July 19, 2019  |  

Precision methylome characterization of Mycobacterium tuberculosis complex (MTBC) using PacBio single-molecule real-time (SMRT) technology.

Tuberculosis (TB) remains one of the most common infectious diseases caused by Mycobacterium tuberculosis complex (MTBC). To panoramically analyze MTBC’s genomic methylation, we completed the genomes of 12 MTBC strains (Mycobacterium bovis; M. bovis BCG; M. microti; M. africanum; M. tuberculosis H37Rv; H37Ra; and 6 M. tuberculosis clinical isolates) belonging to different lineages and characterized their methylomes using single-molecule real-time (SMRT) technology. We identified three (m6)A sequence motifs and their corresponding methyltransferase (MTase) genes, including the reported mamA, hsdM and a newly discovered mamB. We also experimentally verified the methylated motifs and functions of HsdM and MamB. Our analysis indicated the MTase activities varied between 12 strains due to mutations/deletions. Furthermore, through measuring ‘the methylated-motif-site ratio’ and ‘the methylated-read ratio’, we explored the methylation status of each modified site and sequence-read to obtain the ‘precision methylome’ of the MTBC strains, which enabled intricate analysis of MTase activity at whole-genome scale. Most unmodified sites overlapped with transcription-factor binding-regions, which might protect these sites from methylation. Overall, our findings show enormous potential for the SMRT platform to investigate the precise character of methylome, and significantly enhance our understanding of the function of DNA MTase.© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.


July 19, 2019  |  

SMRT genome assembly corrects reference errors, resolving the genetic basis of virulence in Mycobacterium tuberculosis.

The genetic basis of virulence in Mycobacterium tuberculosis has been investigated through genome comparisons of virulent (H37Rv) and attenuated (H37Ra) sister strains. Such analysis, however, relies heavily on the accuracy of the sequences. While the H37Rv reference genome has had several corrections to date, that of H37Ra is unmodified since its original publication.Here, we report the assembly and finishing of the H37Ra genome from single-molecule, real-time (SMRT) sequencing. Our assembly reveals that the number of H37Ra-specific variants is less than half of what the Sanger-based H37Ra reference sequence indicates, undermining and, in some cases, invalidating the conclusions of several studies. PE_PPE family genes, which are intractable to commonly-used sequencing platforms because of their repetitive and GC-rich nature, are overrepresented in the set of genes in which all reported H37Ra-specific variants are contradicted. Further, one of the sequencing errors in H37Ra masks a true variant in common with the clinical strain CDC1551 which, when considered in the context of previous work, corresponds to a sequencing error in the H37Rv reference genome.Our results constrain the set of genomic differences possibly affecting virulence by more than half, which focuses laboratory investigation on pertinent targets and demonstrates the power of SMRT sequencing for producing high-quality reference genomes.


July 19, 2019  |  

Methylation in Mycobacterium tuberculosis is lineage specific with associated mutations present globally.

DNA methylation is an epigenetic modification of the genome involved in regulating crucial cellular processes, including transcription and chromosome stability. Advances in PacBio sequencing technologies can be used to robustly reveal methylation sites. The methylome of the Mycobacterium tuberculosis complex is poorly understood but may be involved in virulence, hypoxic survival and the emergence of drug resistance. In the most extensive study to date, we characterise the methylome across the 4 major lineages of M. tuberculosis and 2 lineages of M. africanum, the leading causes of tuberculosis disease in humans. We reveal lineage-specific methylated motifs and strain-specific mutations that are abundant globally and likely to explain loss of function in the respective methyltransferases. Our work provides a set of sixteen new complete reference genomes for the Mycobacterium tuberculosis complex, including complete lineage 5 genomes. Insights into lineage-specific methylomes will further elucidate underlying biological mechanisms and other important phenotypes of the epi-genome.


July 7, 2019  |  

Complete annotated genome sequence of Mycobacterium tuberculosis (Zopf) Lehmann and Neumann (ATCC35812) (Kurono).

