Proc. Nadl. Acad. Sci. USA Vol. 85, pp. 1917-1921, March 1988 Immunology Immunological significance of Mycobacterium leprae cell walls (cell-mediated immunity/delayed-type hypersensitivty/peptidoglycan-protein complex/leprosy/precursor frequency analysis) JOHANNE MELANCON-KAPLAN*, SHIRLEY Wu HUNTERt, MICHAEL MCNEILt, CAROL STEWARTt, ROBERT L. MODLINf, THOMAS H. REAt, JACINTO CONVIT§, PADMINI SALGAME*, VIJAY MEHRA*, BARRY R. BLOOM*, AND PATRICK J. BRENNANt¶ *Department of Microbiology and Immunology, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY 10461; tDepartment of Microbiology, Colorado State University, Fort Collins, CO 80523; tSection of Dermatology, University of Southern California School of Medicine, Los Angeles, CA 90033; and 1lnstitute de Biomedicine, Caracas 101, Venezuela Contributed by Barry R. Bloom, November 2, 1987 ABSTRACT Cell walls of Mycobacterium leprae, pre- positive healthy contacts of lepromatous patients or from pared by differential solvent extraction, were shown to contain blood or lesions of patients with tuberculoid leprosy, only a arabinogalactan, mycolates, and peptidoglycan. In addition, small proportion of clones tested are responsive to the amino acid analysis revealed the unexpected presence of large known serologically defined recombinant antigens (ref. 5; amounts of protein that retained potent immunological reac- J.M.-K., R.L.M., and B.R.B., unpublished data). tivity. Purified cell walls stimulated proliferation of T cells For many years, significant immune responses to purified from tuberculoid, but not from lepromatous leprosy, patients cell walls of various mycobacteria have been sporadically and elicited delayed-type hypersensitivity skin reactions in reported. For example, cell walls from Mycobacterium guinea pigs and patients sensitized to M. leprae. Analysis ofthe tuberculosis can evoke delayed-type hypersensitivity (DTH) precursor frequency of antigen-reactive human peripheral T responses (6), and, of course, adjuvant activity is associated cells revealed that as many cells (41/6000) proliferate to with the muramyl-dipeptide of the basic peptidoglycan (7). antigen contained in cell walls as to intact M. keprae. Sequen- The present work explores the possibility that M. leprae cell tial removal of mycolates and arabinogalactan resulted in a wall contains antigens important for cell-mediated immunity large peptidoglycan-protein complex that retained all the to this organism. immunological activity. This immunological reactivity and the inherent protein were destroyed by proteolysis. Thus, cell wall protein is a major contributor to cell-mediated immune reac- METHODS AND MATERIALS tivity to this pathogenic mycobacterium. Preparation of Cell Walls of M. eprae. Lyophilized M. leprae (1.42 g) prepared from infected armadillo livers (8) Leprosy is a chronic infectious disease that afflicts 10-13 was exhaustively extracted with CHC13/CH30H (2:1) to million people, primarily living in developing countries (1). remove all lipids, disrupted in a French press followed by Although the incidence of leprosy is declining in many parts sonication and further extraction with refluxing 70% ethanol of the world, largely due to greater control efforts, the cost in water to remove lipoarabinomannan, lipomannan, other of multidrug therapy to combat emergent drug resistance of soluble carbohydrates, and soluble proteins (9). The residue Mycobacterium leprae is high, and the development of an was washed extensively with water to yield 671 mg of cell effective vaccine is widely agreed to offer the best hope for wall insoluble fraction (CWIF). disease eradication (2, 3). Because M. leprae is one of few Cell wall core (CWC) was prepared by stirring CWIF (10 major pathogens of man that has not been successfully mg) in 1-2 ml of 2% NaDodSO4 for 2 hr at 50'C; this cultivated in vitro, the principal available source of antigens extraction was repeated four times. The final insoluble for study and vaccines remains infected tissues of the residue was further extracted 10 times with phosphate- nine-banded armadillo Dasypus novemcinctus, which is in- buffered saline (PBS), followed sequentially by water and evitably limited and costly. Consequently, immunological acetone to yield 6.5 mg of CWC. and molecular biological approaches that permit identifica- Mycolic acids were removed from CWC (10 mg) by tion and production of protective antigens are needed. Thus stirring in 5 ml of 0.5% KOH in C2H5OH at 370C for 48 hr. far, using murine monoclonal antibodies, genes for six major The reaction mixture was centrifuged, and the pellet was antigens of M. leprae have been isolated from Agtll libraries washed three times with 5 ml each of water, ethanol, and (4). ether to yield 6.3 mg of demycolated CWC. To destroy the From a variety of studies, cell-mediated immunity, which covalently bound arabinogalactan, 10 mg of demycolated is regulated by specifically sensitized T lymphocytes, is CWC was stirred into 3 ml of 0.05 M NaIO4 buffered with clearly required for protection and resistance to leprosy (1). 