Comprehensive identification of conditionally essential genes in mycobacteria

Sassetti, Christopher M.; Boyd, Dana; Rubín, Eric J.

doi:10.1073/pnas.231275498

Cited by 566 publications

(517 citation statements)

References 37 publications

(28 reference statements)

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“…Deciphering the M. tuberculosis genome has facilitated the development of screens designed to identify genes implicated in the infection process (Lamichhane et al, 2003;Sassetti et al, 2001;Sassetti & Rubin, 2003). Overall, a significant overlap between genes required for survival of M. tuberculosis in macrophages and those required for full-blown virulence in in vivo infection of mice has been found.…”

Section: Discussionmentioning

confidence: 99%

LspA inactivation in Mycobacterium tuberculosis results in attenuation without affecting phagosome maturation arrest

et al. 2008

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Section: Discussionmentioning

confidence: 99%

LspA inactivation in Mycobacterium tuberculosis results in attenuation without affecting phagosome maturation arrest

et al. 2008

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“…Library-Propagation of the MycoMar transposon phage and preparation of phage lysates have been described previously (22). For phage infection, M. marinum 1218R cells were washed and resuspended in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM MgSO 4 , 2 mM CaCl 2 .…”

Section: Generation and Screening Of M Marinum Mycomar Insertionmentioning

confidence: 99%

PimF, a Mannosyltransferase of Mycobacteria, Is Involved in the Biosynthesis of Phosphatidylinositol Mannosides and Lipoarabinomannan

Alexander

Jones

Tan

et al. 2004

Journal of Biological Chemistry

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Phosphatidylinositol mannosides (PIMs) and their related molecules lipomannan (LM) and lipoarabinomannan (LAM) are important components of the mycobacterial cell wall. These molecules mediate host-pathogen interactions and exhibit immunomodulatory activities. The biosynthesis of these lipoglycans is not fully understood. In this study, we have identified a mycobacterial gene (Rv1500) that is involved in the synthesis of PIMs. We have named this gene pimF. Transposon mutagenesis of pimF of Mycobacterium marinum resulted in multiple phenotypes, including altered colony morphology, disappearance of tetracyl-PIM 7 , and accumulation of tetraacyl-PIM 5 . The syntheses of LAM and LM were also affected. In addition, the pimF mutant exhibited a defect during infection of cultured macrophage cells. Although the mutant was able to replicate and persist within macrophages, the initial cell entry step was inefficient. Transformation of the M. marinum mutant with the pimF homolog of Mycobacterium tuberculosis complemented all of the above mentioned phenotypes. These results provide evidence that PimF is a mannosyltransferase. However, sequence analysis indicates that PimF is distinct from mannosyltransferases involved in the early steps of PIM synthesis. PimF catalyzes the formation of high molecular weight PIMs, which are precursors for the synthesis of LAM and LM. As such, this work marks the first analysis of a mannosyltransferase involved in the later stages of PIM synthesis.The mycobacterial cell wall modulates interactions between the tubercle bacillus and its environment. The cell wall consists of a covalently bound structure, the mycolic acid-arabinogalactan-peptidoglycan complex, and a variety of free lipids that complement the mycolate residues to form an asymmetric bilayer (1-3). Among the cell wall-associated lipids, phosphatidylinositol mannosides (PIMs) 1 and their multiglycosylated counterparts lipomannan (LM) and lipoarabinomannan (LAM), have emerged as major factors in mediating host-pathogen interactions and are presumed to be necessary for the survival and persistence of the pathogen within the host (4). LAM has been implicated in various immunomodulatory effects, including the down-regulation of cell-mediated immunity (5). In addition, the mannose-capped LAM found in the slowly growing mycobacteria, including Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium bovis, and Mycobacterium avium, has been shown to bind cell surface receptors of macrophages and dendritic cells and aid invasion of host cells (6 -8).PIMs are known to mediate the attachment of M. tuberculosis to nonphagocytic cells and have been implicated in the recruitment of natural killer T cells, which affects the granulomatous response (9, 10).Mycobacterial LAMs are lipoglycans composed of three structural components: the membrane anchor, which is a mannosyl-phosphatidyl-myo-inositol; the backbone, which consists of two homopolysaccharides, mannopyranose (Manp) and arabinofuranose (Araf); and the capping motif, which varies among...

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“…Relative abundance of the input and output P T7 transcripts are monitored using an ORF microarray. [82][83][84][85] This method has been applied to Salmonella using a lamba-red recombination method that includes features to minimize polarity and to construct targeted deletion mutants. The P T7 was added to the cassette inserted during mutagenesis and positioned to produce a gene-specific transcript from the genomic sequence adjacent to the insertion.…”

Section: Salmonella Entericamentioning

confidence: 99%

Signature tagged mutagenesis in the functional genetic analysis of gastrointestinal pathogens

Cummins

Gahan

2012

Gut Microbes

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Signature tagged mutagenesis is a genetic approach that was developed to identify novel bacterial virulence factors. It is a negative selection method in which unique identification tags allow analysis of pools of mutants in mixed populations. The approach is particularly well suited to functional genetic analysis of the gastrointestinal phase of infection in foodborne pathogens and has the capacity to guide the development of novel vaccines and therapeutics. In this review we outline the technical principles underpinning signature-tagged mutagenesis as well as novel sequencing-based approaches for transposon mutant identification such as TraDIS (transposon directed insertion-site sequencing). We also provide an analysis of screens that have been performed in gastrointestinal pathogens which are a global health concern (Escherichia coli, Listeria monocytogenes, Helicobacter pylori, Vibrio cholerae and Salmonella enterica). The identification of key virulence loci through the use of signature tagged mutagenesis in mice and relevant larger animal models is discussed.

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Comprehensive identification of conditionally essential genes in mycobacteria

Cited by 566 publications

References 37 publications

LspA inactivation in Mycobacterium tuberculosis results in attenuation without affecting phagosome maturation arrest

LspA inactivation in Mycobacterium tuberculosis results in attenuation without affecting phagosome maturation arrest

PimF, a Mannosyltransferase of Mycobacteria, Is Involved in the Biosynthesis of Phosphatidylinositol Mannosides and Lipoarabinomannan

Signature tagged mutagenesis in the functional genetic analysis of gastrointestinal pathogens

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