Novel 33-kilodalton lipoprotein from Mycobacterium leprae

Abstract

A novel Mycobacterium leprae lipoprotein LpK (accession no. ML0603) was identified from the genomic database. The 1,116-bp open reading frame encodes a 371-amino-acid precursor protein with an N-terminal signal sequence and a consensus motif for lipid conjugation. Expression of the protein, LpK, in Escherichia coli revealed a 33-kDa protein, and metabolic labeling experiments and globomycin treatment proved that the protein was lipidated. Fractionation of M. leprae demonstrated that this lipoprotein was a membrane protein of M. leprae. The purified lipoprotein was found to induce production of interleukin-12 in human peripheral blood monocytes. The studies imply that M. leprae LpK is involved in protective immunity against leprosy and may be a candidate for vaccine design.

FIG. 1.

(A) Comparison of the deduced amino acid of M. leprae lpk (LpK-ML) to its homologue in M. tuberculosis (LpK-MT). The upper amino acid sequence is that of M. leprae, and the lower sequence is that of M. tuberculosis. Identical amino acids at each position are marked by asterisks, conservative substitutions are indicated by one dot, and nonconservative substitutions are indicated by a space. (B) Comparison of the hydropathy plot of M. leprae lpk gene product with its M. tuberculosis homologue by the algorithm of Kyte and Doolittle. Positive regions denote regions of relative hydrophobicity.

FIG. 2.

Expression and purification of the LpK and its fusion products. (A) Western blot with anti-GFP antibody without (lanes 1) and with (lanes 2) IPTG induction of pGFP-lpk-transformed E. coli. (B) Coomassie staining with mock-transformed (lanes 1) and pG-lpk-transformed (lanes 2) E. coli. (C) Western blot of the same protein as that for panel B, using a monoclonal anti-His antibody. (D) Silver staining of the purified protein LpK, a column pass through (lanes 1) and proteins eluted with imidazole (lanes 2).

FIG. 3.

(A) [¹⁴C]glycerol radiolabeling of the expressed protein LpK. E. coli tranformants of lpk were cultured up to an optical density of 0.5; incubated with [¹⁴C]glycerol for 1 h (lanes 1), 2 h (lane 2), or 5 h (lane 3); and then immunoprecipitated. The proteins were run on an SDS-12% polyacrylamide gel, and then autoradiography was performed. (B) Globomycin inhibits the processing of the prolipoprotein to lipoprotein. E. coli were grown in the presence (+) or absence (−) of globomycin to stationary phase. Lysates were run on gel and stained with Coomassie brilliant blue.

FIG. 4.

Presence of the native protein LpK in M. leprae grown in vivo. M. leprae purified from armadillo liver was fractionated into cell wall (lane 1), cell membrane (lane 2), and cytosolic (lane 3) fractions. A Western blot with polyclonal antibody raised against LpK revealed the presence of LpK in the membrane preparation of M. leprae.

FIG. 5.

M. leprae LpK induces IL-12 p40 from human blood monocytes. Peripheral blood monocytes were isolated, and the ability of LpK to stimulate IL-12 p40 was measured as described in Materials and Methods. IL-12 p40 cytokine induction was then assessed, with (▨) or without () polymyxin B, with LpK (columns 1), LpC (ML1699) (columns 2), and LPS (columns 3). lpc was identified from the database as a putative lipoprotein coding gene that was expressed and purified from inclusion bodies as a 39-kDa protein in E. coli. Values are expressed as the mean ± the standard deviation performed in triplicate.