Adaptive surface antigen variation in Mycoplasma bovis to the host immune response

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<ul><li><p>ELSEVIER </p><p>MICROBIOLOGY LETTERS </p><p>FEMS Microbiology Letters 144 (1996) 267-275 </p><p>Adaptive surface antigen variation in Mycoplasma bovis to the host immune response </p><p>Dominique Le Grand &amp;, Michel Solsona b, Renate Rosengarten , Franqois Poumarat bj * </p><p>a EC&amp; Nation&amp; V&amp;tPrinaire de Lyon, Pathologic du B&amp;ail, B.P. 83, 69280 Marcy-IEtoile, France </p><p>b CNEVA-Lyon, Luboratoire de Pathologic Bovine de Lyon, B.P. 7033, 69342 Lyon Cedex 07, France </p><p> Institut fcr Bakteriologie und Tierhygiene, Veteriniirmedizinische Universitiit Wien, Josef-Baumann-Gasse 1, 1210 Vienna, Austria </p><p>Received 14 May 1996; revised 26 August 1996; accepted 9 September 1996 </p><p>Abstract </p><p>The variability of predominant Mycoplasma bovis surface antigens in the presence of specific immune pressure was analyzed in an in vitro assay to determine if M. bovis could escape immune destruction. We have shown that serum antibodies from immunized or experimentally infected calves and monoclonal antibodies which specifically react with previously characterized or as yet undefined major M. bovis membrane surface proteins cause repression of expression or shortening of the target protein, or induce switching to expression of an antigenically distinct variant protein. We have further demonstrated that removal of the inducing antibody results in reversion to the original phenotype. These results suggest that the level of expression and the length of i14. bovis surface antigens in the host is modulated by cognate antibodies. According to the surface antigenic variation systems, random selection of preexisting variants resistant to antibody-mediated inhibition or direct regulation of gene expression may be means by which this organism evades host immune defences. </p><p>Keywords: Mycoplasma bows; Variable surface protein; Antigenic variant; Immune modulation; Immune evasion </p><p>1. Introduction </p><p>Mycoplasma bovis is considered one of the most pathogenic mycoplasma species in cattle. It is well established as the etiologic agent of mastitis [l], ar- </p><p>thritis [2], and pneumonia [3,4], and has also been reported to cause diseases of the genital tract [5], abscesses [6] and meningitis [7]. M. bovis diseases occur all over the world leading to extensive eco- nomic losses for both dairy and meat production </p><p>* Corresponding author. Tel: +33 78 72 65 43; Fax: +33 78 61 91 45. </p><p>[8,9]. Their incidence is growing in Europe with the increase of the beef trade during the past years [9,10]. Due to their resistance to antibiotic therapy and the lack of commercially available sensitive diagnostic tools and effective vaccines, M. bovis-induced dis- eases are difficult to prevent and to control. </p><p>A major characteristic of most mycoplasma infec- tions, including those caused by A4. bovis, is that they are usually chronic in nature. Although the ba- sis for this chronicity is not yet well understood, it has become apparent during the last few years that mycoplasmas may possess immune evasion mechan- isms, which enable them to rapidly change the struc- </p><p>0378-1097 /96/$12.00 Copyright 0 1996 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PIISO378-1097(96)00377-l </p></li><li><p>268 ,!I Lr Grund (1 ul. I FEMS Mrrohwlog~ Lrtten 144 /1996) 267-275 </p><p>a </p><p>CA CA CACACACA </p><p>1 2 3 4 5 6 </p><p>CACACA CACA CACA CA </p><p>1 2 1 2 1 2 1 2 </p><p>Fig. I. In vitro modulation of surface antigen expression in M. hovi~ strain 1067 by serum antibodies from a calf (M019) immunized with the same strain. a: Western blots of total cell protein from a population of organisms resulting from different inocula immunostained with bovine