We report the completely annotated genome sequence of Mycobacterium tuberculosis (Zopf) Lehmann and Neumann (ATCC35812) (Kurono), which is a used for virulence and/or immunization studies. The complete genome sequence of M. tuberculosis Kurono was determined with a length of 4,415,078 bp and a G+C content of 65.60%. The chromosome was shown to contain a total of 4,340 protein-coding genes, 53 tRNA genes, one transfer messenger RNA for all amino acids, and 1 rrn operon. Lineage analysis based on large sequence polymorphisms indicated that M. tuberculosis Kurono belongs to the Euro-American lineage (lineage 4). Phylogenetic analysis using whole genome sequences of M. tuberculosis Kurono in addition to 22 M. tuberculosis complex strains indicated that H37Rv is the closest relative of Kurono based on the results of phylogenetic analysis. These findings provide a basis for research using M. tuberculosis Kurono, especially in animal models. Copyright © 2014 Elsevier Ltd. All rights reserved.


July 7, 2019  |  

Whole-genome sequencing for comparative genomics and de novo genome assembly.

Next-generation sequencing technologies for whole-genome sequencing of mycobacteria are rapidly becoming an attractive alternative to more traditional sequencing methods. In particular this technology is proving useful for genome-wide identification of mutations in mycobacteria (comparative genomics) as well as for de novo assembly of whole genomes. Next-generation sequencing however generates a vast quantity of data that can only be transformed into a usable and comprehensible form using bioinformatics. Here we describe the methodology one would use to prepare libraries for whole-genome sequencing, and the basic bioinformatics to identify mutations in a genome following Illumina HiSeq or MiSeq sequencing, as well as de novo genome assembly following sequencing using Pacific Biosciences (PacBio).


July 7, 2019  |  

Complete genome sequence of the clinical Beijing-like strain Mycobacterium tuberculosis 323 using the PacBio real-time sequencing platform.

We report here the whole-genome sequence of the multidrug-resistant Beijing-like strain Mycobacterium tuberculosis 323, isolated from a 15-year-old female patient who died shortly after the initiation of second-line drug treatment. This strain is representative of the Beijing-like isolates from Colombia, where this lineage is becoming a public health concern. Copyright © 2015 Rodríguez et al.


July 7, 2019  |  

Essential roles of methionine and S-adenosylmethionine in the autarkic lifestyle of Mycobacterium tuberculosis.

Multidrug resistance, strong side effects, and compliance problems in TB chemotherapy mandate new ways to kill Mycobacterium tuberculosis (Mtb). Here we show that deletion of the gene encoding homoserine transacetylase (metA) inactivates methionine and S-adenosylmethionine (SAM) biosynthesis in Mtb and renders this pathogen exquisitely sensitive to killing in immunocompetent or immunocompromised mice, leading to rapid clearance from host tissues. Mtb ?metA is unable to proliferate in primary human macrophages, and in vitro starvation leads to extraordinarily rapid killing with no appearance of suppressor mutants. Cell death of Mtb ?metA is faster than that of other auxotrophic mutants (i.e., tryptophan, pantothenate, leucine, biotin), suggesting a particularly potent mechanism of killing. Time-course metabolomics showed complete depletion of intracellular methionine and SAM. SAM depletion was consistent with a significant decrease in methylation at the DNA level (measured by single-molecule real-time sequencing) and with the induction of several essential methyltransferases involved in biotin and menaquinone biosynthesis, both of which are vital biological processes and validated targets of antimycobacterial drugs. Mtb ?metA could be partially rescued by biotin supplementation, confirming a multitarget cell death mechanism. The work presented here uncovers a previously unidentified vulnerability of Mtb-the incapacity to scavenge intermediates of SAM and methionine biosynthesis from the host. This vulnerability unveils an entirely new drug target space with the promise of rapid killing of the tubercle bacillus by a new mechanism of action.


July 7, 2019  |  

Whole-genome sequences of Mycobacterium tuberculosis TB282 and TB284, a widespread and a unique strain, respectively, identified in a previous study of tuberculosis transmission in central Los Angeles, California, USA.

We report here the genome sequences of two Mycobacterium tuberculosis clinical isolates previously identified in central Los Angeles, CA, in the 1990s using a PacBio platform. Isolate TB282 represents a large-cluster strain that caused 27% of the tuberculosis cases, while TB284 represents a strain that caused disease in only one patient. Copyright © 2017 Zhang and Yang.


July 7, 2019  |  

Complete genome sequences of three representative Mycobacterium tuberculosis Beijing family strains belonging to distinct genotype clusters in Hanoi, Vietnam, during 2007 to 2009.