0.03 M sodium acetate, pH 4.0, and kept under dark condi- The disease encompasses an immunological spectrum. At tions at 250C for 42 hr. The insoluble residue after centrifu- one pole, lepromatous leprosy, patients selectively lack gation at 20,000 x g for 20 min was washed extensively with cell-mediated immunity to antigens of M. leprae and fail to water and reduced with NaBH4 (1.25 mg in 250 Al) at 250C restrict the growth of the pathogen; at the tuberculoid end of for 1 hr. The insoluble residue was again thoroughly washed the spectrum, the patients exhibit cell-mediated immunity to with water, and the residual polyols were removed by gentle antigens of M. leprae and develop one or a few sharply hydrolysis (500 1.l of HCl, pH 1.0, at 150C for 18 hr), defined local lesions that contain only few acid-fast bacilli. followed by repeated washing. Exactly 5 mg (dry weight) of However, when we and other workers derived M. leprae- specific CD4 + (helper T cell) clones from strongly lepromin- Abbreviations: CWIF, cell wall insoluble fraction; CWC, cell wall core; PPC, peptidoglycan-protein complex; DTH, delayed-type The publication costs of this article were defrayed in part by page charge hypersensitivity; GC, gas chromatography; PBS, phosphate- payment. This article must therefore be hereby marked "advertisement" buffered saline. in accordance with 18 U.S.C. §1734 solely to indicate this fact. ITo whom reprint requests should be addressed. 1917 Downloaded by guest on September 26, 2021 1918 Immunology: Melancon-Kaplan et al. Proc. Natl. Acad Sci. USA 85 (1988) this peptidoglycan-protein complex (PPC) free of mycolates Table 1. Chemical composition of the cell wall fractions of and arabinogalactan was recovered. M. leprae Proteolytic digestion of CWIF or CWC was accomplished Cell wall fraction,* weight % by adding protease (either one-half the substrate weight of proteinase K or Pronase from Streptomyces griseus; type Whole Demycolyl Cell wall XXI, Sigma) to the sample in 2 ml of PBS containing 0.5% Component M. leprae CWIF CWC CWC PPC Triton X-100. The mixture was sonicated and incubated at Arabinogalactan 15.9 12.9 22.5 33.1 0.6 37°C for 4 hr and centrifuged at 20,000 x g for 20 min; then Other the pellet was further digested under identical conditions. carbohydrates 20.9 2.0 0 0 0 The pellet was again recovered by centrifugation, washed Mycolates 12.8 35.0 37.0 0 0 repeatedly with water, and Iyophilized to yield 6.7 mg of Peptidoglycan 6.3 6.1 6.9 10.7 18.4 protease-digested CWIF. Protein 50.8 42.8 25.9 38.1 60.6 Analytical Procedures. The neutral sugar content and, *Arabinogalactan was estimated by hydrolysis of cell wall fractions from this content, the arabinogalactan content of cell wall with 2 M CF3COOH and GC of the alditol acetates with inositol fractions were measured by hydrolysis of 100-ug samples added as an internal standard. Arabinose represented 77.3% of the containing 9 of inositol added as internal standard with 2 neutral sugars and galactose represented 18.6% with a trace of pg mannose (0.4%). Mycolates were estimated by weighing the sapon- M CF3COOH, conversion to the corresponding alditol ace- ifiable lipids obtained after treatment of cell wall fractions with tates, and resolution by gas chromatography (GC) on a 0.5% KOH in C2HOH. Peptidoglycan and protein amino acids Durabond-1 fused silica capillary column (30 m x 0.25 mm, were estimated as described in the legend for Fig. 2; peptidoglycan 0.25-,um film thickness; J&W Scientific, Rancho Cordova, content was then calculated using the total weights ofglucosamine, CA) (10). To analyze amino acid and aminoglycoside con- muramic acid, diaminopimelic acid, and the weight of one mol tent, 100-pug samples of cell wall were hydrolyzed with 4 M equivalent of alanine, glycine, and glutamic acid as compared with or 6 M HCl at 115°C for 16 hr and converted to the diaminopimelic acid. corresponding N (O for serine and threonine)-heptafluoro- heptafluorobutyryl isobutylglycoside isobutyl esters for glu- butyryl isobutyl esters, for amino acids, and N- or O- cosamine and muramic acid (11). The derivatives were then A chromatographed on the described Durabond-1 capillary 40-0 column. a-Aminoadipic acid was included in each assay as an internal standard to allow measurement. The amino acid E and aminoglycoside assignments were confirmed by gas 30 chromatography and mass spectrometry using electron- impact ionization. CD20 Lymphocyte Transformation and Precursor Frequency Analysis. The precursor frequency of human peripheral L 0 blood T lymphocytes reactive to various antigen prepara- tions from M. leprae was determined by a modified limiting L00 dilution procedure (12). Briefly, T lymphocytes were iso- lated from the blood of lepromin-positive tuberculoid lep- LL BL BT/11 NORMAL rosy patients or contacts, and replicate cultures were estab- I~ n 27 12 25 12 AMINO ACIDS AND AMINO SUGARS OF A OF M.