ABSTRACT
Improved enzyme-linked immunosorbent assay (ELISA) methods have been developed for the determination of femtomole amounts of mycothiol (MSH), the main low-molecular-weight thiol in mycobacteria. The immunoassays utilize an affinity-purified rabbit polyclonal antibody that is highly specific for the pseudodisaccharide moiety of MSH. MSH was first biotinylated by the thiol-specific reagent 3-(N-maleimidopropionyl)biocytin. The MSH-biotin adduct was then captured with immobilized avidin and detected with anti-MSH antibody (biotin-capture ELISA) or was captured with immobilized anti-MSH antibody and detected with alkaline phosphatase-labelled avidin (MSH-capture ELISA). The MSH-capture ELISA was the most sensitive method, measuring as little as 0.3 fmol of MSH. Methods for biotinylating MSH directly from Mycobacterium spp. are described. The MSH-capture ELISA was tested for the detection of M. avium seeded in human urine or cerebrospinal fluid samples and for screening mutant M. smegmatis strains to detect MSH production.
Subject(s)
Disaccharides/analysis , Mycobacterium Infections, Nontuberculous/cerebrospinal fluid , Mycobacterium smegmatis/chemistry , Pyrazoles , Sulfhydryl Compounds/analysis , Animals , Antibodies , Antibody Specificity , Biotinylation , Cerebrospinal Fluid/microbiology , Cysteine , Enzyme-Linked Immunosorbent Assay/methods , Glycopeptides , Humans , Indicators and Reagents , Inositol , Mycobacterium Infections, Nontuberculous/diagnosis , Mycobacterium smegmatis/growth & development , Mycobacterium smegmatis/isolation & purification , Rabbits , Sensitivity and Specificity , Urine/microbiologyABSTRACT
Mycothiol (MSH) is the major low molecular weight thiol in mycobacteria. Two chemical mutants with low MSH and one with no MSH (strain 49) were produced in Mycobacterium smegmatis mc2155 to assess the role of MSH in mycobacteria. Strain 49 was shown to not produce 1-d-myo-inosityl-2-amino-2-deoxy-alpha-d-glucopyranoside (GlcN-Ins), an intermediate in MSH biosynthesis. Relative to the parent strain, mutant 49 formed colonies more slowly on solid media and was more sensitive to H2O2 and rifampin, but less sensitive to isoniazid. Complementation of mutant 49 with DNA from M. tuberculosis H37Rv partially restored production of GlcN-Ins and MSH, and resistance to H2O2, but largely restored colony growth rate and sensitivity to rifampin and isoniazid. The results indicate that MSH and GlcN-Ins are not essential for in vitro survival of mycobacteria but may play significant roles in determining the sensitivity of mycobacteria to environmental toxins.
Subject(s)
Disaccharides/metabolism , Glucosides/genetics , Mycobacterium smegmatis/genetics , Pyrazoles , Sulfhydryl Compounds/metabolism , Culture Media , Cysteine/biosynthesis , Drug Resistance, Microbial , Genetic Complementation Test , Glucosides/biosynthesis , Glycopeptides , Hydrogen Peroxide/pharmacology , Inositol/analogs & derivatives , Inositol/biosynthesis , Isoniazid/pharmacology , Mutagenesis , Mycobacterium smegmatis/drug effects , Mycobacterium smegmatis/growth & development , Mycobacterium smegmatis/metabolism , Rifampin/pharmacologyABSTRACT
Mycothiol (MSH) is a glycosylated derivative of N-acetylcysteine that may have antioxidant functions in mycobacteria and other actinomycetes. To develop a highly specific assay for MSH, we capitalized on the selective binding of thiols to a maleimide residue linked to bovine serum albumin and employed affinity-purified polyclonal antibody and an enzyme-linked secondary antibody for detection. The assay was shown to be specific and to detect MSH at levels as low as 0.1 pmol when conducted in the form of a microtiter plate-based ELISA. A similar, nitrocellulose membrane-based immunoassay was shown to be useful for qualitative detection of MSH-producing bacterial colonies.
