Chemical definition, cloning, and expression of the major protein of the leprosy bacillus

Abstract

The decline in prevalence of leprosy is not necessarily matched by a fall in incidence, emphasizing the need for new antigens to measure disease transmission and reservoirs of infection. Mycobacterium leprae obtained from armadillo tissues was disrupted and subjected to differential centrifugation to arrive at preparations of cell wall, cytoplasmic membrane, and cytosol. By committing 0.3 g of M. leprae to the task, it was possible to isolate from the cytosol and fully define the major cytosolic protein. Amino-terminus sequencing and chemical and enzymatic cleavage, followed by more sequencing and fast atom bombardment-mass spectrometry of fragments, allowed description of the entire amino acid sequence of a protein of 10,675-Da molecular mass. The sequence derived by chemical means is identical to that deduced previously from DNA analysis of the gene of a 10-kDa protein, a GroES analog. The work represents the first complete chemical definition of an M. leprae protein. PCR amplification of the 10-kDa protein gene, when cloned into Escherichia coli with a pTRP expression vector, allowed production of the recombinant protein. Chemical analysis of the expressed protein demonstrated that it exactly reflected the native protein. The recombinant major cytosolic protein appears to be a promising reagent for skin testing, still probably the most appropriate and pragmatic means of measuring incidence of leprosy.

Characterization of antigens recognized by new monoclonal antibodies raised against culture filtrate proteins of Mycobacterium bovis bacillus Calmette-Guérin

“…BCG subcellular fractions were obtained as previously described, with minor modi¢cations [9,10]. Brie£y, 7-day-old BCG cells were suspended at 0.5 g of wet bacteria ml 3I of lysis bu¡er (PBS, 0.05% Tween 80, 0.8 mg ml 3I phenylmethylsulfonyl £uoride, 156 Wg ml 3I benzamidine, 30 Wg ml 3I pepstatin A, 10 mg ml 3I iodoacetamide, 50 Wg ml 3I NK-ptosyl-L-lysine chloromethyl ketone, 3 mg ml 3I EDTA, and 0.26 mg ml 3I 1,10-phenanthroline).…”

“…The supernatant of the 27 000Ug centrifugation was centrifuged at 100 000Ug for 2 h. The pellet was washed and resuspended in lysis bu¡er (cell membrane-enriched fraction). The 100 000Ug supernatant made up the cytosol-enriched fraction [10].…”

Advances in Proteomics of Mycobacterium leprae

“…Initially, research groups set out to search for new M. leprae proteins with the aim of identifying new target antigens for immunological applications such as use in diagnosis and as vaccines. Employing classical biochemical methodology, recombinant DNA technology, monoclonal antibodies and patients' sera, nearly 37 antigens were documented [31][32][33][34][35][36][37][38][39] prior to the proteomics era. As an extension of this, since 1996 several investigations have described the search for novel proteins, conducted using many different proteomic platforms [12][13][14][15][16][17] including one-dimensional electrophoresis, two-dimensional gel electrophoresis, electroblotting, liquid chromatography for protein separation and Edman sequencing and mass spectrometry analysis for the identification of proteins.…”

“…Past three decades have seen several major developments in elucidation of proteins of M. leprae [31][32][33][34][35][36][37][38][39], primarily, in terms of their immunoreactivities. The major disadvantage of such approaches was missing of non-immunogenic proteins.…”

Leprosy patients with lepromatous disease recognize cross-reactive T cell epitopes in theMycobacterium leprae10-kD antigen

“…Mycobacterium leprae remains one of the few bacterial pathogens of humans that has not been cultivated in vitro and therefore development of a successful vaccine depends on the identification of antigens and epitopes that induce protective responses across the leprosy disease spectrum. Several biochemical, immunological and molecular approaches have been recently used for identification and characterization of protein antigens of the leprosy bacillus [1][2][3][4]. It has long been recognized that leprosy patients with selflimiting tuberculoid leprosy show high T cell reactivity, while patients with disseminated lepromatous form of the disease show absent to low levels of T cell reactivity [5].…”