Role of Complement in Immune Inactivation of Mycopkasma gallisepticum

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1 JOURNAL OF BACTERIOLOGY, Aug. 1967, p Copyright 1967 American Society for Microbiology Vol. 94, No. 2 Printed in U.S.A. Role of Complement in Immune Inactivation of Mycopkasma gallisepticum L. F. BARKER AND J. K. PAIT Laboratory of Virology and Rickettsiology, Division ofbiologics Standards, National Institutes of Health, Bethesda, Maryland Received for publication 31 May 1967 Studies were undertaken to evaluate the role of complement in the interaction between mycoplasmas and antiserum. A suspension of the A-1 strain of Mycoplasma gailisepticum in PPLO broth was incubated at 37 C with rabbit immune serum which had been heated for 30 min at 56 C. Samples were removed from the mixture at timed intervals for 1 hr for titration of the mycoplasmas in broth. When normal guinea pig serum was included in the mixture at a final dilution of 1:40, the titer fell rapidly from 106 to 102 organisms per 0.2 ml. When the guinea pig serum was heated for 30 min at 56 C or was omitted from the mixture, the immune serum did not reduce the titer. The rate of inactivation was related to the final concentration of antiserum and to the incubation temperature. The effect of the guinea pig serum was eliminated by the addition of 0.01 M sodium ethylenediaminetetraacetate or by prior absorption with an unrelated antigen-antibody complex. It was concluded that complement-like substances play an important role in immune inactivation of M. gallisepticum. Inhibition of mycoplasma growth by specific antiserum has been demonstrated on agar (3, 6, 8) and in broth (1, 12; K. E. Jensen, Bacteriol. Proc., p. 70, 1964). As normal serum is an essential component in the growth medium for many Mycoplasma species, it has been difficult to evaluate the role of normal serum factors in the growth inhibition phenomenon. There is some disagreement regarding the need for complement as an accessory factor for growth inhibition. Eaton et al. (5) showed that a heat-labile factor in rabbit antiserum was essential to neutralize M. pneumoniae infectivity for cell cultures. Taylor-Robinson et al. (12) found that antiserum did not inhibit growth of M. pneumoniae in broth if the horse serum in the growth medium had been heated for 1 hr at 56 C; they concluded that a heat-labile factor in normal horse serum was essential for growth inhibition in their system. They also found that the addition of unheated normal guinea pig serum increased the growthinhibiting titer of antiserum against the Negroni agent, a strain of M. pulmonis. Edward and Fitzgerald (6), however, demonstrated inhibition of mycoplasma growth on agar and inactivation of mycoplasmas in broth by antiserum which had been heated for 30 min at 56 C. They concluded that complement was not necessary for either inhibition or inactivation. 403 The experiments reported here were designed to evaluate the role of normal serum factor(s) in the inactivation of mycoplasmas. The results indicated that the rate of inactivation of the A-1 strain of M. gallisepticum (10) by heated antiserum increased considerably when unheated normal guinea pig serum was present in the reaction mixture. The heat-labile serum factor(s) which speeded immune inactivation was found to have several of the characteristics of complement. MATERIALS AND METHODS Mycoplasmas. The A-1 strain of M. gallisepticum was grown in Hayflick's liquid medium (7) supplemented with 0.5% glucose, 200 units of penicillin G per ml, 1:4,000 thallium acetate, and 0.001% phenol red; growth on agar plates has been described previously (10). Titration. Serial 10-fold dilutions of Mycoplasma suspensions were made in growth medium. The highest dilution at which acid production, indicated by a color change of the phenol red dye, occurred during 10 days of incubation at 37 C was taken as the titer. Hereafter titers are given in color-changing units (CCU). Hyperimmune serum. Antiserurn was prepared in rabbits as described elsewhere (10). Guinea pig serum. A single lot of lyophilized pooled normal guinea pig serum (Microbiological Associates, Inc., Bethesda, Md.) was used throughout these studies as a source of complement. Growth-inhibition test. The method of Taylor-

