The interaction between <i>Mycobacterium</i> and the macrophage analyzed by two‐dimensional polyacrylamide gel electrophoresis

Sturgill-Koszycki, Sheila; Haddix, Pryce L.; Russell, David G.

doi:10.1002/elps.1150181411

Cited by 76 publications

(54 citation statements)

References 28 publications

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“…This pathway is required to replenish substrates for the Krebs cycle during growth on fatty acids. The fact that aceA appeared among the selected genes validated our approach since it was shown previously that aceA was upregulated during infection of macrophages (13,23) and that the levels of isocitrate lyase increase during infection (15,31). Significantly, this gene is necessary for virulence in mice (23).…”

Section: Discussionmentioning

confidence: 77%

Mycobacterium tuberculosis Genes Induced during Infection of Human Macrophages

Dubnau¹,

Fontán²,

et al. 2002

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We identified Mycobacterium tuberculosis genes preferentially expressed during infection of human macrophages using a promoter trap adapted for this pathogen. inhA encodes an enoyl-acyl carrier protein reductase that is required for mycolic acid biosynthesis ( Virulence genes are generally defined as genes that are necessary for survival of the pathogen in a host and are involved in pathogenicity but are not necessary for growth in culture medium. Several virulence genes of Mycobacterium tuberculosis have been identified by comparing the pathogenicity of strains with mutations in the genes to the pathogenicity of isogenic strains carrying the wild-type alleles. These genes include katG, encoding a catalase peroxidase (35), hspX (acr), encoding a homologue of ␣-crystallin, (36), and erp, encoding an uncharacterized exported protein (2). Specific modifications of mycolic acids are essential for virulence since strains carrying disruptions in hma, required for the biosynthesis of oxygenated mycolic acids (8), and in pcaA, a gene coding for a mycolic acid cyclopropane synthetase (11), are attenuated in mice. Synthesis of the exported phthiocerol dimycocerosate (3, 6) is also required for virulence in mice. Fatty acid degradation is implicated in pathogenicity since a strain carrying a mutated aceA gene, which is required for synthesis of isocitrate lyase, was attenuated in mice (23). Mahan et al. (19,20) were the first workers to develop a promoter trap, known as an in vivo expression technology (IVET) system, to select for genes of Salmonella enterica serovar Typhimurium specifically induced in host tissues. This method has also been used to study pathogenesis in Staphylococcus aureus, Vibrio cholerae, and other bacteria (4). Using the same approach, we created an IVET method for M. tuberculosis to select for genes specifically upregulated during growth in macrophages derived from the human monocytic cell line THP-1. Our goal was to identify genes expressed during infection of macrophages as a means of analyzing the environmental conditions faced by the pathogen during infection, as well as to identify targets for diagnosis and treatment of tuberculosis. Selection was based upon increased expression of inhA, driven by promoters which are upregulated specifically during infection. inhA codes for an enoyl-ACP reductase, which is required for mycolic acid biosynthesis (26) and is a major target for isoniazid (INH) in M. tuberculosis. Overproduction of this enzyme confers resistance to INH in Mycobacterium smegmatis (1) and in M. tuberculosis (this communication). Specific induction of several candidate genes during growth in macrophages was confirmed by using real-time reverse transcription (RT)-PCR with molecular beacons (mbRT-PCR) (21). MATERIALS AND METHODSConstruction of the promoter trap. The plasmid promoter trap vector pJD32 was constructed by cloning a promoterless PCR-amplified M. smegmatis inhA gene containing an INH resistance mutation (1) into pYUB378, an Escherichia coli-Mycobacterium shuttle vector confe...

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Section: Discussionmentioning

confidence: 77%

Mycobacterium tuberculosis Genes Induced during Infection of Human Macrophages

Dubnau¹,

Fontán²,

et al. 2002

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“…For 2-dimensional (2-D) analysis, proteins of subcellular fractions isolated from infected BMM were separated according to their isoelectric point (pI) by isoelectric focusing as described previously by SturgillKoszycki et al (29). Ampholytes of pH 5 to 8 were used in combination with broad-range ampholytes of pH 3 to 10.…”

Section: Sds-page and Western Blottingmentioning

confidence: 99%

Identification of Mycobacterial Surface Proteins Released into Subcellular Compartments of Infected Macrophages

