Genetic Manipulation of <i>Mycobacterium tuberculosis</i>

Larsen, Michelle H.; Biermann, Karolin; Tandberg, Steven; Hsu, Tsugunda; Jacobs, William R.

doi:10.1002/9780471729259.mc10a02s6

Cited by 146 publications

(142 citation statements)

References 16 publications

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“…These colonies were subjected to a second round of selection and then grown up in 7H9 medium with the corresponding concentration of 14 as a selection pressure. DNA was extracted using cetyltrimethylammonium bromide and lysozyme as described (42). The genomes of the resistant mutants characterized in this study were sequenced using an Illumina HiSeq 2500.…”

Section: Generation Of Constructs For Constitutive Expression Of Genementioning

confidence: 99%

N -methylation of a bactericidal compound as a resistance mechanism in Mycobacterium tuberculosis

Warrier

Kapilashrami

Argyrou

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The rising incidence of antimicrobial resistance (AMR) makes it imperative to understand the underlying mechanisms. Mycobacterium tuberculosis (Mtb) is the single leading cause of death from a bacterial pathogen and estimated to be the leading cause of death from AMR. A pyrido-benzimidazole, 14, was reported to have potent bactericidal activity against Mtb. Here, we isolated multiple Mtb clones resistant to 14. Each had mutations in the putative DNA-binding and dimerization domains of rv2887, a gene encoding a transcriptional repressor of the MarR family. The mutations in Rv2887 led to markedly increased expression of rv0560c. We characterized Rv0560c as an S-adenosyl-L-methionine-dependent methyltransferase that N-methylates 14, abolishing its mycobactericidal activity. An Mtb strain lacking rv0560c became resistant to 14 by mutating decaprenylphosphoryl-β-d-ribose 2-oxidase (DprE1), an essential enzyme in arabinogalactan synthesis; 14 proved to be a nanomolar inhibitor of DprE1, and methylation of 14 by Rv0560c abrogated this activity. Thus, 14 joins a growing list of DprE1 inhibitors that are potently mycobactericidal. Bacterial methylation of an antibacterial agent, 14, catalyzed by Rv0560c of Mtb, is a previously unreported mechanism of AMR.

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Section: Generation Of Constructs For Constitutive Expression Of Genementioning

confidence: 99%

N -methylation of a bactericidal compound as a resistance mechanism in Mycobacterium tuberculosis

Warrier

Kapilashrami

Argyrou

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…High-quality genomic DNA was prepared from PDIM-positive and PDIM-negative isolates of H37Rv by the cetyltrimethylammonium bromide (CTAB)-lysozyme method (17). Genomic DNA fragment sequencing libraries were prepared using a genomic DNA sample prep kit (Illumina) according to the manufacturer's instructions, with 5 g of purified genomic DNA.…”

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confidence: 99%

Spontaneous Phthiocerol Dimycocerosate-Deficient Variants of Mycobacterium tuberculosis Are Susceptible to Gamma Interferon-Mediated Immunity

et al. 2011

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Onset of the adaptive immune response in mice infected with Mycobacterium tuberculosis is accompanied by slowing of bacterial replication and establishment of a chronic infection. Stabilization of bacterial numbers during the chronic phase of infection is dependent on the activity of the gamma interferon (IFN-␥)-inducible nitric oxide synthase (NOS2). Previously, we described a differential signature-tagged mutagenesis screen designed to identify M. tuberculosis "counterimmune" mechanisms and reported the isolation of three mutants in the H37Rv strain background containing transposon insertions in the rv0072, rv0405, and rv2958c genes. These mutants were impaired for replication and virulence in NOS2 ؊/؊ mice but were growth-proficient and virulent in IFN-␥ ؊/؊ mice, suggesting that the disrupted genes were required for bacterial resistance to an IFN-␥-dependent immune mechanism other than NOS2. Here, we report that the attenuation of these strains is attributable to an underlying transposon-independent deficiency in biosynthesis of phthiocerol dimycocerosate (PDIM), a cell wall lipid that is required for full virulence in mice. We performed whole-genome resequencing of a PDIM-deficient clone and identified a spontaneous point mutation in the putative polyketide synthase PpsD that results in a G44C amino acid substitution. We demonstrate by complementation with the wild-type ppsD gene and reversion of the ppsD gene to the wild-type sequence that the ppsD(G44C) point mutation is responsible for PDIM deficiency, virulence attenuation in NOS2 ؊/؊ and wild-type C57BL/6 mice, and a growth advantage in vitro in liquid culture. We conclude that PDIM biosynthesis is required for M. tuberculosis resistance to an IFN-␥-mediated immune response that is independent of NOS2.Pathogenic mycobacteria possess a unique array of complex cell wall-associated lipids. The most abundant of these lipids, the phthiocerol dimycocerosates (PDIMs) (Fig. 1), are among the best characterized (23). PDIMs contain long-chain diols esterified by methyl-branched fatty acid chains. As early as 1974, it was recognized that a spontaneously arising PDIMdeficient variant of the laboratory strain H37Rv was attenuated in a guinea pig model of infection (11). Shortly thereafter, it was shown that the in vivo survival of an avirulent Mycobacterium tuberculosis strain was enhanced by coating the bacteria with cholesterol oleate and purified PDIM (16). A genetic link between PDIM production and virulence was not established until a quarter century later, when a large chromosomal locus was identified as playing an essential role in the biosynthesis and export of PDIM (3,4,6). Transposon insertions within the fadD26, fadD28, mmpL7, and drrC genes, and in the putative transcriptional promoter region upstream of the fadD26 gene, were identified in strains deficient in surface-localized PDIM. The fadD26 and fadD28 mutants apparently fail to synthesize PDIM, whereas the mmpL7 and drrC mutants produce PDIM but accumulate it intracellularly, thus implicating these ...

