Sep 22, 1977 - Division ofInfectious Diseases, Children's Hospital of Michigan, and the ... State University School ofMedicine, Detroit, Michigan 48201.
Vol. 20, No. 1
INFECTION AND IMMUNITY, Apr. 1978, p. 20-24
0019-9567/78/0020-0020$02.00/0 Copyright i 1978 American Society for Microbiology
Printed in U.S.A.
Substances That Interfere with Action of Viridin B, Streptococcus mitis Bacteriocin
a
ADNAN S. DAJANI,* CYNTHIA M. VERES, AND DAVID J. LAW
Division of Infectious Diseases, Children's Hospital of Michigan, and the Department of Pediatrics, Wayne State University School of Medicine, Detroit, Michigan 48201 Received for publication 22 September 1977
Several body fluids and various substances were examined for their effects on the action of viridin B, a Streptococcus mitis bacteriocin. Heme has been shown previously to interfere with viridin B action. In the present study, peroxidase and catalase, but not superoxide dismutase, interfered with the bacteriocin. Sulfhydryl-containing compounds had no effect. Trypsin, protease, and amylase exhibited interfering capacities. Of various body fluids tested, serum, saliva, and leukocyte lysate interfered with viridin B. The activity in leukocyte lysate was probably due to myeloperoxidase. Salivary activity was likely due to both peroxidase and amylase. Interfering activity in serum was neither complement dependent nor antibody mediated and was probably due to a combination of enzymatic activities. Temporal studies with these agents suggested more than one mechanism of interference with viridin B. gel filtration and was used in preference to the latter because of its increased thermal stability. The final concentration of viridin B used in most experiments ranged from 20 to 80 arbitrary units (AU), determined as previously described (4). Assays for interference with viridin B action. The agents described below were tested for interference with viridin B action by two techniques. In the lawn spotting method, doubling dilutions of an agent were prepared in phosphate buffer. The dilutions and a phosphate buffer control were each mixed with an equal volume of a viridin B preparation containing approximately 80 AU. After incubation at 250C for 5 min, the mixtures were spotted onto a tryptic soy agar plate that had been seeded with an indicator strain, and incubated at 370C overnight. Each agent was also spotted on a lawn of the indicator strain to assure that there was no inhibitory effect by the agent. Agents that interfered with viridin B allowed confluent growth of the indicator, whereas the control showed a clear zone of inhibition. The titer of the interfering agent was the highest dilution that allowed growth. The agar well technique (5) was used to assess the temporal effects of interfering agents on viridin B activity. A sterile no. 2 cork bore was used to cut five wells (5 mm in diameter by 5 mm deep) in a tryptic soy agar plate that had been previously seeded with indicator. Equal volumes of the test agent and viridin B were added to the wells as follows: agent followed after 30 min by viridin B and viridin B followed after 30 min by agent and premixed agent and viridin B. Controls of premixed equal volumes of buffer and agent or buffer and viridin B were also used. The 30-min interval was necessary to allow a substance to diffuse into the agar. After overnight incubation at 370C, plates were examined for zones of inhibition around the wells. Human leukocyte lysates. Human leukocytes were harvested by repeated differential centrifugation
Viridin B, a bacteriocin produced by Streptococcus mitis (mitior) 42885, has been isolated and partially characterized (3-5). Several unusual characteristics of viridin B have been described (3-5). One fortuitous observation was that heme interfered with the inhibitory effects of the bacteriocin; however, no interference was detected when substances such as cytochrome c, bilirubin, and ferrous or ferric salts were tested (5). The present investigations were conducted to determine whether other substances have similar interfering properties. Various body fluids, proteolytic enzymes, and substances functionally related to heme were primarily investigated.
