Identification of aMycobacterium tuberculosisGene That Enhances Mycobacterial Survival in Macrophages

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

“…Recombinant protein was then induced, harvested and purified as per the manufacturer's protocol (Novagen). The one step Ni-NTA purification process resulted in a single protein band, of molecular mass similar to that predicted for Eis (42 kDa) (Wei et al, 2000). Purity of the recombinant Eis was greater than 99 %, as determined by scanning densitometry analysis of SDS-PAGE gels, and the His-tagged Eis protein also reacted with the anti-Eis antibody (data not shown).…”

“…For growth on solid culture media, Middlebrook 7H10 (Difco) with 10 % OADC was used. Mycobacterial inocula for infection of U-937 cells with MtbRa/gfp and MtbRaDeis/gfp were prepared as previously described (Wei et al, 2000). For growing Escherichia coli transformants, LuriaBertani (LB) broth or agar with 200 mg hygromycin B ml 21 or 100 mg ampicillin ml 21 was used.…”

“…One-millilitre aliquots of RPMI containing 2610 5 U-937 cells were plated into each well of a 24-well tissue culture plate (Falcon) containing sterile glass coverslips. Cells were infected the next day as described previously (Wei et al, 2000) with MtbRa/gfp and MtbRaDeis/gfp at an m.o.i. of 20.…”

“…U-937 macrophage monolayers were prepared by transformation of cells in T75 flasks (Corning) using phorbol myristic acetate (PMA), as previously described (Wei et al, 2000).…”

“…The eis (enhanced intracellular survival) gene of M. tuberculosis is only found in members of the M. tuberculosis complex and has been shown to confer enhanced survival on Mycobacterium smegmatis in U-937 macrophages and human macrophages (Wei et al, 2000). The Eis protein is a 42 kDa protein that is secreted when M. tuberculosis is grown in liquid culture (Dahl et al, 2001).…”