2 404 BARKER AND PATIO J. BACTERIOL. Robinson et al. (12) was used to test the rabbit hyperimmune serum for growth-inhibiting antibodies. Inactivation test. A suspension containing approximately 106 CCU per 0.2 ml was prepared in PPLO broth (Difco); 3.2-ml samples of this suspension were added to tubes containing 0.4 ml of antiserum and 0.4 ml of normal guinea pig serum. The contents were mixed, and a sample was removed immediately for titration in growth medium. The mixture was then incubated at 37 C except where otherwise noted. At timed intervals during 1 hr of incubation, samples were removed for titration in the growth medium. RESULTS Growth and titration of M. gallisepticum. A sharp drop in the ph of the growth medium from 7.4 to 5.3 followed multiplication of the A-1 strain of M. gallisepticum in the liquid medium (Table 1). In parallel titrations of a suspension of M. gallisepticum in growth medium and on agar plates, the CCU titer (101 per 0.2 ml) closely approximated the CFU titer (3 X 107 to 10 X 107 per 0.2 ml). In 193 titrations of the Mycoplasma suspension used in the inactivation tests at zerotime, the average titer was 106 CCU per 0.2 ml, the range was from 105 to 107 CCU per 0.2 ml, and the 95 % confidence limits on the mean titer were from to CCU per 0.2 ml. Points plotted in the figures which follow represent the average titers from five separate inactivation tests. When duplicate inactivation tests were run, the terminal dilutions at each sampling time almost always fell within one tube of one another. Growth-inhibiting antibody titer of antiserum. Titration of the rabbit antiserum against 103 CCU of the A-1 strain revealed inhibition of acid production at a serum dilution of 1:640. Serum obtained from the same rabbit prior to immunization and the normal guinea pig serum showed no inhibition of growth at a dilution of 1 :10. Inactivation of M. gallisepticum by antiserum. TABLE 1. Relationship ofmycoplasma gallisepticum growth to ph change in growth mediuma Time (days) Titer ph of medium (CCU/0.2 ml) , , , , 5.3 a Incubation at 37 C. When a mycoplasma suspension containing 106 CCU/0.2 ml was incubated with a 1:10 dilution of antiserum at 37 C, there was a gradual fall in the titer of the suspension over 1 hr and a further fall after incubation for 24 hr (Table 2). At a 1:100 dilution of the antiserum, there was a slight decrease in titer over 1 hr and a further decrease over 24 hr. Heating the antiserum for 30 min at 56 C, however, destroyed much of its inactivating activity. Titrations at intervals during incubation for 1 hr of samples from a suspension of mycoplasmas mixed with antiserum diluted 1:1,000 showed a rapid fall in the titer of the organisms from 10 to 102 CCU per 0.2 ml when normal guinea pig serum was included in the reaction mixture at a dilution of 1:40 (Fig. 1). In this and all subsequent experiments, the antiserum was heated for 30 min at 56 C prior to testing. The inactivation rate increased or decreased when the antiserum dilution was changed to 1:100 or 1:10,000, respectively, and the normal guinea pig serum was kept at a 1:40 dilution. Serum from the same rabbit obtained prior to immunization and combined with normal guinea pig serum did not inactivate the mycoplasmas. The inactivation of M. gallisepticum as determined by titration in broth was accompanied by a fall in colonyforming unit (CFU) titer on agar (Table 3). When the normal guinea pig serum was heated for 30 min at 56 C or was omitted, the antiserum at a dilution of 1 :1,000 did not reduce the titer of the Mycoplasma suspension over a 1-hr period (Fig. 2). If the dilution of the antiserum was kept at 1:1,000 and the dilution of normal guinea pig serum was increased at graded intervals from 1:10 to 1:640, the rate of inactivation steadily decreased until there was no perceptible effect at the end of 1 hr (Fig. 3). When the incubation temperature of the reaction mixtures was lowered from 37 to 25 C, the TABLE 2. Effect of unheated and heated antiserum on Mycoplasma gallisepticum CCU titera Antiserum dilution - I hr 24 hr 1: :10, heatedb : :100, heated None a Expressed as log1o/0.2 ml; titer of suspension at time 0 = 106 CCU/0.2 ml; incubation temperature = 37 C. b Heated 30 min at 56 C.

