Veterinary Microbiology, 24 (1990) 29-41 Elsevier Science Publishers B.V., Amsterdam
29
Biochemical and immunochemical characterisation of strains of Treponema
hyodysenteriae Stuart C. Smith 1, Felicity Roddick 1, Soong Ling 1, Norman L. Gerraty 2 and Peter J. Coloe ~ IBiotechnology Unit, Department of Applied Biology, Royal Melbourne Institute of Technology, GPO Box 2476V, Melbourne, Vic. 3001 (Australia) 2E.R. Squibb & Sons Pty. Ltd., Box 39, Noble Park, Vic. 3174 (Australia) (Accepted 4 December 1989 )
ABSTRACT Smith, S.C., Roddick, F., Ling, S., Gerraty, N.L. and Coloe, P.J., 1990. Biochemical and immunochemical characterisation of strains of Treponema hyodysenteriae. Vet. Microbiol., 24:29-41. The protein composition of 18 clinical isolates of Treponema hyodysenteriae from pigs with swine dysentery in Australia were compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis. Coomassie Blue stained SDS-PAGE-profiles of whole cell and outer membrane (OM) proteins demonstrated the same gel pattern among the T. hyodysenteriae isolates, particularly the OM proteins in the molecular mass (Mr) range of 30 kDa to 40 kDa. The T. hyodysenteriae isolates were categorised into two distinct groups (A and B ) based on the strain-variability in the 37 kDa OM protein. Immunoblotting of whole cell proteins after SDS-PAGE using serum from rabbits and pigs immunised with known T. hyodysenteriae serotypes revealed a number of common immunoreactive bands in all isolates. LPS typing of the T. hyodysenteriae isolates by immunoblotting with the rabbit antiserum revealed one additional serotype emphasising the LPS heterogeneity among the strains isolated from geographic locations in Australia, Great Britain and the U.S.A. Immunoblotting of the OM preparations revealed several common immunoreactive polypeptides corresponding to Mr values of 34 kDa to 30 kDa among the T. hyodysenteriae and T. innocens isolates but a distinct 39 kDa found only in the T. hyodysenteriae isolates. Trypsin proteolysis of intact T. hyodysenteriae cells caused selective loss of these and other major abundant proteins identifying the location of the 39 kDa, 36 kDa, 34 kDa and 30 kDa proteins on the cell surface and suggesting a possible role of these proteins in the pathogenesis of swine dysentery.
INTRODUCTION
Treponema hyodysenteriae, a large Gram-negative spirochaete, is the causative agent of swine dysentery (SD), a disease of pigs characterised by in0378-1135/90/$03.50
© 1990 Elsevier Science Publishers B.V.
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S.C. SMITH ET AL.
flammation, oedema and haemorrhage in the large intestine and colon (Taylor, 1970; Harris et al., 1972; Hughes et al., 1975; Baum and Joens, 1979). The disease is highly contagious for breeding and post-weaned pigs in large herds yet little is known of the mechanism of pathogenesis or the host immunological response to the T. hyodysenteriae infection. There are a number of spirochaetes that are often detected as "normal" gastrointestinal flora of the porcine intestinal tract. T. innocens appears to be closely related to T. hyodysenteriae on growth and biochemical characteristics (Hudson et al., 1976 ) but it has not been shown to induce the same pathological lesions as T. hyodysenteriae (Kinyon et al., 1977). Likewise avirulent isolates of T. hyodysenteriae have been found that do not induce swine dysentery (P.J. Coloe, unpublished observations, 1988 ). Pathogenic isolates of T. hyodysenteriae have been classified serologically, on the basis of outer envelope lipopolysaccharide (LPS) (Baum and Joens, 1979; Mapother and Joens, 1985). This LPS may play an important role in the pathogenesis of T. hyodysenteriae in swine dysentery since protection against infection with T. hyodysenteriae appears serotype specific (Joens et al., 1983 ). Moreover, T. innocens may also be differentiated from the pathogenic T. hyodysenteriae by hemolysin production (Kinyon et al., 1977 ). The factors responsible for virulence and protection have not been identified. In many Gram-negative bacterial infections the outer membrane (OM) plays a critical role in the interaction between the pathogen and host (Di Rienzo et al., 1978). The OM components contribute importantly