J. Vet. Med. B 39, 762-766 (1992) 0 1992 Paul Parey Scientific Publishers, Berlin and Hamburg ISSN 0931 - 1793

Department of Veterinary Pathology and Public Health, Massey University, Palmerston North, N e w Zealand

Protein Antigens Secreted by Mycobacterium paratuberculosis P. VALENTIN-WEIGAND~" and K. M. MORIARTY~ Address of authors: 'Arbeitsgruppe Mikrobiologie der GBF, Biozentrum der Technischen Universitat, Konstantin-Uhde-Str. 5, D-3300 Braunschweig, Germany, *Department of Veterinary Pathology and Public Health, Massey University, Palmerston North, New Zealand

With 2 figures (Received for publication July 12, 1991)

Summary Proteins secreted by Mycobacterium paratuberculosis (M.ptb) during short-term cultivations were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western(1mmuno) blotting. Cultivation in a defined medium containing 3 5 s methionine allowed autoradiographic detection of proteins which had been secreted or passively released by actively metabolizing M.ptb organisms. After the first 3 days of cultivation, 4 proteins with molecular weights of approximately 38, 50,65 and 110 kilodaltons (kd) were detected on SDS gels. Longer incubation up to 12 days resulted in an increased concentration of these proteins as well as in appearance of additional proteins ranging from 14 to over 90kd. In long-term (8-10 weeks) culture filtrates only two prominent proteins with molecular weights of 30 and 65kd proteins could be detected. Immunoblot analysis showed that some of the proteins secreted during short-term cultivations were recognized by sera from M.ptb-infected sheep and more significantly by sera from animals which had been immunized with a M.ptb live vaccine strain. The study indicates that during short incubation times M. ptb may secrete immunoreactive proteins which are not dominant in long-term cultures.

Introduction Mycobacterium paratuberculosis (M.ptb) is the causative agent of Johne's disease, a chronic granulomatous enteritis of ruminants. The increasing importance of Johne's disease in cattle and sheep industry and the role of M . p t b as a potential etiologic agent in some cases of Crohn's disease of humans have initiated many recent research activities in this field (5,9). Effective control or eradication of Johne's disease has been hindered by the lack of specific diagnostic methods to identify infected animals. At present, isolation of M.ptb from feces o r tissues is considered the only valid criterion for infection. Recent studies to develop DNA probes for direct detection of the organism and on improved ELISA methods to detect M.ptb specific antibodies showed some promising results (15, 17). However, little is known about protective and/or specific antigens of M . p t b which could be of importance to develope an effective vaccine or improve present diagnostic methods. ;i

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Protein Antigens Secreted by Mycobacteriurn paratuberculosis

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A number of immunological studies on other mycobacterial species like M . tuberculosis and M . bovis indicate that actively metabolizing organisms may induce higher levels of antimycobacterial resistance than dead bacilli (I, 11). It has long been known that aging mycobacteria release antigenic components into the culture medium, which have been isolated as “purified protein derivates” (PPD) and applied to diagnostic methods and vaccine developments (10). However, these preparations are often contaminated with irrelevant polysaccharides and autolytic cell products (13). COLLINS et al. (6) tried to avoid this problem by isolating protein antigens from M . tuberculosis cultures which had been harvested in the mid-logarithmic growth phase. They observed that these proteins were not recognized by a number of monoclonal antibodies directed against M . tuberculosis but et al. were able to stimulate T-cell responses. Similar results were obtained by ABOU-ZEID (2) who reported that short-term cultures of M . tuberculosis secreted antigens which were not recognized by some of the available monoclonal antibodies and discussed the potential importane of these proteins in pathogenicity of mycobacterioses. These studies prompted us to undertake the present investigation in which we could show that actively metabolizing M.ptb are capable of secreting/releasing immunogenic proteins during short-term cultivation. Material and Methods Bacterial cultures A M.ptb isolate (strain 1244) from a M.ptb infected sheep was used throughout this study. The organism had been identified as M.ptb by acid-fast staining, morphological appearance and slow, mycobactin-dependent growth. A commercially available M.ptb vaccine strain (3 19 F, Rhone Merieux Animal Health, Lower Hutt, New Zealand) was used for immunizing adult sheep and lambs (4 weeks old). Herrold’s egg yolk agar served as medium for primary cultivation and maintainance of the organisms. Preparation of secreted proteins M.ptb strain 1244 was subcultured in modified Watson-Reid medium (16) supplemented with 4g/L sodium pyruvate and 4mg/L mycobactin J (Allied Lab., Ames, IA, U.S.A.). Proteins which were synthesized by actively growing mycobacteria were detected according to the method of ABOUZEID et al. (1). For this, %-labeled methionine (Amersham, Buckingshire, U.K.) was added to the bacterial suspensions (containing 108 organisms/ml medium) at 10 pCi/lOO pl. Incubations were carried out in 48-well microdilution plates (Nunc, Roskilde, Denmark) with inoculation volumes of 250 pl/well for 3, 6, 12 and 24 days at 37°C in a humidified atmosphere with 5 YO CO2. Controls included an irradiated M .ptb culture (negative) and a Staphylococcusaureus clinical isolate (positive, incubated for 18h). A long-term culture of M.ptb was prepared by growing similar amounts of the organism as a pellicle on 25ml of the same medium with nonlabeled methionine. The extent of bacterial growth was not monitored during incubations. Bacteria were removed by centrifugation and subsequent filtration of the supernatant through membrane filters (0.2 p, MFS, Dublin, CA, U.S.A.). The filtrates were freeze-dried and stored at -70 “C. The long-term culture filtrate was concentrated 100-fold to directly compare this filtrate with those of short-term cultures. For SDS-PAGE and immunoblot analysis samples were reconstituted in phosphate buffered saline (PBS, 0.15 mol/l, p H 7.4).