2000

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Considerable effort has focused on the identification of proteins secreted from Mycobacterium spp. that contribute to the development of protective immunity. Little is known, however, about the release of mycobacterial proteins from the bacterial phagosome and the potential role of these molecules in chronically infected macrophages. In the present study, the release of mycobacterial surface proteins from the bacterial phagosome into subcellular compartments of infected macrophages was analyzed. Mycobacterium bovis BCG was surface labeled with fluorescein-tagged succinimidyl ester, an amine-reactive probe. The fluorescein tag was then used as a marker for the release of bacterial proteins in infected macrophages. Fractionation studies revealed bacterial proteins within subcellular compartments distinct from mycobacteria and mycobacterial phagosomes. To identify these proteins, subcellular fractions free of bacteria were probed with mycobacteriumspecific antibodies. The fibronectin attachment protein and proteins of the antigen 85-kDa complex were identified among the mycobacterial proteins released from the bacterial phagosome.Mycobacterium spp. are the causative agents of a spectrum of diseases. The success of these pathogens lies in their ability to effectively exploit mononuclear phagocytes, where they invade, replicate, and persist within their mammalian hosts. Within these professional antigen-presenting cells, mycobacteria prevent the normal maturation of their phagosome and remain sequestered from the degradative compartments of the endosome/lysosome continuum (2,4,11,28,35). The mycobacterial phagosome is nonfusigenic with lysosomes (2, 4, 11, 28, 35) and fails to acidify due to lack of accumulation of proton ATPase complexes (30). This is a deviation from the normal maturation process, in which phagosomes of macrophages differentiate into acidic compartments containing proteases, as well as molecules promoting antigen presentation (see reference 21 for a recent review).Numerous proteins that are secreted from Mycobacterium have been described, and many of these have been postulated to contribute to the development of protective immunity (7,8,10,15,16,33,34). Surface proteins and proteins secreted by mycobacteria are likely preferential targets for the immune system early in infection. However, during chronic infection, in the absence of cell lysis and dispersal of killed bacteria, it is not clear what antigens, if any, are processed and presented for recognition by T cells. Limited acidification of the mycobacterial phagosome and its anomalous distribution of lysosomal markers indicate that it is not an optimal compartment for antigen processing (5, 31). It has previously been shown that mycobacterial lipids are actively released from the mycobacterial phagosome and traffic within the endocytic network of the host macrophage (3, 35). Mycobacterial proteins may have the same fate, providing an alternate mechanism by which bacterial proteins may intersect the antigen-processing pathway of the macrophage. ...

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“…Bacteria harvested from infected tissues preferentially metabolize fatty acids over carbohydrates (13) and the icl1 and icl2 genes, which are required for growth on lipids as a sole carbon source, are required for growth in vivo (14,15). Furthermore, icl1 and icl2, along with genes involved in the ␤-oxidation of fatty acids, are up-regulated during M. tuberculosis infection (16)(17)(18)(19)(20)(21).…”

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confidence: 99%

Lipidomics reveals control of Mycobacterium tuberculosis virulence lipids via metabolic coupling

Jain

Kim

Schelle³

et al. 2007

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

193

203

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Mycobacterium tuberculosis synthesizes specific polyketide lipids that interact with the host and are required for virulence. Using a mass spectrometric approach to simultaneously monitor hundreds of lipids, we discovered that the size and abundance of two lipid virulence factors, phthiocerol dimycocerosate (PDIM) and sulfolipid-1 (SL-1), are controlled by the availability of a common precursor, methyl malonyl CoA (MMCoA). Consistent with this view, increased levels of MMCoA led to increased abundance and mass of both PDIM and SL-1. Furthermore, perturbation of MMCoA metabolism attenuated pathogen replication in mice. Importantly, we detected increased PDIM synthesis in bacteria growing within host tissues and in bacteria grown in culture on odd-chain fatty acids. Because M. tuberculosis catabolizes host lipids to grow during infection, we propose that growth of M. tuberculosis on fatty acids in vivo leads to increased flux of MMCoA through lipid biosynthetic pathways, resulting in increased virulence lipid synthesis. Our results suggest that the shift to host lipid catabolism during infection allows for increased virulence lipid anabolism by the bacterium.lipid virulence factor ͉ metabolic flux ͉ pathogenesis ͉ PDIM ͉ sulfolipid-1

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The interaction between Mycobacterium and the macrophage analyzed by two‐dimensional polyacrylamide gel electrophoresis

Cited by 76 publications

References 28 publications

Mycobacterium tuberculosis Genes Induced during Infection of Human Macrophages

Mycobacterium tuberculosis Genes Induced during Infection of Human Macrophages

Identification of Mycobacterial Surface Proteins Released into Subcellular Compartments of Infected Macrophages

Lipidomics reveals control of Mycobacterium tuberculosis virulence lipids via metabolic coupling

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