serum M019: both lanes (1) corresponding to culture from 10 CFU/ml inoculum; (2), lOa CFUlmI inoculum; (3) lo3 CFUl ml inoculum; (4), lo5 CFU/ml inoculum; (5) lo6 CFUlml inoculum: (6) IO7 CFU/ml inoculum. Lanes A (assay): culture with MO19 se- rum antibodies; lanes C (control): culture with bovine control serum. The position and size (in kDa) of immunostained proteins are indi- cated (right panel). b, c, d, e: Western blots of total cell protein from a population of organisms resulting from a 10 CFU/ml (lanes 1) and 10 CFUlml (lanes 2) inocula, respectively immunostained with mAbs: lA1 (b), lE5 (c), 5D7 (d) and Ia (e). Lanes A: culture with MO19 serum antibodies: lanes C: culture with bovine control serum. The position and size (in kDa) of immunostained proteins are indi- cated (right panel). </p></li><li><p>D. Le Grand et al. I FEMS Microbiology Letters 144 (1996) 267-275 269 </p><p>ture and expression of some of their membrane sur- face proteins exposed to the host immune system [l l-131. Our previous investigations have shown that M. bovis employs two surface antigenic varia- tion systems which could act independently to facil- itate immune evasion by this organism [14-171. The first represents a family of membrane surface lipo- proteins designated Vsps (variable surface proteins) [16], with as yet three defined members (VspA, VspB, VspC) which undergo a high rate of phase and size variation in vitro. A second, unrelated membrane surface protein designated pMB67 [17] proved to be as highly variable as the Vsps. Both types of membrane proteins are predominant antigens recog- nized during M. bovis infection and disease [14,15,17-l However, neither in M. bovis nor in other mycoplasma species in which variable surface pro- teins have been identified, has the function of these proteins in immune evasion in the host been unequi- vocally demonstrated. Only very recently, Citti and Wise [l&amp;19] have reported that antigenic variants of the swine pathogen M. hyorhinis are very likely to be subject to random selection, i.e., they arise sponta- neously and independently of the host immune re- sponse, but their subsequent survival is determined by immune selection. </p><p>In the present study we have explored the possibil- ity that M. bovis may be able to avoid the host immune system by changing its surface antigenic mo- saic as soon as the immune system recognizes it, i.e., whether an effective humoral immune response in the host can repress expression of the target protein or induce switching to expression of an antigenically distinct protein. To address this issue, we have devel- oped an immune pressure assay to examine the effect of sera from calves immunized or experimentally in- fected with M. bovis and of specific monoclonal anti- bodies (mAbs) on the pattern of surface antigen ex- pression in vitro. </p><p>2. Materials and methods </p><p>2.1. Monoclonal antibodies </p><p>Four mAbs directed toward M. bovis variable sur- face antigens were used in this study (as ascites fluids or hybridoma culture supematants). The construc- </p><p>tion and characteristics of mAb lE5 have recently been described in detail [ 151. It is an immunoglobulin (Ig) M isotype and recognizes a surface epitope on VspA, VspB and VspC [15]. mAbs 5D7 [14] and 1Al (IgGl) have been developed within a collaborative research project of the CNEVA Lyon and the Istitu- to Zooprofilattico Sperimentale in Brescia (Italy). Both mAbs were prepared against M. bovis type strain PG45 and react with VspA and VspC (mAb lA1) and, respectively, other as yet undefined pro- teins of the Vsp family (mAbs 1Al and 5D7; Rosen- garten, Poumarat, Le Grand and Yogev, unpub- lished results), which have been previously classified as antigen cluster I [15]. mAbs 12 and Ns have been constructed by Vetoquinol Biotechnologie in colla- boration with CNEVA Lyon [14]. Both mAbs