Plainsman Research Center at Walsh, Colorado, 2002

Abstract

Kevin Larson shares the results of his research at a field day, August 29, 2002 at the Plainsman Research Center at Walsh, Colorado.

The progression of leprosy disease highly correlates with predominant type 1/type 2 cytokines and proliferation of Th1/Th2 cells in response to M. leprae. Th1 immunity is thought to be protective to M. leprae infection but Th2 immunity is pathogenic. The primary objective of this dissertation was to identify and characterize a panel of new protein based T cell antigens, particularly those that induce Th1 immunity, in order to understand host-M. leprae interaction and to develop diagnostic tests for leprosy. Cellular mediated immunity (CMI) based in vitro interferon-γ released assay (IGRA) to M. leprae specific antigens is known as the most promising diagnostic test to detect those individuals in the early stages of M. leprae infection. Here, we identified two classes of antigens using advanced genomic and proteomic approaches. Comparative bioinformatic analysis among mycobacterial genomes identified potential M. leprae unique proteins called "hypothetical unknowns" from the M. leprae genome. We performed cDNA based quantitative real time PCR to investigate the expression status of 136 putative open reading frames (ORFs) encoding hypothetical unknowns. Twenty six of M.leprae specific antigen candidates showed significant levels of gene expression compared to that of ESAT-6 (ML0049) which is an important T cell antigen of low abundance. Fifteen out of 26 selected antigen candidates were expressed and purified in E. coli. Serological analysis using the sera obtained from lepromatous leprosy patients and cavitary tuberculosis patients clearly indicated that 9 out of 26 selected proteins elicited M .leprae specific immune responses. M. leprae membrane antigen (MLMA) induces a strong Th1 immune response. However, the large amount of lipomannans/lipoarabinomannans (LMs/LAMs) content hindered the further processing of this fraction and the optimal use of this fraction as diagnostic reagents in a CMI based assay. A simple sodium carbonate treatment successfully enriched membrane proteins into MLMA-SP (alkali soluble proteins of MLMA) and excluded the most hydrophobic lipids. Subsequent IFN-γ release assay (IGRA) of peripheral blood mononuclear Cells (PBMCs) from 16 mycobacterial laboratory/leprosy clinic workers revealed that MLMA-SP induced higher levels of IFN-γ secretion in the group exposed to leprosy patients, as compared to the group with environmental/professional mycobacterial exposure. Therefore, MLMA-SP and 9 of the hypothetical unknowns may be good diagnostic reagents to improve both sensitivity and specificity in detection of individuals with asymptomatic leprosy. Bacterial lipoproteins, containing an N-acyl di-O-acylglyceryl-cysteine unit, have been known to induce strong CMI response to bacterial infections through Toll like receptor (TLR-2) signaling. CD1-restricted T cells in response to mycobacterial lipid antigens efficiently and rapidly induce cytolytic T cell response to kill intracellular pathogens. In this work, we developed simple proteomic and chemical methodologies to enrich lipoproteins of low abundance from MLMA. T cell reactivity to these lipoprotein enriched fractions combined with proteolysis provided the first evidence that bacterial lipoproteins are a new class of CD1a restricted antigens recognized by a CD1a restricted T cell line derived from skin lesions of a leprosy patient. Further proteomic and genomic analyses identified lipoproteins, ML1086, ML2095 and ML2446, as CD1a antigen candidates. However, these three lipoproteins expressed in recombinant form in M. smegmatis were unable to activate a CD1a restricted T cell line (LCD4.15) though the acylation of all recombinant proteins was biochemically verified. The possible reasons for this result are: 1) that the antigen recognized by LCD4.15 would be another lipoprotein of the least abundance making it difficult to identify using current proteomic analysis; 2) that the bacterial lipoproteins of in vitro grown M. smegmatis could present different acylation patterns from those of in vivo grown M. leprae. The presence of acylated cysteine at the N terminus of lipoprotein is primarily predicted by bioinformatic analysis but rarely verified by biochemical analysis. Lipopeptides of mycobacterial lipoproteins were prepared by proteolysis and organic extraction, and analyzed by gas chromatography-mass spectrometry (GC-MS) and quadrapole time of flight liquid chromatography/mass spectrometry (Q-TOF LC/MS). GC-MS results revealed that the major fatty acids of the glyceryl portion of the lipoproteins are tuberculostearic acid, stearic acid, oleic acid and palmitic acid, which are also the dominant fatty acids of mycobacterial phospholipids. The N-linked fatty acid of the cysteine residue is predominantly palmitic acid. Interestingly, Q-TOF LC/MS data showed that mycobacterial lipoproteins appeared as di- and tri-acylated mature forms, but also had an array of fatty acids in various combinations. Di-acylated lipopeptides appeared more frequently than tri-acylated lipopeptides. The present study demonstrated a distinct fatty acid composition of the lipid portion of mycobacterial lipoproteins compared to other bacterial lipoproteins. Moreover, we defined the full spectrum of acylation pattern in tri- or di-acyled mycobacterial lipoproteins, the impact of which in host-bacteria responses is subject to further immunological investigation. Here, we have identified and characterized a panel of new M. leprae derived protein antigens by developing novel biochemical and proteomic techniques. The knowledge and methodologies presented in this dissertation will allow for further understanding of the physiology and pathogenicity of M. leprae in human hosts.