SDA-PAGE and Western blotting Culture filtrates were separated by SDS-PAGE in 12 % gels under denaturing conditions according to LAEMMLI(8). Similar amounts of each sample were loaded. Gels were stained with Coomassie blue to visualize molecular weight standards, dried and exposed to an X-Ray film for 5 days at -70°C to detect %-labeled proteins. Immuno blotting from SDS-gels was performed essentially according to BURNETTE(4) for 1 h at 100 V in a trans-blot cell (BioRad, Northcote, New Zealand). After transfer, membranes were soaked in 10 % (w/v) skimmed milk in PBS for 1h to block nonspecific binding sites. One part of the blot containing molecular weight standards was stained with india ink before blocking to calculate molecular weights. Membranes were then cut into 5 mm strips and incubated for 1 h at 37°C with different immune sera. Sera were obtained from M.ptb-infected sheep (histopathological and clinical evidence of paratuberculosis) and from adult sheep and lambs

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which had been immunized with live M.ptb vaccine strain 319 F. Sera from sheep with no evidence of M.ptb infection served as controls, All sera were used at 1/100 dilutions in blocking buffer. After washings with PBS, bound antibodies were deteted by incubation with peroxidase-conjugated rabbit anti-sheep IgG (at 1/2,000 in blocking buffer, 1 h at 37°C) and diaminobenzidine (DAB, Sigma) as substrat. Membranes were then sealed in plastic bags and exposed to an X-ray film for 5 days at -70°C to visualize total radiolabeled (secreted) proteins and relate them to the immunoreactive proteins.

Results and Discussion A number of recent studies revealed that secreted antigens might be important immunogens of M . tuberculosis, M . leprae and M . bovis (2,3, 7). In this study we observed that M.ptb is also capable of secreting proteins some of which are recognized by antibodies present in serum of M.ptb-infected animals. However, it has to be considered that the method used according to ABOU-ZEIDet al. (1) did not allow to differentiate between actively secreted and passively released proteins. We incubated cultures of a clinical isolate of M.ptb for 3 to 24 days (which for this organism is still a relatively short period) in microdilution plates. After the first 3 days, 4 proteins with molecular weights of approximately 38, 50, 65 and 110kd could be identified (Fig. 1).After longer incubation for 6 and 12 days, concentrations of these proteins increased and additional proteins with molecular weights ranging from 14 to over 90 kd appeared at detectable concentrations (Fig. 1). When M.ptb was grown for 24 days, no difference of secreted protein composition or concentrations was observed. However, at this time the medium might have been exhausted. The proteins which appeared in the filtrates were synthesized by actively growing organisms since we could not detect any labeled proteins in filtrates of irradiated M.ptb cultures included as negative controls in these experiments. Some of the proteins secreted by M.ptb were recognized by different immune sera. Reactivities in Western blots were detected in the range of 27, 31 to 43, 65 and 90kd (Fig.2). However, in the range of 31 to 43 kd no

MW 3 6 12 2 4 d 97 66 43

--

31

21

14-

Fig. 1. Autoradiograph showing profiles of %-labeled proteins in culture supernatants of M .ptb grown for 3, 6 , 12 and 24 days. Proteins were separated by SDS-PAGE, blotted onto nitrocellulose membranes and exposed to an X-ray film for 5 days. Molecular weight standards (MW) are given in kilodalton

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a b c d a b c d a b c d

A

B

C

Fig.2. Immunoblot analysis of filtrates from M . p t b grown for 3 (a), 6 (b), 12 (c) and 24 (d) days. Proteins were separated by SDS-PAGE, blotted onto nitrocellulose membranes and probed with serum from sheep immunized with live M . p t b bacilli (A = adult sheep; B = lambs) and from M . p t b infected sheep (C). Bars in the first lane indicate molecular weight standards in kilodalton

individual proteins were distinguishable which was propably due t o contamination with an immunoreactive carbohydrate. Comparing sera from M,ptb-infected sheep with those from sheep immunized with a live M.ptb vaccine strain, only the latter reacted with the proteins in the range of 27, 65 and 90kd (Fig.2). Interestingly, recent studies in our laboratory revealed that a 27 kd protein reacted specifically in Western blotting analysis and ELISA with sonicated M.ptb (14). We also compared SDS-PAGE profiles of shortand long-term cultures, since filtrates of the latter have often been used as so-called “PPD” in diagnostic and vaccine development studies. We found that, in contrast t o the large number of proteins in the short-term culture filtrates, supernatants of M.ptb grown for 10 weeks contained only 2 prominent proteins with MWs of 30 and 65 kd respectively (data not shown). Concluding, our study showed that M.ptb is capable of secreting proteins during short-term cultivations some of which are specifically recognized by sera from infected and vaccinated sheep and, thus, could be of pathogenic interest. However, more detailed studies are needed to characterize the secreted proteins of M.ptb and relate them t o similiarsized ones described as protective or common antigens of other mycobacteria/ bacteria (12).

Acknowledgements One of us (PVW) was supported by a grant from Deutsche Forschungsgemeinschaft (DFG), Bonn, FRG.

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