were prepared against M. bovis strain 1067 (see below). While mAb Is (IgGl) reacts with a surface epitope on the phase- and size-variant Vsp-unrelated surface protein pMB67 [17], mAb Ns recognizes a surface protein of 45 kDa that is present in most, but not all M. bovis strains tested [14]. </p><p>2.2. Bovine sera </p><p>Two calves, designated MO19 and M021, were immunized with M. bovis strain 1067 as follows. Briefly, each calf was injected intramuscularly with 1 X lOlo formalized mycoplasmas emulsified in alumi- num hydroxide as adjuvant, followed by a second injection of the same dose and by the same route 4 weeks later. Three weeks after the second injection, the calves were challenged by inoculation of 2 x lo7 viable mycoplasmas into the carpal joint [20]. In par- allel experiments, two other calves which were not immunized, designated MO1 5 and M017, received the same dose. Corresponding sera, designated M019, M021, MO15 and M017, respectively, were obtained 15 days after inoculation, Serum anti- body titers were determined by the indirect hemag- glutination test (IHT) [21,22]. IHT titers of the end- point dilution were 1:640, 1:640, 1:80 and 1:40, re- spectively, for the four sera. </p><p>2.3. Mycoplasmas and culture conditions </p><p>M bovis strain 1067 was isolated in 1983 from a case of bovine mastitis [24] and successfully used to </p></li><li><p>270 D. Le Grand et al. I FEMS Microbiology Letters 144 (IYY6) 267-275 </p><p>experimentally induce reproducible arthritis in calves [20]. Clonal variants of M. bovis type strain PG45 expressing a single size variant of VspA (65 and 63 kDa), VspB (46,44.5,43 or 42 kDa) or Vsp C (79 or 75 kDa), or lacking all three Vsps (designated ABC- OK) have been previously described [16]. Each organ- ism was propagated at 37C in a standard mycoplas- ma medium [23]. Stocks were prepared from mid- exponential phase cultures and stored at -80C. </p><p>2.4. Immune pressure test </p><p>Fresh, broth-grown organisms from primary pas- sages of stocks (late exponential phase) containing approximately log CFU/ml were serially diluted lo- fold to lo-. 160-ml aliquots of each dilution were distributed in duplicate in 96-well microtiter plates. To each test well, 40 ml of IHT-positive bovine sera or 40 ml of ascites or hybridoma culture superna- tants containing mAbs were added. Similarly, 40 ml of IHT-negative bovine serum or of mAb-nega- tive ascites was added to each control well. Bovine test and control sera were heat-inactivated at 56C for 30 min prior to use to destroy any complement- mediated mycoplasmacidal effect. Growth was di- rectly estimated by measuring medium optical den- sity at 460 nm. After incubation at 37C for 7 days, each well content was prepared for Western immu- noblot analysis as described below. </p><p>2.5. Western blot analysis </p><p>Organisms from each well (200 ml of culture) were harvested by centrifugation at 13 000 X g for 30 min at 4C. The procedures for sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent Western blotting of mycoplasma proteins have been previously described [14]. For SDS- PAGE, cell pellets were heated at 100C for 5 min under reducing conditions and samples correspond- ing to 20 ml of culture (containing total proteins from approximately lo7 CFU) loaded onto 10% polyacrylamide gels. For Western blot analysis, blots were blocked for 50 min with Tris-buffered saline (TBS; Tris-HCl, 0.01 M; NaCI, 0.15 M; pH 7.2) containing 10% (v/v) horse serum, washed once with TBS containing 0.05% (v/v) of Tween 20 and once with TBS only, and then incubated for 2 h at </p><p>33C with the primary antibodies diluted 1:50 (bo- vine sera), 1 :lOO (mAb lE5), 1:400 (mAb 5D7), I:500 (mAb Nz), 1: 1000 (mAb 12) or 1: 1500 (mAb 1Al) in TBS supplemented with 5% (v/v) horse ser- um (TBS-HS). After washing as before, blots were incubated for 1 h at 33C in