Gene Expression Profile and Immunological Evaluation of Unique Hypothetical Unknown Proteins of Mycobacterium leprae by Using Quantitative Real-Time PCR

Department of Microbiology, Immunology and Pathology. Colorado State University, Fort Collins, Colorado 80523-2025.
Clinical and vaccine Immunology: CVI (Impact Factor: 2.47). 12/2012; 20(2). DOI: 10.1128/CVI.00419-12
Source: PubMed


Cell mediated immunity (CMI) based in vitro interferon-γ released assay (IGRA) to Mycobacterium leprae specific antigens has potential as a promising diagnostic means to detect those individuals in the early stages of M. leprae infection. Diagnosis of leprosy is a major obstacle toward ultimate disease control, and has been compromised in the past by the lack of specific markers. Comparative bioinformatic analysis among mycobacterial genomes identified potential M. leprae unique proteins called "hypothetical unknowns". Due to massive gene decay and the prevalence of pseudogenes, it is unclear whether any of these proteins are expressed or are immunologically relevant. Here, we performed cDNA based quantitative real time PCR to investigate the expression status of 131 putative open reading frames (ORFs) encoding hypothetical unknowns. Twenty six of the 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 in M. leprae. Fifteen out of 26 selected antigen candidates were expressed and purified in Escherichia coli. The seroreactivity to these proteins using the pooled sera of lepromatous leprosy patients and cavitary tuberculosis patients revealed that 9 out of 15 recombinant hypothetical unknowns elicited M. leprae specific immune responses. These nine proteins may be good diagnostic reagents to improve both sensitivity and specificity in detection of individuals with asymptomatic leprosy.

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    ABSTRACT: Langerhans cells participate in the immune response in leprosy by their ability to activate T cells that recognize the pathogen, Mycobacterium leprae, in a langerin dependent manner. We hypothesized that langerin, the distinguishing C-type lectin of Langerhans cells, would recognize the highly mannosylated structures in pathogenic Mycobacterium spp. The coding region for the extracellular and neck domain of human langerin was cloned and expressed to produce a recombinant active trimeric form of human langerin (r-langerin). Binding assays performed in microtiter plates, by 2-D Western blot, and by surface plasmon resonance demonstrated r-langerin possessed carbohydrate dependent affinity to glycoproteins in the cell wall of M. leprae. This lectin, however, yielded less binding to mannose capped lipoarabinomannan (ManLAM) and even lower levels of binding to phosphatidylinositol mannosides. However, the superoxide dismutase C (SodC) protein of the M. leprae cell wall was identified as a langerin reactive ligand. Tandem mass spectrometry verified the glycosylation of a recombinant form of M. leprae SodC (rSodC) produced in Mycobacterium smegmatis. Analysis of r-langerin affinity by surface plasmon resonance revealed a carbohydrate dependent affinity of rSodC (KD=0.862 μM) that was 20-fold greater than for M. leprae ManLAM (KD=18.69 μM). These data strongly suggest that a subset of the presumptively mannosylated M. leprae glycoproteins act as ligands for langerin and may facilitate the interaction of M. leprae with Langerhans cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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