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MSystems Feb 2023Tolerance of Mycobacterium tuberculosis to antibiotics contributes to the long duration of tuberculosis (TB) treatment and the emergence of drug-resistant strains. M....
Tolerance of Mycobacterium tuberculosis to antibiotics contributes to the long duration of tuberculosis (TB) treatment and the emergence of drug-resistant strains. M. tuberculosis drug tolerance is induced by nutrient restriction, but the genetic determinants that promote antibiotic tolerance triggered by nutrient limitation have not been comprehensively identified. Here, we show that M. tuberculosis requires production of the outer membrane lipid phthiocerol dimycocerosate (PDIM) to tolerate antibiotics under nutrient-limited conditions. We developed an arrayed transposon (Tn) mutant library in M. tuberculosis Erdman and used orthogonal pooling and transposon sequencing (Tn-seq) to map the locations of individual mutants in the library. We screened a subset of the library (~1,000 mutants) by Tn-seq and identified 32 and 102 Tn mutants with altered tolerance to antibiotics under stationary-phase and phosphate-starved conditions, respectively. Two mutants recovered from the arrayed library, ::Tn and ::Tn, showed increased susceptibility to two different drug combinations under both nutrient-limited conditions, but their phenotypes were not complemented by the Tn-disrupted gene. Whole-genome sequencing revealed single nucleotide polymorphisms in both the ::Tn and ::Tn mutants that prevented PDIM production. Complementation of the ::Tn Q291* mutant with restored PDIM production and antibiotic tolerance, demonstrating that loss of PDIM sensitized M. tuberculosis to antibiotics. Our data suggest that drugs targeting production of PDIM, a critical M. tuberculosis virulence determinant, have the potential to enhance the efficacy of existing antibiotics, thereby shortening TB treatment and limiting development of drug resistance. Mycobacterium tuberculosis causes 10 million cases of active TB disease and over 1 million deaths worldwide each year. TB treatment is complex, requiring at least 6 months of therapy with a combination of antibiotics. One factor that contributes to the length of TB treatment is M. tuberculosis phenotypic antibiotic tolerance, which allows the bacteria to survive prolonged drug exposure even in the absence of genetic mutations causing drug resistance. Here, we report a genetic screen to identify M. tuberculosis genes that promote drug tolerance during nutrient starvation. Our study revealed the outer membrane lipid phthiocerol dimycocerosate (PDIM) as a key determinant of M. tuberculosis antibiotic tolerance triggered by nutrient starvation. Our study implicates PDIM synthesis as a potential target for development of new TB drugs that would sensitize M. tuberculosis to existing antibiotics to shorten TB treatment.
Topics: Humans; Membrane Lipids; Mycobacterium tuberculosis; Tuberculosis; Drug Resistance, Bacterial
PubMed: 36598240
DOI: 10.1128/msystems.00699-22 -
Revista Medica de Chile Dec 2011Mycobacterium tuberculosis, the etiological agent of human tuberculosis, causes annually three million deaths and latently infects about two billion people.... (Review)
Review
Mycobacterium tuberculosis, the etiological agent of human tuberculosis, causes annually three million deaths and latently infects about two billion people. Immunodeficiency caused by malnutrition, senescence or co-infection with HIV enhances the risk of developing active tuberculosis, either from a primary infection or by reactivation of a latent infection. The increasing appearance of multidrug-resistant strains to existing drugs is worrisome, since it leaves patients practically without treatment options. The understanding of the mechanisms of transmission, pathogenesis and virulence of M. tuberculosis is important. The analysis of its genome shows the presence of alternative sigma factors, transcriptional repressors and activators, two component signaling systems, metabolic enzymes and cellular secretory systems, that are associated with virulence in a series of pathogenic micro-organisms. Environmental stimuli such as pH, temperature, osmolality, oxygen availability are processed, activating or repressing virulence genes. The molecular mechanisms of action of these genes have been elucidated in in vitro and in vivo models.