3 VOL. 94, 1967 IMMUNE INACTIVATION OF M. GALLISEPTICUM z z 0 -j Lr-4 Pre Serum, O-O Pre Serum, 1:1000 < Post Serum, 1: 10,000 < h\a PostSerum, 1:I000 r PPost Serum, 1:I FIG. 1. Relationship of antiserum dilution to inactivation ofmycoplasma gallisepticum in the presence of complement (1: 40). Initial titer ofmycoplasmas was 106 CCU/0.2 ml with 95% confidence limits indicated between dotted lines. Pre serum was obtained prior to immunization; incubationz was at 37 C. TABLE 3. Inactivation ofmycoplasma gallisepticum by antiserum plus complementa determined by titration in broth and on agar Time (min) Broth (CCU) Titer Agar (CFU) b 1 X X X X , 2 X X 102 a Antiserum dilution, 1:1,000; complement dilution, 1:40. b Titers expressed per 0.2 ml. rate of inactivation was slower for equivalent concentrations of antiserum in the presence of normal guinea pig serum diluted 1:40 (Fig. 4). It the mixture was incubated at 4 C, no inactivation occurred over a 1-hr period, and there was very little inactivation over 24 hr. At 37 and 25 C, there was always a persistent fraction of approxi- 3 0.I FIG. 2. Effect ofheating (56 C for 30 min) or omitting complement (C') on immune inactivation of Mycoplasma gallisepticum. Antiserum dilution was 1:1,000; initial titer ofmycoplasmas was 106 CCU/0.2 ml (95% confidence limits between dotted lines); incubation was at 37 C. mately 10'-1 mycoplasmas which survived during 24 hr of incubation. When the Mycoplasma suspension was mixed with normal guinea pig serum at a dilution of 1: 40 without antiserum, no inactivation took place over 24 hr (Table 4). At a dilution of 1 :10, however, the normal guinea pig serum produced a gradual decrease in the titer of the mycoplasmas; this effect was eliminated by heating the normal guinea pig serum for 30 min at 56 C. When sodium ethylenediaminetetraacetate, 0.01 M, was included in the reaction mixture (ph 7.4), no inactivation by the combination of antiserum (1:100) and normal guinea pig serum (1:10) took place over a 1-hr period (Fig. 5). The guinea pig serum was absorbed with a suspension of killed bacteria (Proteus OX-19) which had been sensitized with specific agglutinating antiserum and then washed with saline. After removal of the antibody-coated bacteria by centrifugation, the hemolytic complement activity of the guinea pig serum was reduced by more than 90% when compared with unabsorbed guinea pig serum or serum absorbed with unsensitized bacteria and handled in the same manner. The decomplemented guinea pig serum c < r-

4 406 BARKER AND PATT J. BACTERIOL. - _-ff - did not enhance the rate of immune inactivation of the mycoplasmas, in contrast with the unab sorbed serum and the serum absorbed with bacteria alone (Fig. 6). Absorption of the guinea pig serum with M. 5T \-_ 10 gailisepticum did not remove its enhancing effect on immune inactivation. After absorption of the rabbit antiserum with M. gallisepticum, however, it had no inactivwting aptivitv_ -F ~~~~~~~~~~~z \ o4-0- \ ~~~~~~C' 1:160c 0 -e C' 1:40 < 8 C' 1: M INUTES FIG. 3. Effect of complement (C') dilution on immune inactivation of Mycoplasma gallisepticum. Antiserum dilution was 1:1,000; initial titer ofmycoplasmas was 106 CCU/0.2 ml (95% confidence limits between dotted lines); incubation was at 37 C. I \ \ *~0-* AB1: 100, 25C 5 -\ \ \ -U AB 1:100,37C LI AB1 1000, 25C o~~~~~~~- AB1:I0, 37Cn 100 TABLE 4. Effect of complement (guinea pig serum) alone on Mycoplasma gallisepticum at 1, 2, and 24 hr Dilution of complement CCU titera 1 hr 2 hr 24 hr 1: :10, heatedb : :40, heated None a Expressed as log1o/0.2 ml; titer of suspension at time 0 = 106 CCU/0.2 ml; incubation temperature = 37 C. b Heated for 30 min at 56 C o4_ \ ~~~~~~~~~ \ ~~~~~~~~~ z *-v NoC', EDTA O.IM n --_ C' 1: I0 EDTA 0.0I M < -[}O C' 1: 10, No EDTA r FIG. 4. Relationship of incubation temperature to rate of inactivation of Mycoplasma gallisepticum by antiserum (AB) plus complement (1:40). Initial titer of mycoplasmas was 106 CCU/0.2 ml (95% confidence limits between dotted lines). FiG. 5. Effect of ethylenediaminetetraacetate (EDTA), 0.01 m, on inactivation of Mycoplasma gallisepticum by antiserum (1:100) plus complement (C'). Initial titer ofmycoplasmas was 106 CCU/0.2 ml (95% confidence limits between dotted lines); incubation was at 37 C.