to host cell attachment, initiation of infection, resistance to phagocytosis and virulence. Recent studies using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting have identified the protein composition of a number of T. hyodysenteriae isolates from locations in the United States and Great Britain with respect to T. innocens (Joens and Marquez, 1966; Chatfield et al., 1988 ). To better define the extent of heterogeneity of T. hyodysenteriae from geographical locations the present study was undertaken to examine by SDSPAGE the polypeptide profiles of whole cell and OM enriched fractions of clinical isolates from Australia with reference to known T. hyodysenteriae strains, to determine by immunoblotting the LPS-serotypes of isolates from Australia; and to evaluate the surface distribution and immunogenicity of the OM involved in the immunological response to infection by clinical isolates from Australia. MATERIALS AND METHODS
Bacterial strains and growth conditions The T. hyodysenteriae strains used in this study were all isolated from pigs suffering from natural outbreaks of swine dysentery and were identified as T.
IMMUNOREACTIVE PROTEINS OF TREPONEMA HYOD YSENTERIAE
31
hyodysenteriae on the basis of their biochemical reactions and hemolytic activity on horse blood agar plates. The T. hyodysenteriae strains 70A, 5380, 924, 933, 947, 1155 and 4943, 508, 3636, 1415, were isolated from naturally occurring SD in Victoria. Strains 2612 and 2482 were from New South Wales, strain WA 2528 from Western Australia, and strains L7273, Q5363, Q5392, Q4662 and Q4374 from Queensland. Strains $78 and $82 were donated by R.J. Lysons, Institute for Research on Animal Diseases, Compton, Great Britain. The reference strains 31287, 31212 and 27164 were obtained as stock cultures from the American Type Culture Collection (ATCC), U.S.A. Serotype isolates B78, B204, B 169 and A 1, which were representatives of serotype I, II, III and IV, respectively were donated by D. Hampson, Murdoch University, Perth, Western Australia. T. innocens strains 9509 and 9510, 9690, 8841 and 8441 were isolated from pigs and identified on the basis of biochemical tests (Smibert, 1985 ). T. innocens strains B256 was donated by D. Hampson. A selection of bacterial isolates held in the Royal Melbourne Institute of Technology laboratory culture collection including Escherichia coli strain JP777, Salmonella typhimurium strain V27a, Yersinia enterocolitica strain 430-1, Campylobacter jejuni strains F1 and F14, and Campylobacterfetus subsp, fetus strain 45 were used as representatives of other Gram-negative bacteria. All isolates were stored at - 7 0 ° C . When required, the frozen isolates were thawed at 37 °C and cultivated on 5% horse blood agar at 37 °C in air or at 37°C or 42°C in an anaerobic jar in an atmosphere of H2/CO2 1 : 1. Cells of each culture were removed from the blood agar plates with sterile phosphate-buffered saline (PBS; 0.15 M sodium chloride, 10 mM sodium phosphate, pH 7.4 ), washed four times by repeated centrifugation (8000 ×g, 10 min, 4°C) and resuspension in 0.1 M Tris-HC1 (pH 8.3). If the cell mass was not used immediately it was stored at - 2 0 ° C until extractions were performed. Preparation of whole-cell and outer membrane extracts and peptidoglycan associated proteins Whole T. hyodysenteriae cells were dissolved in sodium dodecyl sulfate (SDS) buffer ( 125 mM Tris-HC1 (pH 6.9), 2% (w/v) SDS, 10% (w/v) glycerol, 5 ¢tM phenylmethyl sulphonyl fluoride (PMSF)) and heated in a waterbath for 5 min at 100 ° C. Outer membranes of T. hyodysenteriae, T. innocens and other Gram-negative bacteria were isolated from harvested cells after cell disruption, removal of whole-cell debris by centrifugation and differential solubilisation of the cell envelope with sodium N-lauryl sarcosinate (Sarkosyl; Sigma Chemical Co. ) according to the method of Achtman et al. ( 1983 ). Enriched OM and Sarkosyl-soluble inner membranes (IM) were separated by centrifugation ( 100 000 × g, 1 h, 4 ° C) and the OM suspended in SDS buffer prior to storage at - 2 0 ° C . Peptidoglycan-associated proteins were prepared by preferential solubilisation of the cell envelope in 2% (w/v) SDS at 37°C for 30 min (Nakae, 1976).