MATERIALS AND METHODS Bacterial strains and media. S. mitis (mitior) 42885, which produces viridin B, Neisseria sicca 15362, which is killed by viridin B, and coagulasenegative Staphylococcus strain 23709, which is only inhibited by viridin B, have been described in detail previously (4). Media used were tryptic soy broth and tryptic soy agar (Difco, Detroit, Mich.). Sorensen phosphate buffer (0.067 M, pH 7.2) was generally used. When trypsin was tested for interference, the buffer pH was 8.2. Bacteriocin preparation. Viridin B was prepared by ammonium sulfate fractionation of supernatants from mechanically disrupted S. mitis cells by modification of a previously described technique (4). A 40 to 60% saturated ammonium sulfate fraction contained maximal viridin B activity and contained less inactive proteins than the one-step 80% precipitates previously used. The dialyzed ammonium sulfate fraction behaved identically to preparations further purified by 20
VOL. 20, 1978
INTERFERENCE WITH VIRIDIN B ACTION
of leukocyte-rich blood fractions obtained from the Southeast Michigan chapter of the American Red Cross, Detroit, Mich. When erythrocyte contamination was minimal, an erythrocyte-lysing solution [0.75% ammonium chloride, 0.15 M tris(hydroxymethyl)aminomethane-hydroc-Moride, pH 7.65] was added to the pellet. After 10 min of incubation at 370C, erythrocyte lysis was complete, and the leukocytes were washed twice in normal saline. Washed leukocytes were then suspended in phosphate buffer and lysed by repeated freeze-thaw in acetone-dry ice. Cell fragments were removed by centrifugation at 10,000 x g for 1 h at 4VC. The leukocyte lysate was sterilized by membrane filtration (0.45 um, Millipore Corp., Bedford, Mass.) and stored at -20'C until use. Nonimmune sera. Nineteen cord sera were obtained through the courtesy of the Newborn Service of Hutzel Hospital, Detroit, Mich. Sera from 40 children were obtained from the Serology Laboratory at Children's Hospital of Michigan. Staff and laboratory personnel were the source of 20 adult sera. Twelve albino rabbits also served as a source of nonimmune sera. Since heme interfered with viridin B action (5), any sera showing hemolysis were excluded from the study. Sera that inhibited indicators were not used. All sera were tested without treatment and then after heat inactivation at 560C for 30 min. If sera could not be tested immediately, they were frozen at -20'C. Rabbit complement, bovine serum albumin, fetal bovine serum, and immunoglobulin-free horse serum (Gibco Diagnostics, Madison, Wis.) were tested also. Fractionation of serum. Nonimmune human sera were fractionated by gel filtration on Sephadex G-200 (2). Sephadex was equilibrated with 0.005 M tris(hydroxymethyl)aminomethane buffer containing 0.001 M CaCl2 and 0.001 M MgCl2 (pH 7.0) and was packed into a column (1.5 by 90 cm) with a void volume of 140 ml. Serum was dialyzed against the same buffer overnight at 4°C, and a 2-ml sample was placed on the column. Fractions of 5 ml were collected at a flow rate of 0.23 ml/min, monitored for absorbance at 280 nm, and tested for interference with viridin B. Serum was also subjected to ammonium sulfate fractionation, from 30 to 90% saturation in 10% increments. Each redissolved, dialyzed fraction was tested as above. Immunization of rabbits. Adult albino rabbits were immunized with viridin B by a previously described technique (2). A partially purified bacteriocin preparation containing 160 AU was mixed with an equal volume of Freund complete adjuvant (Difco, Detroit, Mich.) and emulsified by sonic oscillation. Each of nine rabbits received a single 2.0-ml subcutaneous injection weekly for up to 6 months. Also, three rabbits were immunized with a Formalin-treated whole-cell vaccine prepared from the bacteriocin-producing organism. Preparation of the vaccine and immunization schedule were by the method of Lancefield (11). Sera were obtained before immunization and at weekly intervals and were tested as described above. Other body fluids. Unstimulated mixed saliva was collected from six laboratory personnel by the method of Hamon and Klebanoff (9). Specimens were centrifuged at 1,600 x g for 15 min and sterilized by membrane filtration (0.45 1m, Millipore Corp.). Each spec-