5 VOL. 94, 1967 IMMUNE INACTIVATION OF M. GALLISEPTICUM V a l O \ \ 10-C C' 140, AG-AB ABS z 0 4-*-* C' 1:40, AG ABS -J C' 40, No ABS 3 WX FIG. 6. Effect of absorbing complement (C') with an unrelated antigen-antibody complex on inactivation of Mycoplasma gallisepticum by antiserum (1:1,000) plus C' (1:40). Initial titer of mycoplasmas was 106 CCU/0.2 ml (95% confidence limits between dotted lines); incubation was at 37 C. DIscUSSION Specific antibody plus complement can produce death or lysis of gram-negative bacteria, treponemes, leptospires, protoplasts, spheroplasts, and mammalian erythrocytes and nucleated cells (9). Since mycoplasma cell membranes are morphologically similar to mammalian cell membranes (4), it seemed reasonable to postulate that complement might play an important role in immune inactivation of these organisms. In favor of this hypothesis are the experiments of Eaton et al. (5) in cell cultures and of Taylor-Robinson et al. (12) in broth. Our results indicated that at a low dilution of antiserum a heat-labile factor greatly increased the rate of immune inactivation of M. gallisepticum. At higher dilutions of antiserum or with heated antiserum, little or no inactivation took place unless unheated normal guinea pig serum was added to the reaction mixture. The inactivating effect of unheated guinea pig serum alone at a 1:10 dilution cannot be adequately explained at present. We might postulate the presence of heat-labile cross-reacting or natural antibodies in low concentration in the normal guinea pig serum. Such antibodies could be directed against either Mycoplasma or growth medium antigens, as the latter have been shown to be incorporated into the antigenic structure of M. gallisepticum (11). Another possibility is that nonspecific attachment of y-globulin from the serum in the growth medium to the mycoplasma cell membranes sensitized the organisms for the destructive action of complement. Specific antibodies in the guinea pig serum could not be demonstrated in the growth-inhibition test of Taylor-Robinson et al. (12), nor could the activity of the unheated guinea pig serum be removed by absorption with M. gallisepticum. As in the well-described phenomenon of immune hemolysis, the rate of immune inactivation of M. gallisepticum was directly related to the concentration of antibody and of complement in the reaction mixture. The heat-labile factor in guinea pig serum was more active at 37 C than at 25 or 4 C, and it could be removed by absorption with an unrelated antigen-antibody complex which removed complement activity from the guinea pig serum. The enhancing effect of guinea pig serum on immune inactivation did not occur if ethylenediaminetetraacetate was included in the reaction mixture. These characteristics led us to conclude that serum complement was largely responsible for the guinea pig serum effect. In the case of antibody-sensitized gram-negative bacteria, complement results in the release of ribonucleic acid and deoxyribonucleic acid, presumably by disrupting permeability control mechanisms (J. K. Spitznagel, Bacteriol. Proc., p. 51, 1964). Guinea pig complement produces holes or lesions in the cell membranes of sheep erythrocytes which have been sensitized with specific antibody (2). It seems likely that the antibody-complement system is functioning against the mycoplasmas in a manner similar to the well-known bactericidal and hemolytic systems. If so, the hypothetical mechanism for immune inactivation would be sensitization of the mycoplasmas by antibody for the damaging effect of complement on the cell membranes, resulting in increased permeability, loss of essential internal components, and inability of affected organisms to multiply. ACKNOWLEDGMENTS The technical assistance of B. R. Prather is gratefully acknowledged. LrrERATURE CITED 1. BAILEY, J. S., H. W. CLARK, W. R. FELTS, R. C. FOWLER, AND T. McP. BROWN Antigenic properties of pleuropneumonia-like organisms from tissue cell cultures and the human genital area. J. Bacteriol. 82:

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