Topics: Genome, Bacterial; Humans; Mycobacterium tuberculosis; Tuberculosis Vaccines; Vaccines, Synthetic; Virulence Factors
PubMed: 22446709
DOI: No ID Found -
Revista de Salud Publica (Bogota,... 2018Due to the emergence of multi-drug resistant (MDR-MTB) and extensively drug-resistant (XDR-MTB) Mycobacterium tuberculosis (MTB) isolates, the failure rates of standard... (Review)
Review
Due to the emergence of multi-drug resistant (MDR-MTB) and extensively drug-resistant (XDR-MTB) Mycobacterium tuberculosis (MTB) isolates, the failure rates of standard treatment regimens are high, thus becoming a major public health challenge worldwide. Resistance to anti-tuberculous (anti-TB) drugs is attributed mainly to specific mutations in target genes; however, a proportion of drug-resistant MTB isolates do not have mutations in these genes, which suggests the involvement of other mechanisms, such as the low permeability of the mycobacterial cell wall, enzymatic modification and/or efflux pumps. Clinical drug resistance to anti-TB drugs occurs largely as a result of the selection of resistant mutants caused by poor patient adherence to treatment, inappropriate follow-ups and prescriptions, suboptimal doses of drugs and poor access to health services and treatment. Major advances in molecular biology tools and the availability of the complete genome sequences of MTB have contributed to improve understanding of the mechanisms of resistance to the main anti-TB drugs. Better knowledge of the drug-resistance of MTB will contribute to the identification of new therapeutic targets to design new drugs, develop new diagnostic tests and/or improve methods currently available for the rapid detection of drug-resistant TB. This article presents an updated review of the mechanisms and molecular basis of drug resistance in MTB.
Topics: Antitubercular Agents; Humans; Mycobacterium tuberculosis; Tuberculosis, Multidrug-Resistant
PubMed: 30843986
DOI: 10.15446/rsap.V20n4.50575 -
Microbiology Spectrum Oct 2014Discontinuity of both strands of the chromosome is a lethal event in all living organisms because it compromises chromosome replication. As such, a diversity of DNA... (Review)
Review
Discontinuity of both strands of the chromosome is a lethal event in all living organisms because it compromises chromosome replication. As such, a diversity of DNA repair systems has evolved to repair double-strand DNA breaks (DSBs). In part, this diversity of DSB repair systems has evolved to repair breaks that arise in diverse physiologic circumstances or sequence contexts, including cellular states of nonreplication or breaks that arise between repeats. Mycobacteria elaborate a set of three genetically distinct DNA repair pathways: homologous recombination, nonhomologous end joining, and single-strand annealing. As such, mycobacterial DSB repair diverges substantially from the standard model of prokaryotic DSB repair and represents an attractive new model system. In addition, the presence in mycobacteria of a DSB repair system that can repair DSBs in nonreplicating cells (nonhomologous end joining) or when DSBs arise between repeats (single-strand annealing) has clear potential relevance to Mycobacterium tuberculosis pathogenesis, although the exact role of these systems in M. tuberculosis pathogenesis is still being elucidated. In this article we will review the genetics of mycobacterial DSB repair systems, focusing on recent insights.
Topics: DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Repair Enzymes; Homologous Recombination; Mycobacterium tuberculosis; Recombinational DNA Repair; Virulence Factors
PubMed: 26104351
DOI: 10.1128/microbiolspec.MGM2-0024-2013 -
PloS One 2011Mycobacterium tuberculosis dosRS two-component regulatory system controls transcription of approximately 50 genes including hspX, acg and Rv2030c, in response to hypoxia...
Mycobacterium tuberculosis dosRS two-component regulatory system controls transcription of approximately 50 genes including hspX, acg and Rv2030c, in response to hypoxia and nitric oxide conditions and within macrophages and mice. The hspX lies between acg and Rv2030c. However, the functions of the dosR regulated genes in vitro and in vivo are largely unknown. Previously, we demonstrated that deletion of hspX gene produced a mutant which grew faster in macrophages and in mice. In this study, we attempted to determine the functions of acg and Rv2030c by gene inactivation. We demonstrate that Rv2030c is dispensable for virulence and growth. However, deletion of acg produced a mutant which is attenuated in both resting and activated macrophages and in acute and persistent murine infection models. Surprisingly, deletion of acg did not compromise the viability of the mutant to nitrosative and oxidative stresses in vitro and in vivo. In addition, when the WT and the acg mutants were treated with antibiotics such as the prodrugs nitrofurantoin and nitrofuran, the acg mutant became more sensitive than the WT strain to these drugs. This suggests that Acg may not function as a nitroreductase. These data indicate that acg encodes an essential virulence factor for M. tuberculosis and enables it to grow and survive in macrophages and in mouse organs.
