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  • Vol. 13, No. 2JOURNAL 0F CLINICAL MICROBIOLOGY, Feb. 1981, p. 335-343 0095-1137/81/020335-09$02.00/0

    Phospholipase D Activity of Corynebacterium pseudotuberculosis (Corynebacterium ovis) and

    Corynebacterium ulcerans, a Distinctive Marker Within the Genus Corynebacterium

    LANE BARKSDALE,* REGINA LINDER, IOAN T. SULEA,t AND MARJORIE POLLICEt Department ofMicrobiology, New York University School ofMedicine and Medical Center, New York,

    New York 10016

    A search has been made for corynebacterial phospholipase D, "ovis toxin," a sphingomyelinase (phosphatidylcholine phosphohydrolase, EC 3.1.4.4), among a wide variety of corynebacteria. Phospholipase D activity has been found in strains exhibiting the biochemical properties characteristic of Corynebacterium pseudo- tuberculosis or of Corynebacterium ulcerans and in no other species of Coryne- bacterium. Methods for the assay of phospholipase D as a sphingomyelinase and methods for screening for phospholipase D in the presence of Corynebacterium equi on washed sheep blood agar are discussed.

    Certain strains of Corynebacterium pseudo- tuberculosis (Corynebacterium ovis), when ly- sogenic for a tox+-carrying phage, produce both diphtherial toxin (3, 30) and a second toxic sub- stance, a sphingomyelinase (phosphatidylcho- line phosphohydrolase, EC 3.1.4.4, a phospholi- pase D [PLD]) capable of hydrolyzing sphingo- myelins to N-acylsphingosyl phosphates (39,40). When the gene tox is absent from either of these corynebacteria, PLD is the characteristic toxin produced. Over a period of 55 years what came to be called "ovis toxin" gave rise to much speculation (2, 6, 28), because sometimes it con- sisted of a mixture of diphtherial toxin and PLD, but most often it consisted of only the latter (34). Once the matter ofthe two toxic substances was resolved (12, 26, 27), there was a need to investigate the relationship between the PLD activity of the toxin and "associated substances" which under certain conditions caused the he- molysis of erythrocytes and which blocked the hemolytic activity of the fl-toxin ofStaphylococ- cus aureus. Over the last 15 years, through the efforts of Soucek and co-workers (36-38), Goel and Singh (17), and Lovell and Zaki (27), it has become clear that the hemolytic activity (14, 15, 46) of C. pseudotuberculosis (and by analogy that of Corynebacterium ulcerans), the capacity of products of these two corynebacterial species to block the hemolytic activity of the ,B-lysin (phospholipase C) of S. aureus (13-15, 18) and that of the a-toxin of Clostridium perfringens

    t Present address: Institute of Rehabilitation Medicine, New York University Medical Center, New York, NY 10016.

    t Present address: Department of Medicine, New York Hospital, Cornell Medical Center, New York, NY 10021.

    (38), and their PLD activity are all one and the same (27, 37, 39). More recently, Linder and Bernheimer purified to near homogeneity the PLD from C. pseudotuberculosis, finding its mo- lecular weight to be around 31,000 with a pI of about 9.8. They assessed its activity as sphin- gomyelinase by employing radioactively labeled sphingomyelin (24, 25). The hemolytic activity early ascribed to C.

    pseudotuberculosis required its growth on a blood agar plate in reasonably close proximity to a colony of Corynebacterium equi (14), and Fraser suggested that the hemolysis observed resulted from the conjoint action on sheep eryth- rocytes of products diffusing from these two bacterial species (14, 15). Bernheimer et al. (8) have investigated the conjoint hemolysis of sheep erythrocytes by the PLD of C. pseudotu- berculosis and a protein, equi factor, from C. equi. They have purified equi factor and found it to contain a phospholipase C with a require- ment for activity of either Mg2e or Ca2", to have the capacity to cleave sphingomyelin to cera- mide and phosphoryl choline, and, further, to be capable of hydrolyzing the phosphate group from ceramide phosphate. The greatest activity was found when Triton X-100 was included in the assay mixture. Among the other phospholip- ids hydrolyzed by equi factor were phosphati- dylcholine, phosphatidylserine, and phospha- tidic acid. In cooperatively bringing about the lysis of sheep erythrocytes, PLDs of C. pseudo- tuberculosis make available, to equi factor, cer- amide phosphate split from sphingomyelin at the surface of the erythrocyte. It has been sug-

    335

  • 336 BARKSDALE ET AL.

    gested that phospholipase C of equi factor hy- drolyzes ceramide phosphate, modifying the in- tegrity of the cell membrane so that it becomes sensitive to physical effects such as chilling, and when these occur, lysis ensues (8). There are available, then, two reliable meth-

    ods for the detection of the PLDs of C. pseudo- tuberculosis and of C. ulcerans: (i) an assay for sphingomyelinase activity by using radiolabeled sphingomyelin and (ii) an assay for hemolytic activity in the presence of C. equi. Up to the present only a few strains of C. pseudotubercu- losis and of C. ulcerans have been examined for PLD activity. We have employed these two sys- tems for detecting PLD activity among a num- ber of bacteria labeled as C. pseudotuberculosis and C. ulcerans and for ascertaining the distri- bution of PLD among representative species belonging to the genus Corynebacterium. Unlike diphtherial toxin, which occurs in only about 50% of field isolates of Corynebacterium diph- theriae (35, 42), PLD appeared to be character- istic of most (if not all) strains of C. pseudotu- berculosis and C. ulcerans, and it was not found in any other corynebacteria.