We present here three complete genome sequences of Mycobacterium tuberculosis Beijing family strains isolated in Hanoi, Vietnam. These three strains were selected from major genotypic clusters (15-MIRU-VNTR) identified in a previous population-based study. We emphasize their importance and potential as reference strains in this Asian region. Copyright © 2017 Wada et al.


July 7, 2019  |  

Complete genome sequence of a Mycobacterium tuberculosis strain belonging to the East African-Indian family in the Indo-Oceanic lineage, isolated in Hanoi, Vietnam.

The East African-Indian (EAI) family of Mycobacterium tuberculosis is an endemic group mainly observed in Southeast Asia. Here, we report the complete genome sequence of an M. tuberculosis strain isolated as a member of the EAI family in Hanoi, Vietnam, a country with a high incidence of tuberculosis. Copyright © 2017 Wada et al.


July 7, 2019  |  

Phenotypic and genomic comparison of Mycobacterium aurum and surrogate model species to Mycobacterium tuberculosis: implications for drug discovery.

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.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.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.


July 7, 2019  |  

Comparative genomic analysis of Mycobacterium tuberculosis Beijing-like strains revealed specific genetic variations associated with virulence and drug resistance.

Isolates of the Mycobacterium tuberculosis lineage 2/East-Asian are considered one of the most successful strains due to their increased pathogenicity, hyper-virulence associated with drug resistance, and high transmission. Recent studies in Colombia have shown that the Beijing-like genotype is associated with multidrug-resistance and high prevalence in the southwest of the country, but the genetic basis of its success in dissemination is unknown. In contribution to this matter, we obtained the whole sequences of six genomes of clinical isolates assigned to the Beijing-like genotype. The genomes were compared with the reference genome of M. tuberculosis H37Rv and 53 previously published M. tuberculosis genomes. We found that the six Beijing-like isolates belong to a modern Beijing sub-lineage and share specific genomic variants: i.e. deletion in the PPE8 gene, in Rv3806c (ubiA) responsible of high ethambutol resistance and in Rv3862c (whiB6) which is involved in granuloma formation and virulence, are some of them. Moreover, each isolated has exclusively single nucleotide polymorphisms (SNPs) in genes related with cell wall processes and cell metabolism. We identified polymorphisms in genes related to drug resistance that could explain the drug-resistant phenotypes found in the six isolates from Colombia. We hypothesize that changes due to these genetic variations contribute to the success of these strains. Finally, we analyzed the IS6110 insertion sequences finding very low variance between them, suggesting that SNPs is the major cause of variability found in Beijing-like strains circulating in Colombia. Copyright © 2017 Elsevier B.V. All rights reserved.


July 7, 2019  |  

Comparative genomic analysis of two clonally related multidrug resistant Mycobacterium tuberculosis by Single Molecule Real Time Sequencing.

Background: Multidrug-resistant tuberculosis (MDR-TB) is posing a major threat to global TB control. In this study, we focused on two consecutive MDR-TB isolated from the same patient before and after the initiation of anti-TB treatment. To better understand the genomic characteristics of MDR-TB, Single Molecule Real-Time (SMRT) Sequencing and comparative genomic analyses was performed to identify mutations that contributed to the stepwise development of drug resistance and growth fitness in MDR-TB underin vivochallenge of anti-TB drugs.Result:Both pre-treatment and post-treatment strain demonstrated concordant phenotypic and genotypic susceptibility profiles toward rifampicin, pyrazinamide, streptomycin, fluoroquinolones, aminoglycosides, cycloserine, ethionamide, and para-aminosalicylic acid. However, although both strains carried identical missense mutations atrpoBS531L,inhAC-15T, andembBM306V, MYCOTB Sensititre assay showed that the post-treatment strain had 16-, 8-, and 4-fold elevation in the minimum inhibitory concentrations (MICs) toward rifabutin, isoniazid, and ethambutol respectively. The results have indicated the presence of additional resistant-related mutations governing the stepwise development of MDR-TB. Further comparative genomic analyses have identified three additional polymorphisms between the clinical isolates. These include a single nucleotide deletion at nucleotide position 360 ofrv0888in pre-treatment strain, and a missense mutation atrv3303c(lpdA)V44I and a 6-bp inframe deletion at codon 67-68 inrv2071c(cobM)in the post-treatment strain. Multiple sequence alignment showed that these mutations were occurring at highly conserved regions among pathogenic mycobacteria. Using structural-based and sequence-based algorithms, we further predicted that the mutations potentially have deleterious effect on protein function.Conclusion:This is the first study that compared the full genomes of two clonally-related MDR-TB clinical isolates during the course of anti-TB treatment. Our work has demonstrated the robustness of SMRT Sequencing in identifying mutations among MDR-TB clinical isolates. Comparative genome analysis also suggested novel mutations atrv0888, lpdA, andcobMthat might explain the difference in antibiotic resistance and growth pattern between the two MDR-TB strains.