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as was a pooled sample consisting of equal portions of each specimen. In addition, a portion of each specimen was heated at different temperatures before testing. Pooled, unheated saliva was also tested for interference after dialysis (12,000-molecular-weight cutoff, Arthur H. Thomas Co., Philadelphia, Pa.) against phosphate buffer. Urine specimens from seven laboratory personnel were collected, adjusted to pH 7.2 with 1 N NaOH, and filter sterilized. Pooled cerebrospinal fluids from 10 patients were obtained from the Chemistry Laboratory, Children's Hospital of Michigan. Only clear, blood-free specimens were used. Enzymes. The proteolytic enzymes protease (type V, Sigma Chemical Co., St. Louis, Mo.) and trypsin (Grand Island Biological Co., Grand Island, N.Y.) were tested for interference with viridin B action. Peroxidase (horseradish) type II, catalase, superoxide dismutase, phospholipase A, and a-amylase type VI-A (Sigma) were similarly tested. All enzymes were used at an initial concentration of 1 mg/ml. Sulfhydryl-containing compounds. Cystamine dihydrochloride, L-cystine, andf-mercaptoethylamine hydrochloride (Sigma), L-cysteine (Nutritional Biochemicals Corp., Cleveland, Ohio), and dithiothreitol (P-L Biochemicals Inc., Milwaukee, Wis.) were tested for interference as described above, at a 1-mg/ml initial concentration. Enzymes and protein assays. Peroxidase activity (purpurogallin units per milliliter) was estimated in saliva and in leukocyte lysate by the assay procedure recommended by Sigma Chemical Co., St. Louis, Mo. Amylase activity (1 amylase unit is equivalent to 10 mg of starch hydrolyzed at 37°C for 30 min) was assayed in saliva with an Amylo-tube set (Biomedix, Inc., Princeton, N.J.). Saliva was diluted in distilled water to be within the sensitivity limits of the test. Protein determinations were performed with the Folin-Ciocalteau or biuret reagent (1).
RESULTS Human leukocyte lysate. Human leukocyte lysate interfered with viridin B action. This interfering activity was non-dialyzable but passed through a 0.45-,um filter. Activity was not affected by heating at 65°C for 10 min but was abolished when heated at 80°C or more for 10 min. Treatment with trypsin or protease did not alter the activity. Leukocyte lysate, filtered and dialyzed against phosphate buffer, was fractionated by gel filtration on a Bio-Rad P-200 column calibrated for molecular weight determination (5). The leukocyte lysate activity was contained in a single peak, with a corresponding molecular weight of ca. 150,000. Sera. Unheated sera from 79 human subjects were tested. All but one interfered with the bactericidal effect of viridin B. The level of the interfering activity (titers) varied among individuals and age groups (Fig. 1). Mean activity in the sera of children was highest, cord sera contained less such activity, and adult sera had the imen was tested for interference with viridin B action, lowest mean. In serum from one of the adults,
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DAJANI, VERES, AND LAW
INFECT. IMMUN.
noted above, heat-stable interfering activity was lacking in 79 human sera tested. Other body fluids. Human cerebrospinal *000 ) 128 fluids and urines had no effect on the inhibitory action of viridin B. Unstimulated mixed saliva, however, did interfere with viridin B action. All *000 64 six salivary specimens tested had interfering ac(484) (47.0) tivity that was filterable but non-dialyzable. The 32 activity was not affected by heating at 560C for 30 min but was lost after heating at 650C or (18.9) higher for 10 min. 16 Enzymes and sulfhydryl-containing substances. It has been shown previously (4) that the proteolytic enzymes trypsin and protease interfere with the action of viridin B. In the (5.9) present studies, amylase, catalase, and peroxidase were found to interfere with the bacterio4 cin. No interference was noted when L-cystine, L-cysteine, cystamine, dithiothreitol, 8-mercap00*0e 0@ 2 toethylamine, or superoxide dismutase was used at the concentrations indicated in Materials and Methods.
