Effect of p-Glucuronidase Inhibitor from Mycobacterium tuberculosis Against Microbicidal Activity in Phagocytes of Guinea

j3-Glucuronidase inhibitor extracted and purified from culture filtrate of Mycobacterium tuberculosis H37Rv significantly reduced the microbicidal activity of guinea pig peritoneal macrophages against Candida parapsilosis. It did not have any effect on the antimicrobial action of polymorphonuclear cells against Staphylococcus aureus 209P. A lot of studies have been made on biologically active substances in cell constituents of Mycobacterium tuberculosis, especially on lipid fractions. However, little is known of substances excreted from the cells with the exception of the purified protein derivative0 . When mycobacteria are cultured in liquid media, proteinaceous substances are secreted into the media, and the amount is greater with M tuberculosis than with mycobacteria other than tubercle bacilli. We attempted to separate any biologically active substances in the culture filtrate of M. tuberculosis, and found a substance possessing an inhibitory activity against several lysosomal enzymes'. Recently, we isolated another proteinaceous substance with a molecular weight of approximately 25, 0004'. The inhibitory action is non-competitive against j3-glucuronidase obtained from polymorphonuclear leukocytes (PMNs) an:l peritoneal macrophages (PM¢s) of guinea pigs. Of interest is the fact that the activity is optimum at pH of about 4. 5 which is almost the same as the pH within the phagolysosome. The present short communication describes the effect of this particular jS-glucuronidase inhibitor on survival of microbes ingested by cultured phagocytes. The inhibitor was isolated from unheated culture filtrates of ]yf. tubercilosis H37Rv by the method described previously>. PMNs were prepared according to the method of Simmons et al. 8' using a male guinea pig, Hartley strain, weighing approximately 400 g. Eighteen hr after intraperitoneal mJection with 12% casein-saline (pH 7. 4) in a dose of 12 ml/kg, peritoneal exudate cells (PMNs) were collected, washed, and adjusted to 5 x 10 cell/ml in Hanks balanced salt solution containing 0. 1 % gelatin (gelatin-Hanks). The bactericidal activity was determined according to the methods of Cohn et al. ' and Quie et al. 7' Briefly, a 50μ1 of 2x10 cocci/ml suspension of Staphylococcus aureus FAD 209P JC-1 freshly cultivatd in Tryptosoy broth(Eiken Chemical, Tokyo) was mixed with an aliquot of the inhibitor, to which was added 4-fold diluted fresh guinea pig serum in gelatin-Hanks solution. After 10 min opsonization in a shaking water bath at 37°C, the mixture was added with 500 μl of the PMNs suspension, and was reincubated for 0, 30, 60 and 120 min with shaking. At the indicated incubation. period, a portion of 10 pl of the mixture was withdrawn. Sedimente::l PMNs were washed with saline and added with 0. 1% Triton X-100 to make cell lysate, which was usd for measuring j3-glucuronidase activity of the cells. Bacillary counts were carried out on the cell lysate as well as the supernatant of the mixture. *) EBtR:i~ff§, fi!f:'§-Jljl, i~~~~: ~+~M&l*O) ~-glucuronidase inhibitor 0)-:i:-;v-:i:~ r 1Ulll~§O)~iff'fffl~;:.:X;J-t