Adherence of Mycobacterium lepraemurium to tissue culture cells.

Adherence of Mycobacterium lepraemurium to tissue culture cells was examined and compared with that of M microti. M lepraemurium used in the present study was maintained on 1% Ogawa egg yolk medium. This microbe adhered to HEp-2 cells much more than A31 or McCoy cells, though the adherence was unusually low as compared with the large infectious dose. Frequency distribution of the number of bacteria agreed nearly with Polya-Eggenberger distribution. The pretreatment of M lepraemurium with heat or protease increased the adherence of the bacteria to HEp-2 cells, whereas the pretreatment with lipase or hyaluronidase retained the adhering ability. The pretreatment of M microti with heat or protease produced the same effect on the adherence as that of M lepraemurium. These results suggest that adherence of M lepraemurium to HEp-2 cells is prevented by protein-like material on the surface of the bacteria, and that the adherence is independent of specific adhesin-receptor interaction. to tissue culture cells. INTRODUCTION MATERIALS AND ME1HODS 285 Bacterial adherence to tissue cell surface is of importance at the first step of the infectious process. The adherence has a more or less different meaning according to the habitat of microbes. For obligate intracellular parasites, the adherence is followed by their invasion into and multiplication within the host cells without colonization on the surface, whereas colonization generally occurs in common bacteria such as Escherichia coli and Streptococcus. Although M. lepraemurium is not an 'obligate' intracellular parasite because of its capability to grow on artificial culture media, its infectious process is very close to that of obligate intracellular parasites. Bacteria. Since the bacillary preparation of in vivo grown M. lepraemurium is contaminated by host tissue components and various treatments for taking off the contamination modify the surface structure of the organisms, the bacillary suspension was prepared from in vitro grown organisms. In spite of many studies on the adherence of various microbes, no report has been made on that of M lepraemurium. The present paper deals with a preliminary experiment on the adherence of M lepraemurium and M microti Seven strains of in vitro grown M lepraemurium (strains Hawaii, Kurume, Osaka, Kumamoto, Keishicho, Douglas and Odessa) were maintained on 1% Ogawa egg yolk medium. Unless mentioned otherwise, the 78th passaged Hawaiian strain was used throughout the experiments. M microti (TC-48 strain) was maintained on 1% Ogawa whole egg medium. Cell culture. Established cell lines used were HEp-2 (derived from human epiglottis carcinoma), A31 (a cloned strain of the 3T3 cell derived from a BALB/c mouse embryo) and 286 M. Katoh and Y. Matsuo McCoy cells (derived from human synovial fluid). The former two are epithelial-like cells, and the latter is a fibroblastic cell. They have been routinely cultured in -Dulbecco's modified Eagle mediu~, (D-MEMi Ni~sui Seiyaku Co., Ltd., Japan) supplemented with 10% caILserum-(Grand Island Biological Co., U.S. A.), per ml 100 u of penicillin and 100 pg of streptomycin. Preparation of bacillary suspension. The bacteria grown on the medium · fot 1 to 3 months were washed twice with Dulbecco's phosphate buffered-saline (PBS) and resuspended in antibiotic-free D-MEM. The bacillary suspension was gently dispersed with a teflon homogenizer and centrifuged at 180 x g for 5 · min. The supernatant fluid was decanted and adjusted to 0. D. 0. 30 at 540 nm with Coleman Junior spectrophotometer. Adherence assay. · The cells at a concentration of 2 x 104 were introduced into each chamber of four-chambered tissue culture slides (Lab-Tek Products, Miles Laboratories, Inc., U. S. A.) and cultivated overnight. The medium of cultured cells was replaced with the bacillary suspension, and the. cells were further inc;:ubated statically at 37° C. Thr~e h after -incubation, the cells were washed with D-MEM, fixed with methanol and acid-fast stained. The number of bacteria attached was counted tor the total number of 200 cells under a light microscope ( x 1, 000). The area to read was a center portion on each sample where the distribution of cells was relatively even. Adherence was given as the mean number of bacteria per cell and standard deviation (S. D.). Pretreatment of bacteria. a) Enzymatic treatments of bacteria were carried out according to the method by Saunders and Miller5> : PBSwashed bacteria were suspended in each buffered enzyme solution and incubated at 37° C for 60 min. The enzymes utilized are presented in Table 1. Table 1. Incubation conditions for enzymatic pretreatment of bacteria Enzyme Concentration Incubation buffer Protease (B. subtilis; Sigma, type VIII) 10 U/ml 0. 046 M Tris-l!Cl (pH 7. 5) with 0. 0115 M CaCl2 Trypsin (Bovine pancreas; Sigma, type III) 10, 000 BAEE U/mla) 0. 046 M Tris-HCl (pH 7. 5) with 0. 0115 M CaCl2 Lipase (wheat germ; Sigma, type I) lOU/ml Hyaluronidase (bovine testes; Sigma, type IV) 980 NF U /mlb) 0.15 M Na acetate (pH 6. 5) 0.1 M Na phosphate (pH 6. 5) · with 0. 15 M NaCl PBS-washed bacteria were suspended in each buffered enzyme solution indicated, and incubated at 37° C for 60 min. ' ' ' ' a) N-Benzoyl'-L-arginine ethyl ester units. b) National Formulary units. b) The heating of bacteria was done in boiling water for 30 min. After e_ach pretreatment, the bacteria were washed 3 times with PBS and resuspended in D-MEM. Statistical methods. Statistical analyses wer~ made with the Student' t-test and/or by the Chi-squared test.