32
s.c, SMITHETAL.
The insoluble peptidoglycan and associated proteins (including porins) were separated from the soluble material by centrifugation ( 100 000 × g, 1 h, 4 °C ) and the peptidoglycan-associated proteins suspended in SDS buffer prior to storage at - 2 0 ° C. The protein content of the peptidoglycan-associated proteins and the outer membrane and whole-cell extracts was determined by a modification of the Lowry method (Markwell et al., 1978 ).
SDS-PAGE Samples ( 10-100 pg) were solubilised by heating at 100 ° C for 3 min in an equal volume of SDS sample buffer (SDS buffer containing 10% (w/v) 2-mercaptoethanol, (Sigma Chemical Co. ), and 0.01% (w/v) bromophenol blue (Sigma)). SDS-PAGE was performed as described by Laemmli (1970) with 12% (w/v) resolving gels and 4.75% (w/v) stacking gels. Gels were stained with either Coomassie Brilliant blue to visualise proteins or silver nitrate to visualise LPSs (Hitchcock and Brown, 1983) and the molecular masses (Mr) of the proteins determined using low Mr standards (Pharmacia LKB, Uppsala, Sweden).
Transfer and immunodetection of cellular and outer membrane proteins and LPS T. hyodysenteriae and T. innocens whole cell and OM proteins were electrophoretically transferred from one-dimensional SDS-polyacrylamide gels to nitrocellulose membranes (pore size, 0.2/~m) (Bio-Rad Laboratories) according to the Western blotting method of Towbin et al. ( 1979 ). For optimal detection of antigen-specific antibodies, blocking of non-specific binding sites and washing of zetaprobe membranes was performed in 5% (w/v) skim milk in TST buffer (10 mM Tris/HC1, 10 mM-NaC1 0.1% (w/v), Tween 20 pH 7.4 ), and all antibodies were diluted in TST buffer. Antibody adsorption was performed for 2 h using hyperimmune or pre-immune antisera from pigs or rabbits at a 1:200 dilution. Antibody binding was detected with either [ 35S ] protein A (0.1/tCi/ml, Amersham) and subsequent autoradiography, or horse-radish peroxidase-conjugated anti-rabbit or anti-porcine immunoglobulin ( 1 : 400, Cappel Laboratories) using 4-chloro- 1-naphthol (Sigma Chemical Co., St. Louis, MO) as a chromogenic substrate. Serotyping of T. hyodysenteriae LPS from 20 clinical isolates was determined by comparing control whole cell extracts (containing LPS ) with pronase treated extracts after SDS-PAGE and electrophoretic transfer to nitrocellulose. LPS was detected immunologically by using hyperimmune rabbit serum against defined serotypes I (B78), II (B204), III (B169), IV (A1) and Australian isolates 5380 and 70A at 1:200 dilution and antibody detection as above.
IMMUNOREACTIVE PROTEINS OF TREPONEMA HYODYSENTERIAE
33
Preparation of antisera Antiserum against whole cells of T. hyodysenteriae strains 5380, 70A, and known serotypes B78, B204, B 169 and A 1 were produced in rabbits or in pigs by intramuscular injection of whole T. hyodysenteriae cells ( 108 cells per ml) emulsified in an equal volume of Freund's incomplete adjuvant (CSL, Parkville). Booster injections were administered intramuscularly at 2, 4 and 8 weeks. Serum was collected prior to immunisation and 2 weeks after the last injection, and titrated by immunoblot analysis.
