Mycobacterium tuberculosis Genes Induced during Infection of Human Macrophages

Abstract

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...

Expression, production and release of the Eis protein by Mycobacterium tuberculosis during infection of macrophages and its effect on cytokine secretion

“…To discount the possibility that the presence of Eis in the macrophage cytoplasm may be due to intracellular lysis of phagocytosed bacteria, the cytoplasmic fraction was probed by Western blot analysis for the presence of the 16 kDa alpha crystallin protein (Acr) at 48 h after infection. Acr is a mycobacterial protein encoded by hspX that is upregulated in M. tuberculosis during infection of macrophages and is known not to be secreted from the bacteria (Dubnau et al, 2002). Acr was not detected in the cytoplasmic fraction of macrophages infected with M. tuberculosis by Western blot analysis.…”

Unraveling the Secret Lives of Bacteria: Use of In Vivo Expression Technology and Differential Fluorescence Induction Promoter Traps as Tools for Exploring Niche-Specific Gene Expression

“…The glyoxylate pathway enables bacteria to grow on acetate. Interestingly, two genes, aceA and aceB, of which the corresponding gene products catalyze subsequent steps of the glyoxylate pathway, were isolated with IVET from the animal pathogens Mycobacterium tuberculosis (56) and Yersinia enterocolitica (300), respectively. An independent SCOTS experiment equally showed that Mycobacterium tuberculosis cells contain more aceA transcript during macrophage infection (86).…”

“…Several authors adapted the auxotrophy-based selection strategy and used other essential metabolic genes (Table 1): panB, involved in pantothenate biosynthesis (218); dapB or asd, involved in diaminopimelate biosynthesis (63,78,95,224,245); metXW (159) or trpEG (26), necessary for methionine and tryptophan biosynthesis, respectively; inhA, required for mycolic acid biosynthesis (56); pyrB or thyA, necessary for de novo biosynthesis of pyrimidine nucleotides (140,156); galU, involved in galactose metabolism (133); or ribBAH, involved in riboflavin biosynthesis (76). IVET was also applied to study infection of mice by the pathogenic fungus Histoplasma capsu-latum.…”

P27-PPE36 (Rv2108) Mycobacterium tuberculosis Antigen – Member of PPE Protein Family with Surface Localization and Immunological Activities

“…However, this variability can not be extended to all pe/ppe family members since some are in fact conserved across strains and species (Cubillos-Ruiz et al, 2008). It has then been suggested that the PPE proteins may play a role in the virulence of Mtb (Rindi et al, 1999;Li et al, 2005), in the maintenance of bacterial growth in macrophages (Camacho et al,1999;Dubnau et al, 2002;Hou et al, 2002;Li et al, 2005;Sassetti et al, 2003) and in the regulation of bacterial iron starvation and oxidative stress responses (Rodriguez at al., 1999;Rodriguez at al., 2002). In addition, PPE might be a target for the protective immune response in experimental mouse models (Skeiky et al, 2000).…”