Topics: Animals; Female; Gene Deletion; Genes, Bacterial; Inflammation; Macrophages; Mice; Mice, Inbred BALB C; Mycobacterium tuberculosis; Nitroreductases; Stress, Physiological
PubMed: 21687631
DOI: 10.1371/journal.pone.0020958 -
Protein & Cell Sep 2011Biotin is an important micronutrient that serves as an essential enzyme cofactor. Bacteria obtain biotin either through de novo synthesis or by active uptake from... (Review)
Review
Biotin is an important micronutrient that serves as an essential enzyme cofactor. Bacteria obtain biotin either through de novo synthesis or by active uptake from exogenous sources. Mycobacteria are unusual amongst bacteria in that their primary source of biotin is through de novo synthesis. Here we review the importance of biotin biosynthesis in the lifecycle of Mycobacteria. Genetic screens designed to identify key metabolic processes have highlighted a role for the biotin biosynthesis in bacilli growth, infection and survival during the latency phase. These studies help to establish the biotin biosynthetic pathway as a potential drug target for new anti-tuberculosis agents.
Topics: Biotin; Carbon-Carbon Ligases; Carrier Proteins; Cell Membrane; Coenzymes; Fatty Acids; Genes, Bacterial; Genome, Bacterial; Metabolic Networks and Pathways; Molecular Structure; Mycobacterium Infections; Mycobacterium tuberculosis; Virulence
PubMed: 21976058
DOI: 10.1007/s13238-011-1100-8 -
BMC Genomics Jul 2017Tuberculosis (TB) is caused by Mycobacterium tuberculosis and represents one of the major challenges facing drug discovery initiatives worldwide. The considerable rise... (Comparative Study)
Comparative Study
BACKGROUND
Tuberculosis (TB) is caused by Mycobacterium tuberculosis and represents one of the major challenges facing drug discovery initiatives worldwide. The considerable rise in bacterial drug resistance in recent years has led to the need of new drugs and drug regimens. Model systems are regularly used to speed-up the drug discovery process and circumvent biosafety issues associated with manipulating M. tuberculosis. These include the use of strains such as Mycobacterium smegmatis and Mycobacterium marinum that can be handled in biosafety level 2 facilities, making high-throughput screening feasible. However, each of these model species have their own limitations.
RESULTS
We report and describe the first complete genome sequence of Mycobacterium aurum ATCC23366, an environmental mycobacterium that can also grow in the gut of humans and animals as part of the microbiota. This species shows a comparable resistance profile to that of M. tuberculosis for several anti-TB drugs. The aims of this study were to (i) determine the drug resistance profile of a recently proposed model species, Mycobacterium aurum, strain ATCC23366, for anti-TB drug discovery as well as Mycobacterium smegmatis and Mycobacterium marinum (ii) sequence and annotate the complete genome sequence of this species obtained using Pacific Bioscience technology (iii) perform comparative genomics analyses of the various surrogate strains with M. tuberculosis (iv) discuss how the choice of the surrogate model used for drug screening can affect the drug discovery process.
CONCLUSIONS
We describe the complete genome sequence of M. aurum, a surrogate model for anti-tuberculosis drug discovery. Most of the genes already reported to be associated with drug resistance are shared between all the surrogate strains and M. tuberculosis. We consider that M. aurum might be used in high-throughput screening for tuberculosis drug discovery. We also highly recommend the use of different model species during the drug discovery screening process.
Topics: Drug Discovery; Drug Resistance, Bacterial; Genomics; Molecular Sequence Annotation; Mycobacterium tuberculosis; Phenotype; Species Specificity
PubMed: 28705154
DOI: 10.1186/s12864-017-3924-y -
Trends in Microbiology Mar 1998Mycobacterium tuberculosis can persist within the human host for years without causing disease, in a syndrome known as latent tuberculosis (TB). As one-third of the... (Review)
Review
Mycobacterium tuberculosis can persist within the human host for years without causing disease, in a syndrome known as latent tuberculosis (TB). As one-third of the world population has latent TB, placing them at risk for active TB, the mechanisms by which M. tuberculosis establishes a latent metabolic state, eludes immune surveillance and responds to triggers that stimulate reactivation are a high priority for the future control of TB.