Protease treatment T. hyodysenteriae cells were harvested and washed as described above, and resuspended in PBS buffer at a concentration of 1 × 109 organisms per ml. To 1 ml of cell suspension was added either 50/~g of trypsin or 200 #g of proteinase K (Boehringer, Mannheim). A cell suspension without protease treatment was included as control. After incubation for 30 min at room temperature, cells were washed twice in PBS containing 5/~M PMSF (Sigma) and the pellets suspended in SDS sample buffer and subjected to SDS-PAGE. RESULTS
Comparison ofT. hyodysenteriae strains by SDS-PAGE SDS-PAGE profiles of polypeptides of whole-cell lysates from 18 isolates of T. hyodysenteriae from pigs with clinical SD in Australia, two isolates from the United Kingdom, three ATCC type strains and four serotype strains of T. hyodysenteriae were grown at 37 °C and compared as an initial step towards identifying potential immunogenic treponenal antigens. The majority of T. hyodysenteriae strains, including the serotype strains, revealed similar protein banding profiles to that of type strain ATCC 31287 (Fig. 1, lane 9) with respect to the major abundant proteins in the Mr range of 30 kDa to 40 kDa and at least 30 additional less intense Coomassie blue stained proteins. A typical profile is demonstrated in Fig. 1. The 36 kDa proteins were detected as closely migrating doublets in most isolates. Additional diffuse bands present in most strains in the Mr region 16-25 kDa were identified as complexes of LPS on the basis of silver staining and nitrocellulose binding characteristics (data not shown). Type strain ATCC 27164 (Fig. 1, lane 8) and Great Britain strain $78 (Fig. 1, lane 10) and $82 (data not shown) demonstrated identical polypeptide profiles to strains ATCC 31287 except that they did not express the 39 kDa protein doublet. After numerous passages on growth medium different preparations of whole-cell lysates from the same T. hyodysenteriae strains ( 5380 70A, 31287, 31212 ) showed identical polypeptide profiles on SDS-PAGE demonstrating that the stable protein composition of the isolates did not vary (data not shown). Furthermore the protein profile of strain 5380 (Fig. 1, lane 2) re-
34 ii~Da
S.C. SMITH ¸
i
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i!!!~!~~iii~,~,~, ~i¸~!~ii~,iii ii~i,~
!
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,,i,~ ~'~i~!'¸...... ¸ ~, ii ...... ~,~
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ET AL.
~,
i,,,~, ¸t~!~ ~,,~,~,,~,~! ,ii
Fig. 1. S D S - P A G E profiles o f w h o l e cell lysates o f isolates a n d reference s t r a i n s o f T. hyodysenteriae. L a n e 1, 70A; lane 2, 5380; lane 3, L7273; lane 4, Q 5 3 6 3 ; lane 5, 2612; lane 6 , 5 0 8 ; lane 7, 3630; lane 8, A T C C 27164; lane 9, A T C C 31287; lane 10, $78; lane 11, 9690; lane 12, 8841: lane 13, 8441.
mained stable after in vivo challenge and subsequent reisolation from several pigs. When T. hyodysenteriae strains 5380 and 70A were grown at 42 °C there were no major differences in the abundant proteins in the Coomassie blue stained protein profiles compared with the whole cell-lysates from cells grown at 37°C (data not shown).
Detection of immunogenic proteins and LPS heterogeneity of T. hyodysenteriae strains Using the Western immunoblotting procedure (Towbin et al., 1979 ) rabbit h y p e r i m m u n e serum raised against whole T. hyodysenteriae 5380 cells recognised at least 20 immunoreactive polypeptides with apparent molecular masses in the 20 kDa to 70 kDa Mr range (Fig. 2). No proteins were recognised by the pre-immune control serum. Rabbit h y p e r i m m u n e serum raised against T. hyodysenteriae cells from serotypes 1 to 4 and strain 70A revealed similar immunoblot profiles for each of the isolates with strong immunoreactivity with proteins in the Mf range 30 kDa to 47 kDa. The majority of immunoreactive proteins appear conserved in all isolates tested but some differences were detected in the immunoreactive proteins recognised in the T. innocens isolates, this is consistent with the data of Chatfield et al. ( 1988 ). Immunoblotting of the whole cell lysates with h y p e r i m m u n e porcine serum revealed the same bands were recognised by pigs as rabbits.