July 7, 2019  |  

Comparative whole-genomic analysis of an ancient L2 lineage Mycobacterium novel phylogenetic clade and common genetic determinants of hypervirulent strains.

Background: Development of improved therapeutics against tuberculosis (TB) is hindered by an inadequate understanding of the relationship between disease severity and genetic diversity of its causative agent, Mycobacterium tuberculosis. We previously isolated a hypervirulent M. tuberculosis strain H112 from an HIV-negative patient with an aggressive disease progression from pulmonary TB to tuberculous meningitis—the most severe manifestation of tuberculosis. Human macrophage challenge experiment demonstrated that the strain H112 exhibited significantly better intracellular survivability and induced lower level of TNF-a than the reference virulent strain H37Rv and other 123 clinical isolates. Aim: The present study aimed to identify the potential genetic determinants of mycobacterial virulence that were common to strain H112 and hypervirulent M. tuberculosis strains of the same phylogenetic clade isolated in other global regions. Methods: A low-virulent M. tuberculosis strain H54 which belonged to the same phylogenetic lineage (L2) as strain H112 was selected from a collection of 115 clinical isolates. Both H112 and H54 were whole-genome-sequenced using PacBio sequencing technology. A comparative genomics approach was adopted to identify mutations present in strain H112 but absent in strain H54. Subsequently, an extensive phylogenetic analysis was conducted by including all publically available M. tuberculosis genomes. Single-nucleotide-polymorphisms (SNPs) and structural variations (SVs) common to hypervirulent strains in the global collection of genomes were considered as potential genetic determinants of hypervirulence. Results: Sequencing data revealed that both H112 and H54 were identified as members of the same sub-lineage L2.2.1. After excluding the lineage-related mutations shared between H112 and H54, we analyzed the phylogenetic relatedness of H112 with global collection of M. tuberculosis genomes (n = 4,338), and identified a novel phylogenetic clade in which four hypervirulent strains isolated from geographically diverse regions were clustered together. All hypervirulent strains in the clade shared 12 SNPs and 5 SVs with H112, including those affecting key virulence-associated loci, notably, a deleterious SNP (rv0178 p. D150E) within mce1 operon and an intergenic deletion (854259_ 854261delCC) in close-proximity to phoP. Conclusion: The present study identified common genetic factors in a novel phylogenetic clade of hypervirulent M. tuberculosis. The causative role of these mutations in mycobacterial virulence should be validated in future study.


July 7, 2019  |  

Genomic and transcriptomic analysis of the streptomycin-dependent Mycobacterium tuberculosis strain 18b.

The ability of Mycobacterium tuberculosis to establish a latent infection (LTBI) in humans confounds the treatment of tuberculosis. Consequently, there is a need to discover new therapeutic agents that can kill M. tuberculosis both during active disease and LTBI. The streptomycin-dependent strain of M. tuberculosis, 18b, provides a useful tool for this purpose since upon removal of streptomycin (STR) it enters a non-replicating state that mimics latency both in vitro and in animal models.The 4.41 Mb genome sequence of M. tuberculosis 18b was determined and this revealed the strain to belong to clade 3 of the ancient ancestral lineage of the Beijing family. STR-dependence was attributable to insertion of a single cytosine in the 530 loop of the 16S rRNA and to a single amino acid insertion in the N-terminal domain of initiation factor 3. RNA-seq was used to understand the genetic programme activated upon STR-withdrawal and hence to gain insight into LTBI. This revealed reconfiguration of gene expression and metabolic pathways showing strong similarities between non-replicating 18b and M. tuberculosis residing within macrophages, and with the core stationary phase and microaerophilic responses.The findings of this investigation confirm the validity of 18b as a model for LTBI, and provide insight into both the evolution of tubercle bacilli and the functioning of the ribosome.


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