberculosis (MDR-TB) represents a major health problem worldwide. Drug efflux and the activity of efflux transporters likely play important roles in the development of drug-tolerant and drug-resistant mycobacterial phenotypes. This study is focused on the action of a mycobacterial efflux pump as a mechanism of drug resistance. Previous studies demonstrated up-regulation of the TetR-like transcriptional regulator MSMEG_3765 in Mycobacterium smegmatis and its ortholog Rv1685c in Mycobacterium tuberculosis (Mtb) in acid-nitrosative stress conditions. MSMEG-3765 regulates the expression of the MSMEG_3762/63/65 operon, and of the orthologous region in Mtb (Rv1687c/86c/85c). MSMEG-3762 and Rv1687c are annotated as ATP-binding proteins, while MSMEG-3763 and Rv1686c are annotated as trans-membrane polypeptides, defining an ABC efflux pump in both M. smegmatis and Mtb. The two putative efflux systems share a high percentage of identity. To examine the role of the putative efflux system MSMEG-3762/63, we constructed and characterized a MSMEG-3763 deletion mutant in M. smegmatis (∆ MSMEG_3763). By comparative analysis of wild type, knockout, and complemented strains, together with structural modeling and molecular docking bioinformatics analyses of the MSMEG-3763 trans-membrane protein, we define the protein complex MSMEG-3762/63 as an efflux pump. Moreover, we demonstrate involvement of this pump in biofilm development and in the extrusion of rifampicin and ciprofloxacin (CIP), antimicrobial drugs used in first- and second-line anti-TB therapies. [ABSTRACT FROM AUTHOR]

Bacteria undergo adaptive morphological changes to survive under stress conditions. The present work documents the morphological changes in Mycobacterium tuberculosis (Mtb) cells cultured under hypoxic condition using Wayne's in vitro hypoxia model involving non-replicating persistence stages 1 and 2 (NRP stage 1 and NRP stage 2) and reveals their physiological significance. Transmission electron microscopy of the NRP stage 2 cells showed uneven but thick outer layer (TOL), unlike the evenly thin outer layer of the actively growing mid-log phase (MLP) cells. On the contrary, the saprophytic Mycobacterium smegmatis NRP stage 2 cells lacked TOL. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) of the Mtb NRP stage 2 cells confirmed the rough uneven surface unlike the smooth surface of the MLP cells. Zeta potential measurements showed high negative charge on the surface of NRP stage 2 cells and polysaccharide specific calcofluor white (CFW) staining of the cells revealed high content of polysaccharide in the TOL. This observation was supported by the real-time PCR data showing high levels of expression of the genes involved in the synthesis of sugars, such as trehalose, mannose and others, which are implicated in polysaccharide synthesis. Experiments to understand the physiological significance of the TOL revealed restricted entry of the biologically low-active 5-carboxyfluorescein-rifampicin (5-FAM-RIF), at concentrations equivalent to microbicidal concentrations of the unconjugated biologically active rifampicin, into the NRP stage 2 cells, unlike in the MLP cells. Further, as expected, mechanical removal of the TOL by mild bead beating or release of the NRP stage 2 cells from hypoxia into normoxia in fresh growth medium also significantly increased 5-FAM-RIF permeability into the NRP stage 2 cells to an extent comparable to that into the MLP cells. Taken together, these observations revealed that Mtb cells under hypoxia develop TOL that helps in restricting rifampicin entry, thereby conferring rifampicin tolerance. [ABSTRACT FROM AUTHOR]

The Mce systems are complex ABC transporters that are encoded by different numbers of homologous operons in Actinobacteria. While the four Mce systems of Mycobacterium tuberculosis are all energized by a single ATPase, MceG, each system appears to import different fatty acids or sterols. To explore if this behaviour can be extended to saprophytic mycobacteria, whose more complex genomes encode more Mce systems, we have identified and characterized the MceG orthologue of Mycobacterium smegmatis. This bacterium relies on MceG to energize its six Mce systems that contribute to a variety of cellular functions including sterol uptake and cell envelope maintenance. In the absence of MceG, M. smegmatis was not able to utilize cholesterol or phytosterols as carbon sources implying that this ATPase is necessary to energize the Mce4-sterol transport system. Other phenotypic alterations observed in the ΔMceG mutant, such as cell envelope modifications, suggest a pleiotropic functionality of the Mce systems that are particularly important for stress responses. Several ΔMceG phenotypes were recapitulated in a strain lacking only the unique C-terminal region of MceG, suggesting an important functional or regulatory function for this domain. [ABSTRACT FROM AUTHOR]