peroxidase-labelled sec- ondary antibody against mouse IgM, mouse IgG or bovine immunoglobulins, each diluted 1:600 in TBS- HS. After repeated washing, the blots were devel- oped using 4-chloro- 1 -naphthol as chromogenic sub- strate. </p><p>3. Results </p><p>3.1. lZJkct of bovine serum antibodies jiom immunized or experimentally infected calves on the pattern of surface antigen expression in M. bovis strain 1067 </p><p>When organisms of M. bovis strain 1067 were cul- tivated in the presence of M. bovis-specific bovine immune sera lacking complement activity, there was independently from the mycoplasma inoculum size and the serum tested never any growth inhibiting effect observed. To determine whether the serum antibodies could induce alterations in expression of surface antigens, the mycoplasma cells were har- vested and the patterns of antigen expression exam- ined in Western immunoblot with MO19 serum anti- bodies. The antigen pattern of such a culture of strain 1067 grown in the presence of MO19 serum antibodies (A) compared to a control culture (C) is shown in Fig. la. When the initial CFU/ml value (inoculum) of the mycoplasma culture was 107-lo, no significant alteration in expression of immuno- dominant (antibody binding) antigens (with a major antigen migrating at 50 kDa) were detected (Fig. la, lanes 3-6). However, when the inoculum size was only 10 CFU/ml, the exposure of strain 1067 to M. bovis-specific serum antibodies resulted in ex- pression of a 45 kDa major immunogenic antigen (Fig. la, lane lA), while the 50 kDa major reactive band of the original strain (data not shown) and of the control culture (Fig. la, lane 1C) disappeared. The same differences in the antigen profile of strain 1067 were seen when identical blots of total protein were immunostained with Vsp-specific mAbs 1Al </p></li><li><p>D. Le Grand et al. IFEMS Microbiology Letters 144 (1996) 267-275 271 </p><p>a </p><p>-60 </p><p>-17 </p><p>CACACACA </p><p>-50 </p><p>-45 </p><p>Fig. 2. In vitro modulation of pMB67 and Vsp surface antigen </p><p>expression in M. bovis strain 1067 by mAbs Is (a) and 5D7 (b). Western blots of total cell protein from a population of organ- </p><p>isms resulting from different inocula were immunostained with mAbs Is (a) and 5D7 (b). Lanes A: culture grown in the pres- ence of mAb; lanes C: control culture grown in the absence of mAb. a: both lanes (1) corresponding to culture from 10s CFUl </p><p>ml inoculum; (2), lo4 CFU/ml; (3), lo3 CFU/ml. A second step of immune pressure (4) was performed from cuhure shown on </p><p>lane A2 as indicated by arrows. b: both lanes (1) corresponding to culture from lo6 CFUlml inoculum; (2), lo5 CFU/ml; (3), lo4 </p><p>CFU/ml. A second step of immune pressure (4) was performed from culture shown on lane A2 as indicated by arrows. After im- munostaining with mAb Is (a) or 5D7 (b), the blots were re- stained with mAb 1Al (lower panels) to ensure equivalent amounts of total protein per channel. The position and size (in kDa) of immunostained proteins are indicated (right panels). c </p><p>(Fig. lb) or lE5 (Fig. lc), indicating that the variant antigens recognized by the bovine immune serum (Fig. la) are members of the Vsp surface lipoprotein family, which under the influence of specific antibod- ies either decrease in length or are replaced by an antigenically distinct variant Vsp protein. In con- trast, when mAbs 5D7 (Fig. Id) and Is (Fig. le) were used as probes in replicate blots, one other effect of serum antibodies on the antigen profile of M. bovis was detected, namely the repression of ex- pression of certain surface antigens, which involve Vsp proteins (as identified by mAb 5D7, Fig. Id, lanes 1A and 2A), as well as the Vsp-unrelated sur- face protein pMB67 that occurs in strain 1067 in a 36 kDa size form (Fig. le, lanes 1A and 2A). While the various alte...</p></li></ul>