Topics: Animals; Disease Models, Animal; Genes, Bacterial; Humans; Mycobacterium tuberculosis; Time Factors; Tuberculosis, Pulmonary
PubMed: 9582936
DOI: 10.1016/s0966-842x(98)01216-5 -
International Journal of... Mar 2016Mycobacterium tuberculosis (MTB) causes active tuberculosis (TB) in only a small percentage of infected people. In most cases, the infection is clinically latent, where...
OBJECTIVE/BACKGROUND
Mycobacterium tuberculosis (MTB) causes active tuberculosis (TB) in only a small percentage of infected people. In most cases, the infection is clinically latent, where bacilli can persist in human hosts for years without causing disease. Surprisingly, the biology of such persister cells is largely unknown. This study describes the isolation, identification, and whole-genome sequencing (WGS) of latent TB bacilli after 782days (26months) of latency (the ability of MTB bacilli to lie persistent).
METHODS
The in vitro double-stress model of latency (oxygen and nutrition) was designed for MTB culture. After 26months of latency, MTB cells that persisted were isolated and investigated under light and atomic force microscopy. Spoligotyping and WGS were performed to verify the identity of the strain.
RESULTS
We established a culture medium in which MTB bacilli arrest their growth, reduce their size (0.3-0.1μm), lose their acid fastness (85-90%) and change their shape. Spoligopatterns of latent cells were identical to original H37Rv, with differences observed at spacers two and 14. WGS revealed only a few genetic changes relative to the already published H37Rv reference genome. Among these was a large 2064-bp insertion (RvD6), which was originally detected in both H37Ra and CDC1551, but not H37Rv.
CONCLUSION
Here, we show cell-wall free cells of MTB bacilli in their latent state, and the biological adaptation of these cells was more phenotypic in nature than genomic. These cell-wall free cells represent a good model for understanding the nature of TB latency.
Topics: Cell Wall; Genome, Bacterial; High-Throughput Nucleotide Sequencing; Humans; Latent Tuberculosis; Mycobacterium tuberculosis
PubMed: 26927992
DOI: 10.1016/j.ijmyco.2015.12.001 -
Microbial Genomics Mar 2021is a known human pathogen that causes the airborne infectious disease tuberculosis (TB). Every year TB infects millions of people worldwide. The emergence of multi-drug...
is a known human pathogen that causes the airborne infectious disease tuberculosis (TB). Every year TB infects millions of people worldwide. The emergence of multi-drug resistant (MDR), extensively drug resistant (XDR) and totally drug resistant (TDR) strains against the first- and second-line anti-TB drugs has created an urgent need for the development and implementation of new drug strategies. In this study, the complete genomes of 174 strains of are analysed to understand the evolution of molecular drug target (MDT) genes. Phylogenomic placements of strains depicted close association and temporal clustering. Selection pressure analysis by deducing the ratio of non-synonymous to synonymous substitution rates () in 51 MDT genes of the 174 . strains led to categorizing these genes into diversifying (D, >0.70), moderately diversifying (MD, =0.35-0.70) and stabilized (S, <0.35) genes. The genes and were identified as diversifying, and , and were identified as stabilized genes. Furthermore, sequence similarity networks were drawn that supported these divisions. In the multiple sequence alignments of diversifying and stabilized proteins, previously reported resistance mutations were checked to predict sensitive and resistant strains of . Finally, to delineate the potential of stabilized or least diversified genes/proteins as anti-TB drug targets, protein-protein interactions of MDT proteins with human proteins were analysed. We predict that (=0.29), a stabilized gene that encodes the most host-interacting protein, KasA, should serve as a potential drug target for the treatment of TB.
Topics: Antitubercular Agents; Bacterial Proteins; Biological Evolution; Drug Resistance, Multiple, Bacterial; Genome, Bacterial; Humans; Mutation; Mycobacterium tuberculosis; Phylogeny; Sequence Analysis, DNA; Tuberculosis
PubMed: 33750515
DOI: 10.1099/mgen.0.000542