Kdp-ATPase is an inducible high affinity potassium uptake system that is widely distributed in bacteria, and is generally regulated by the KdpD/KdpE two-component system (TCS). In this study, conducted on Mycobacterium smegmatis, the kdpFABC (encoding Kdp-ATPase) expression was found to be affected by low concentration of K+, high concentrations of Na+, and/or NH+ 4 of the medium. The KdpE was found to be a transcriptional regulator that bound to a specific 22-bp sequence in the promoter region of kdpFABC operon to positively regulate kdpFABC expression. The KdpE binding motif was highly conserved in the promoters of kdpFABC among the mycobacterial species. 5'-RACE data indicated a transcriptional start site (TSS) of the kdpFABC operon within the coding sequence ofMSMEG_5391, which comprised a 120-bp long 5'-UTR and an open reading frame of the 87-bp kdpF gene. The kdpE deletion resulted in altered growth rate under normal and low K+ conditions. Furthermore, under K+ limiting conditions, a single transcript (kdpFABCDE) spanning kdpFABC and kdpDE operons was observed. This study provided the first insight into the regulation of kdpFABC operon by the KdpD/KdpE TCS in M. smegmatis. [ABSTRACT FROM AUTHOR]

Mycobacterium smegmatis strain MC2 155 is an attractive model organism for the study of M. tuberculosis and other mycobacterial pathogens, as it can grow well using cholesterol as a carbon resource. However, its global transcriptomic response remains largely unrevealed. In this study, M. smegmatis MC2 155 cultivated in androstenedione, cholesterol and glycerol supplemented media were collected separately for a RNA-Sequencing study. The results showed that 6004, 6681 and 6348 genes were expressed in androstenedione, cholesterol and glycerol supplemented media, and 5891 genes were expressed in all three conditions, with 237 specially expressed in cholesterol added medium. A total of 1852 and 454 genes were significantly up-regulated by cholesterol compared with the other two supplements. Only occasional changes were observed in basic carbon and nitrogen metabolism, while almost all of the genes involved in cholesterol catabolism and mammalian cell entry (MCE) were up-regulated by cholesterol, but not by androstenedione. Eleven and 16 gene clusters were induced by cholesterol when compared with glycerol or androstenedione, respectively. This study provides a comprehensive analysis of the cholesterol responsive transcriptome of M. smegmatis. Our results indicated that cholesterol induced many more genes and increased the expression of the majority of genes involved in cholesterol degradation and MCE in M. smegmatis, while androstenedione did not have the same effect. [ABSTRACT FROM AUTHOR]

Objectives The current method for testing new drugs against tuberculosis in vivo is the enumeration of bacteria in organs by cfu assay. Owing to the slow growth rate of Mycobacterium tuberculosis (Mtb), these assays can take months to complete. Our aim was to develop a more efficient, fluorescence-based imaging assay to test new antibiotics in a mouse model using Mtb reporter strains. Methods A commercial IVIS Kinetic® system and a custom-built laser scanning system with fluorescence molecular tomography (FMT) capability were used to detect fluorescent Mtb in living mice and lungs ex vivo. The resulting images were analysed and the fluorescence was correlated with data from cfu assays. Results We have shown that fluorescent Mtb can be visualized in the lungs of living mice at a detection limit of ∼8 × 107 cfu/lung, whilst in lungs ex vivo a detection limit of ∼2 × 105 cfu/lung was found. These numbers were comparable between the two imaging systems. Ex vivo lung fluorescence correlated to numbers of bacteria in tissue, and the effect of treatment of mice with the antibiotic moxifloxacin could be visualized and quantified after only 9 days through fluorescence measurements, and was confirmed by cfu assays. Conclusions We have developed a new and efficient method for anti-tuberculosis drug testing in vivo, based on fluorescent Mtb reporter strains. Using this method instead of, or together with, cfu assays will reduce the time required to assess the preclinical efficacy of new drugs in animal models and enhance the progress of these candidates into clinical trials against human tuberculosis. [ABSTRACT FROM PUBLISHER]

Mycobacteria, including most of all MTB (Mycobacterium tuberculosis), cause pathogenic infections in humans and, during the infectious process, are exposed to a range of environmental insults, including the host's immune response. From the moment MTB is exhaled by infected individuals, through an active and latent phase in the body of the new host, until the time they reach the reactivation stage, MTB is exposed to many types of DNA-damaging agents. Like all cellular organisms, MTB has efficient DNA repair systems, and these are believed to play essential roles in mycobacterial pathogenesis. As different stages of infection have great variation in the conditions in which mycobacteria reside, it is possible that different repair systems are essential for progression to specific phases of infection. MTB possesses homologues of DNA repair systems that are found widely in other species of bacteria, such as nucleotide excision repair, base excision repair and repair by homologous recombination. MTB also possesses a system for non-homologous end-joining of DNA breaks, which appears to be widespread in prokaryotes, although its presence is sporadic within different species within a genus. However, MTB does not possess homologues of the typical mismatch repair system that is found in most bacteria. Recent studies have demonstrated that DNA repair genes are expressed differentially at each stage of infection. In the present review, we focus on different DNA repair systems from mycobacteria and identify questions that remain in our understanding of how these systems have an impact upon the infection processes of these important pathogens. [ABSTRACT FROM AUTHOR]