IMMUNOREACTIVE PROTEINS OF TREPONEMA HYODYSENTER1AE
35
Fig. 2. Western immunoblot analyses of T. hyodysenteriae whole cell solubilised proteins using specific rabbit serum. Cell lysates were prepared as described in the text, separated by SDSPAGE and transferred to nitrocellulose for Western blot analyses. Antigens were detected using pre-immune control serum (lane 1 ) or polyvalent rabbit anti-T, hyodysenteriae (isolate 5380) serum (lanes 2 to 14) diluted 1:200. Lanes 1 and 2, 5380; lane 3, L7273; lane 4, Q5363; lane 5, Q5392; lane 6, Q4662; lane 7, Q9374; lane 8, $825; lane 9, $78.4; lane 10, 508; lane 11, 3636; lane 12, 1415; lane 13, 2612; lane 14, 2482. Bound antibody was detected using [ 35S] Protein A and subsequent autoradiography. Arrows indicate the position of LPS and the 39 kDa and 36 kDa polypeptides.
Differences between the T. hyodysenteriae clinical isolates were observed in the immunoreactivity of the antigen in the 16 kDa to 20 kDa region to the strain-specific porcine or rabbit hyperimmune serums raised against whole T. hyodysenteriae cells. This antigen was determined to be LPS complex by using silver nitrate staining (Hitchcock and Brown, 1983) and by examining control whole cell lysates with pronase-treated cell lysates prior to SDS-PAGE and immunoblotting. Of 18 field isolates of T. hyodysenteriae probed with rabbit hyperimmune serum raised against the known serotypes of T. hyodysenteriae, only six strains (508, 3636, 2612, 924, 933 and 4943 ) had LPS immunoreactive with antiserotype II (strain B204) serum and five strains ($82, $78, 1415, WA2528 and L7273 ) immunoreactive with anti-serotype I (strain B78 ) serum. Rabbit anti-serotype III (strain B 169 ) serum did not recognise the LPS complex from any of the Australian isolates tested. The majority of Australian field isolates examined, reacted strongly with hyperimmune serum raised against T. hy-
36
s.c. SMITH ET AL.
odysenteriae strains 5380 and 70A and to a lesser extent with serum raised against serotype IV (strains A~ ). This diversity suggests further heterogeneity in the LPS complexes within serotype 4 in the field isolates from Australia. OM protein profiles ofT. hyodysenteriae Cell envelope fractions from the field isolates and type strains of T. hyodysenteriae and two isolates of T. innocens were extracted with Sarkosyl and the Sarkosyl-insoluble, OM protein-enriched preparations resolved by SDSPAGE (Fig. 3 ). The major OM proteins present in T. hyodysenteriae type and field strains corresponded to the major abundant proteins migrating in the M r range 30 kDa to 40 kDa in the cellular protein preparations (refer to Fig. 1 ). The OM protein profiles revealed all isolates expressed common 36 kDa, 34 kDa, 33 kDa, 31.5 kDa and 30 kDa OM proteins when grown at 37°C (Fig. 3, lanes 1 to 6 ) or 42 ° C (data not shown) but of the 18 clinical isolates tested 11 expressed a major 37 kDa OMP and 7 expressed a major 37.5 kDa OMP (see Fig. 3, lanes 1 and 2, respectively). These isolates have been designated groups A and B, respectively based upon the variable migration on kDa
Fig. 3. SDS-PAGE profiles ofSarkosyl-insoluble OM preparations. Ten pg of OM proteins from T. hyodysenteriae isolates. Lane 1, 70A; lane 2, ATCC 31287; lane 3, ATCC 27164; lane 4, Q5363; lane 5, 508; lane 6, 5380; lane 7, T. innocens 9690; lane 8, T. innocens 8841; lane 9, T. innocens 8441; lane 10, T. innocens 9509; lane 11, T. innocens 9510; lane 12, 5380 whole cell lysate. Asterisks indicate the position of the 37 kDa and 37.5 kDa polypeptides.