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“…Escherichia coli strains were routinely grown in Luria-Bertani broth at 37 uC, with 100 mg ampicillin ml 21 or 150 mg hygromycin ml 21 used for selection. High titres of phages were prepared, and phage titres were determined, according to protocols described by Larsen et al (2007). Sensitivity of M. smegmatis strains to different phages was determined by spotting 10 ml of a serial 10-fold dilution (10…”

mentioning

confidence: 99%

“…The cell pellet was washed four times with PBS containing 0.1 % Tween-80. Total DNA was then extracted from the washed cell pellet using standard protocols (Larsen et al, 2007), and it was used as template for PCR detection of intracellular phage I3 DNA. The primers I317kD-F (59-GTAC-AACCCGCCAACCCAC-39) and I317kD-R (59-CAGGCGGACG-AGATAGGTG-39), designed to amplify part (461 bp) of a phage I3 gene encoding a 17 kDa structural protein (Ramesh & Gopinathan, 1994), were used for PCR amplification.…”

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confidence: 99%

“…Temperature-sensitive transposon-delivery phages phAE185 (containing Tn5370; McAdam et al, 2002) and phAE181 (containing Tn5371; Kriakov et al, 2003) were used to construct transposon mutant libraries of M. smegmatis mc 2 155, according to protocols described by Larsen et al (2007). For isolation of phage-I3-resistant mutants, individual transposon mutants were pre-inoculated in 96-well plates, and then replicated with a 96-spike replicator onto rectangular plates containing TSB agar pre-seeded with a suspension of I3 phage (10 10 p.f.u.…”

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confidence: 99%

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Defects in glycopeptidolipid biosynthesis confer phage I3 resistance in Mycobacterium smegmatis

et al. 2009

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Mycobacteriophages have played an important role in the development of genetic tools and diagnostics for pathogenic mycobacteria, including Mycobacterium tuberculosis. However, despite the isolation of numerous phages that infect mycobacteria, the mechanisms of mycobacteriophage infection remain poorly understood, and knowledge about phage receptors is minimal. In an effort to identify the receptor for phage I3, we screened a library of Mycobacterium smegmatis transposon mutants for phage-resistant strains. All four phage I3-resistant mutants isolated were found to have transposon insertions in genes located in a cluster involved in the biosynthesis of the cell-wall-associated glycopeptidolipid (GPL), and consequently the mutants did not synthesize GPLs. The loss of GPLs correlated specifically with phage I3 resistance, as all mutants retained sensitivity to two other mycobacteriophages: D29 and Bxz1. In order to define the minimal receptor for phage I3, we then tested the phage sensitivity of previously described GPL-deficient mutants of M. smegmatis that accumulate biosynthesis intermediates of GPLs. The results indicated that, while the removal of most sugar residues from the fatty acyl tetrapeptide (FATP) core of GPL did not affect sensitivity to phage I3, a single methylated rhamnose, transferred by the rhamnosyltransferase Gtf2 to the FATP core, was critical for phage binding. INTRODUCTIONPhages that infect mycobacteria have played an important role in the development of genetic tools for mycobacteria, particularly Mycobacterium tuberculosis, which is the causative agent of tuberculosis Jacobs et al., 1991;Lee et al., 2004;Raj & Ramakrishnan, 1970;Snapper et al., 1988;van Kessel et al., 2008). Recombinant mycobacteriophages have also been used for phage typing of clinical isolates (Kubica, 1982;Rado et al., 1975), in diagnostics Hazbon et al., 2003; Jacobs et al., 1993;McNerney & Traore, 2005;Piuri et al., 2009;Riska et al., 1999), and as therapeutic agents for mycobacterial diseases (Broxmeyer et al., 2002;Mankiewicz & Beland, 1964). For the further development of mycobacteriophages as genetic tools for manipulation of mycobacteria, and as diagnostic and therapeutic agents, it is necessary to understand the mechanism of interactions between mycobacteriophages and mycobacteria. Attachment of a phage by binding to its receptor on the mycobacterial cell surface is important for initiating infection, and the study of phage-resistant mutants defective in phage adsorption is a useful approach to identifying these receptors. While a number of cell wall components have been implicated in roles as phage receptors (Besra et al., 1994;Bisso et al., 1976;Dhariwal et al., 1986;Furuchi & Tokunaga, 1972), no defined mycobacteriophage-resistant mutant with a missing phage receptor has been characterized to date. The only report of a mycobacteriophage-resistant mutant describes the appearance of a new lipid species (rather than the disappearance of an existing component) in a Mycobacterium smegmatis transposon mutant tha...

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Genetic Manipulation of Mycobacterium tuberculosis

Cited by 146 publications

References 16 publications

N -methylation of a bactericidal compound as a resistance mechanism in Mycobacterium tuberculosis

N -methylation of a bactericidal compound as a resistance mechanism in Mycobacterium tuberculosis

Spontaneous Phthiocerol Dimycocerosate-Deficient Variants of Mycobacterium tuberculosis Are Susceptible to Gamma Interferon-Mediated Immunity

Defects in glycopeptidolipid biosynthesis confer phage I3 resistance in Mycobacterium smegmatis

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