The resistance of mycobacteria to the clinical applied antibiotics poses a serious problem to deal with the infections they cause. So, the search for new antibiotics active against these bacteria becomes urgent. We report here the isolation from a Moroccan biotope of a bacterial strain secreting an active substance of protein nature that inhibits the growth of several mycobacterial species ( Mycobacterium smegmatis; M. aurum A+; M. vaccae; M. bovis BCG and M. kansasii). PCR amplification and DNA sequecing of the 16S ribosomal RNA gene allowed the identification of this strain as Staphylococcus haemolyticus. Moreover, the substance produced by this strain was able to lyse the wall of M. smegmatis and to extract its genomic DNA indicating that it acts probably, like others anti-mycobacterial antibiotics, on this envelope. The identification and characterisation of the active substance would open the way for further technological and therapeutic investigations. [ABSTRACT FROM AUTHOR]

The use of molecular techniques to study the mycobacteria has advanced greatly since the first genomic libraries of Mycobacterium tuberculosis and M. leprae were constructed in 1985. However, there are still pitfalls for the unwary. Most of the problems associated with the use of molecular techniques to study mycobacteria can be related to one of the following problems: slow growth rate causing problems with contamination; the formation of macroscopic clumps when grown in culture; resistance to standard chemical lysis procedures; the requirement for containment facilities for pathogenic species; the lack of suitable genetic vectors; and the problems of spontaneous antibiotic resistance. Despite these problems, considerable progress has been made and standard techniques have been developed for the preparation of protein, nucleic acids (DNA and RNA) and cell wall components, chemical and transposon mutagenesis and gene replacement methods, the use of reporter genes and expression vectors, and improved detection and drug sensitivity testing. [ABSTRACT FROM AUTHOR]

KstR2 (Rv3557c) is one of two TetR-family transcriptional repressors of cholesterol metabolism in Mycobacterium tuberculosis. The ability to degrade cholesterol fully is important for pathogenesis, and therefore this repressor was expressed, purified and crystallized. Crystals of KstR2 diffracted to better than 1.9 Å resolution and belonged to space group C2, with unit-cell parameters a = 72.3, b = 90.3, c = 49.7 Å, α = γ = 90, β = 128.2°. [ABSTRACT FROM AUTHOR]

An important prerequisite for the effective control, timely diagnosis, and successful treatment of mycobacterial infections in both humans and animals is a rapid, specific, and sensitive detection technique. Culture is still considered the gold standard in the detection of viable mycobacteria; however, mycobacteria are extremely fastidious and slow-growing microorganisms, and therefore cultivation requires a very long incubation period to obtain results. Polymerase Chain Reaction (PCR) methods are also frequently used in the diagnosis of mycobacterial infections, providing faster and more accurate results, but are unable to distinguish between a viable and non-viable microorganism, which results in an inability to determine the success of tuberculosis patient treatment or to differentiate between an active and passive infection of animals. One suitable technique that overcomes these shortcomings mentioned is the phage amplification assay (PA). PA specifically detects viable mycobacteria present in a sample within 48 h using a lytic bacteriophage isolated from the environment. Nowadays, an alternative approach to PA, a commercial kit called Actiphage™, is also employed, providing the result within 6–8 h. In this approach, the bacteriophage is used to lyse mycobacterial cells present in the sample, and the released DNA is subsequently detected by PCR. The objective of this review is to summarize information based on the PA used for detection of mycobacteria significant in both human and veterinary medicine from various kinds of matrices. [ABSTRACT FROM AUTHOR]

Roughly a third of the world's population is estimated to have latent Mycobacterium tuberculosis infection, being at risk of developing active tuberculosis (TB) during their lifetime. Given the inefficacy of prophylactic measures and the increase of drug-resistant M. tuberculosis strains, there is a clear and urgent need for the development of new and more efficient chemotherapeutic agents, with selective toxicity, to be implemented on patient treatment. The component enzymes of the shikimate pathway, which is essential in mycobacteria and absent in humans, stand as attractive and potential targets for the development of new drugs to treat TB. This review gives an update on published work on the enzymes of the shikimate pathway and some insight on what can be potentially explored towards selective drug development. [ABSTRACT FROM AUTHOR]