37
IMMUNOREACTIVEPROTEINSOF TREPONEMAHYODYSENTERIAE
SDS-PAGE of the 37 kDa protein. The OM protein-enriched composition of T. hyodysenteriae was readily distinguished from that of T. innocens (Fig. 3, lanes 7 to 11 ) and the OMP-enriched preparations from E. coli, S. typhimurium, Y. enterocolitica, C. jejuni and C. fetus subsp, fetus.
Detection of immunoreactive OM proteins ofT. hyodysenteriae When Sarkosyl-insoluble OM preparations from seven T. hyodysenteriae (four group A and two group B) isolates and two T. innocens isolates were separated by SDS-PAGE and immunoblotted and probed with porcine antiT. hyodysenteriae strain 5380 hyperimmune serum, the pattern of reactivity was similar in almost all isolates except for a small degree of strain-variation in the proteins in the 38 kDa to 40 kDa molecular mass range (Fig. 4). Included for comparative purposes was the pattern of reactivity with isolate 5380 whole-cell lysate (Fig. 4, lanes 1 and 5 ). The OM proteins from groups A and B and the T. innocens isolates were reactive with antibodies present in A
B
kDa 94
.
43
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34
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.......
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..........
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1
2
3
4
5
6
7
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9
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Fig. 4. Western blot profiles of OM proteins of T. hyodysenteriae and T. innocens isolates using porcine anti-T, hyodysenteriae serum. OM proteins were prepared by Sarkosyl solubilisation. Panel A, immune blot with pre-immune control sera; panel B, immune blot with hyper-immune sera. Serum was diluted 1 : 100. Lanes 1 and 5, T. hyodysenteriae 5380 cell extract; lanes 2 and 6, 5380 OMP; lanes 3 and 7, ATCC 31287 OMP; lanes 4 and 8, 70A OMP; lane 9, Q5363 OMP; lane 10, 508 OMP; lane 11, ATCC 27164 OMP; lane 12, T. innocens 8841 OMP; lane 13, T. in nocens 9510 OMP. Antibody binding was detected by incubation with [35S] Protein A and subsequent autoradiography.
38
S.C. SMITH ET AL.
the hyperimmune sera but not with prechallenge control serum (Fig. 4, lanes 1 to 4). In all T. hyodysenteriae isolates, strongly immunoreactive common OM proteins were observed at 34 kDa and 30 kDa. The 34 kDa and 30 kDa bands were also reactive in the T. innocens isolates (Fig. 4, lanes 12 and 13 ). The major 40 kDa in the T. innocens isolate 8841 (Fig. 4, lane 12), the 38 kDa and 38.5 kDa protein bands in the T. innocens isolate 9510 (Fig. 4, lane 13) and the specific 39 kDa band conserved in T. hyodysenteriae groups A and B isolates (Fig. 4, lanes 6 to 11 ) were also immunoreactive. A similar antibody response was observed when OM proteins were probed with rabbit anti- T. hyodysenteriae hyperimmune serum. Immunoblotting of the OM proteins from other Gram-negative organisms with the same porcine anti-T, hyodysenteriae hyperimmune serum revealed no detectable antibody crossreactivity.