    MATERIALS AND METHODS Bacterial strains. The sources of bacterial strains

    employed in this study are listed in Table 1. Production of PLD. Cultures of bacteria were

    inoculated into 500 ml of medium (in 2-liter flasks) containing: 220 ml of yeast extract diffusate (prepared as described by Bernheimer and Schwartz [9]), 20 g of Casamino Acids (Difco Laboratories), 33 ,ug of thia- mine, 1.2 mg of nicotinic acid, and distilled water to 1 liter (pH 7.2). Cells were grown to an optical density of 6.0 at 650 nm, and the supernatant was collected after centrifugation at 8,000 x g for 1 h at 4°C. The pooled culture supernatants were made 80% saturated with (NH4)2SO4 at room temperature and left over- night at 4°C. The centrifuged precipitate was washed with and resuspended in 80% saturated (NH4)2SO4. The suspension from 680 ml of culture was centri-

    fuged, the supernatant was discarded, and the pellet was taken up in approximately 3 ml of 0.1 M sodium phosphate buffer (pH 6.0) containing 0.25 M NaCl and 5% (vol/vol) glycerol.

    Estimation of PLD activity of C. pseudotuber- culsis. PLD (sphingomyelinase) activity was esti- mated with radioactively labeled sphingomyelin sus- pensions (1 mg/ml) containing 1 ,uCi of N-[methyl- '4C]sphingomyelin (60 Ci/mol, Radiochemical Centre, Amersham, United Kingdom) per ml as previously described (24). Incubation mixtures employed in esti- mating sphingomyelinase activity contained: 5 pl of ['4C]sphingomyelin suspension (6.7 nmol), 20 pl of 0.1 M tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 9.2) containing 0.24 M NaCl-5.5 mM MgCl2 and 10 p1 of diluted enzyme preparation. Incubation was at 37°C for 30 min. After incubation, the mixtures were spotted at the origin of chromatograms of What-

    man no. 1 paper and developed for 5 h in ii-butanol- glacial acetic acid-water (2:1:1, vol/vol) to resolve sphingomyelin from free choline. ['4C]choline liber- ated in the reaction was quantified by cutting out the areas corresponding to sphingomyelin and free choline and counting in a Nuclear Chicago Mark I scintillation spectrometer in aqueous counting scintillation fluid from Amersham Corp. (Arlington Heights, Ill.). One unit of activity is defined as the amount of enzyme releasing 1 nmol of choline in 30 min.

    Cooperative production of hemolysis by PLD of C. pseudotuberculosis and equi factor. When either C. pseudotuberculosis, C. ulcerans, or C. equi was grown on a neopeptone agar (Difco) containing 2% (vol/vol) washed sheep erythrocytes there was very little evidence of hemolysis: at the edge of the growth of C. pseudotuberculosis there was sometimes a faint clearing, and at the margins of the growth of C. ulcerans there was a slight greening. However, when either C. ulcerans or C. pseudotuberculosis was grown in the presence of C. equi, there appeared clear zones of hemolysis between the two species (see Fig. 1A). This cooperative hemolysis was the result of the con- joint action of the equi factor and the PLDs of C. pseudotuberculosis and C. ulcerans.

    The first step in carrying out the tests for coopera- tive hemolysis involved the growth of cultures over- night on chocolate agar slants. A mass of overnight growth was taken up on a sterile cotton swab and deposited (smudged) on a sheep blood agar plate ac- cording to the patterns shown in Fig. 1A, where C. equi was inoculated in the center of the plate, and the cultures of C. pseudotuberculosis and C. ulcerans were inoculated radial to the center. The inoculated plates were incubated for 48 h (37°C) and scored for hemolysis. As many as eight strains were tested per plate.

    Inhibition of the cooperative hemolysis pro- duced by staphylococcal 8-lysin and a protein from Streptococcus agalactiae. When fB-lysin-pro- ducing strains of Staphylococcus aureus are grown on sheep blood agar plates near a strain of Streptococcus agalactiae (at 37°C), an area of clear hemolysis de- velops between the two strains (the CAMP reaction, the name originating from the first letters of Christie, Atkins, and Munch-Petersen, 13). This cooperative hemolysis is subject to interference from the PLD of C. pseudotuberculosis or that of C. ulcerans (39) (Fig. 1B). Since PLD i

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