Surface location of protease-sensitive OM proteins To further characterise the T. hyodysenteriae antigens important in the immunological response, the OM proteins recognised in the Western blots were A
B
kDa 94
-
67
_
3
.-
2:0.1
--
Fig. 5. Effect of protease digestion on T. hyodysenteriae isolate 5380 whole cells. Intact cells were incubated with 50 #g/ml of trypsin (lane A) or with PBS buffer (lane B). After 30 min, reactions were stopped by the addition of PMSF and cells solubilised in SDS sample buffer prior to SDS-PAGE. Arrows indicate the position of the Coomassie blue-stained proteins sensitive to proteolysis.
IMMUNOREACTIVE PROTEINS OF TREPONEMA HYODYSENTERIAE
39
correlated with proteins located on the surface of T. hyodysenteriae. Intact T. hyodysenteriae strain 5380 whole cells were treated with trypsin and the proteolysis stopped with PMSF. Both protease-treated and control T. hyodysenteriae 5380 cells were solubilised in SDS sample buffer and subjected to SDSPAGE (Fig. 5 ). Trypsin proteolysis caused selective loss of the protein bands at Mr values of 39 kDa, 34 kDa (doublet), and 30 kDa indicating that these proteins extend out through the OM to the treponemal cell surface. The same pattern of proteolysis was observed when proteinase K treatment of the T. hyodysen teriae 5380 cells was used rather than trypsin (data not shown ). The 37.5 kDa protein band was shown to be a peptidoglycan-associated protein based upon the resistance of this outer membrane protein to solubilisation by 2% (w/v) SDS at 37 °C (Nakae, 1976 ). Solubilisation at 37 °C resulted in the absence of the 37 kDa protein from the outer membrane profile from T. hyodysenteriae strain 5380 and the appearance of apparent higher molecular weight proteins which may represent oligomeric forms of this protein due to the anomalous migration of such complexes on SDS-PAGE (data not shown ). DISCUSSION In the present study we have examined by SDS-PAGE the whole cell proteins of 18 strains of T. hyodysenteriae isolated from pigs from several locations in Australia and compared those with isolates from the United States and Great Britain. The high degree of uniformity in the whole cell polypeptide profiles of the T. hyodysenteriae isolates is in agreement with observations made by another research group (Chatfield et al., 1988) and demonstrates the usefulness of SDS-PAGE for differentiation of T. hyodysenteriae from the commensal spirochaete T. innocens. OM proteins from the 18 field T. hyodysenteriae isolates demonstrated minor differences in the OM proteins especially the strain variable 37 kDa OM protein suggesting the subdivision of the T. hyodysenteriae isolates into two groups, A and B. E. coli OM protein profiles have been used as epidemiologic markers in the study of disease (Achtman et al., 1983; Barenkamp et al., 1981 ) and OM protein patterns can be used to characterise T. hyodysenteriae strains. However, there was no clear correlation between groups A and B OM patterns and LPS serotype, indicating that the two features do not appear closely related. These resuits suggest further evaluation of the OM proteins is required to determine the role of these OM proteins and how they interact with LPS in pathogenicity. SDS-PAGE and immunoblotting demonstrated a heterogeneous response to LPS but a conserved protein response, with three strongly immunoreactive proteins of 39 kDa, 34 kDa and 30 kDa being recognised by anti-T, hyodysenteriae sera, irrespective of serotype. Several of these OM proteins were crossreactive to antisera raised against T. hyodysenteriae and T. innocens (data not shown) suggesting that the 39 kDa OM protein appears specific to T. hyodys-
40
S.C. SMITH ET AL.
enteriae. Joens and Marquez (1986) and Chatfield et al. ( 1988 ) have shown other major polypeptide antigens apparently specific to T. hyodysenteriae Chatfield et al. (1988) has shown that the predominant polypeptides between 30 kDa and 36 kDa are associated with the cell envelope fraction. In this study we have shown, by using the sodium N-lauryl sarcosinate (Sarkosyl) method of Achtman et al. ( 1983 ) which selectively solubilises the inner membrane and enriches for the OM Proteins that these same cell-envelopeassociated polypeptides reside in the OM of T. hyodysenteriae and are situated on the cell surface, and sensitive to trypsin proteolysis in situ, indicating that they must have surface-exposed epitopes. These major immunoreactive polypeptides may be targets for a protective porcine immune response against SD. The strain variable 37 kDa OM protein in both group A and B T. hyodysenteriae isolates did not appear strongly immunoreactive due to the fact that it appears to be a peptidoglycan-associated protein (data not shown) and is not degraded by trypsin treatment. This protein may represent an integral membrane protein due to its selective release from the cell membrane by Triton X-114 (M. Alderton, personal communication), a detergent used to extract integral membrane proteins (Bordier, 1981 ). By analogy with other enteric porins the 37 kDa OM protein may serve an important role in the synthesis of capsular polysaccharides (Paakkanen et al., 1979; Achtman et al., 1983 ) but not in the sero-specificity of the T. hyodysenteriae LPS. When isolates from varied geographical locations in Australia were typed using hyperimmune rabbit serum against four known T. hyodysenteriae serotypes (I to IV) and two Australian isolates, at least one additional LPS serotype, specific to Australia, was observed, confirming and extending the evidence of LPS heterogeneity among the T. hyodysenteriae isolates examined (Baum and Joens, 1979; Mapother and Joens, 1985; Chatfield et al., 1988). This serotype did not react with hyperimmune rabbit serum raised against serotypes 1, 2 or 3 (Baum and Joens, 1979) and is different to serotypes 5, 6 or 7 since it did not cross-react with serotypes 1 and 2 antisera, a feature of these serotypes (Mapother and Joens, 1985 ). Hampson et al. (1989) has proposed a revised typing scheme for T. hyodysenteriae isolates placing isolates which share common LPS antigens into serogroups A to E with serotypes within each serogroup. Serogroups A to D correspond to the serotypes 1 to 4 in the typing scheme of Baum and Joens ( 1979 ) while serogroup E is a novel group. Hampson et al. ( 1989 ) has placed Western Australia isolates of T. hyodysenteriae into the serogroups A, B and E. Additional studies will be carried out to determine if the additional LPS serotype found herein may represent another serogroup. In this paper we have demonstrated that the major polypeptides of T. hyodysenteriae are strong immunogens and are conserved with the various serotypes whereas there is considerable diversity in the antigenicity of LPS between those same serotypes.
IMMUNOREACTIVE PROTEINS OF TREPONEMA HYOD YSENTER1AE
41
REFERENCES Achtman, M., Mercer, A., Kusecek, B., Pohl, A., Heuzenroder, M., Aaronson, A., Sutton, A. and Silver, R.P., 1983. Six widespread bacterial clones among Escherichia coli K1 isolates. lnfect. Immun., 39:315-335. Barenkamp, S.J., Munson, R.S. and Granoff, D.M., 1981. Subtyping isolates of Haemophilus influenzae type b by outer membrane protein profiles. J. Infect. Dis., 143: 668-676. Baum, D.M. and Joens, L.A., 1979. Serotypes of beta-hemolytic Treponema hyodysenteriae. Infect. Immun., 25: 792-796. Bordier, C., 1981. Phase separation of integral membrane proteins in Triton X-114 solution. J. Biol. Chem., 256: 1604-1607. Chatfield, S.N., Fernie, D.S., Penn, C. and Dougan, G., 1988. Identification of the major antigens of Treponema hyodysenteriae and comparison with those of Treponema innocens. Infect. Immun., 56: 1070-1075. DiRienzo, J.M., Nakamura, K. and Inouye, M., 1978. The outer membrane proteins of Gramnegative bacteria: biosynthesis, assembly and function. Annu. Rev. Biochem., 47:481-532, Hampson, D.J., Mhorna, J.R.L,, Combs, B. and Buddle, J.R., 1989. Proposed revisions to the serological typing system for Treponema hyodysenteriae. Epidemiol. Infect., 102: 75-84. Harris, D.L., Glock, R.D., Christensen, C.R. and Kinyon, J.M., 1972. Swine dysentery. I. Inoculation of pigs with Treponema hyodysenteriae (new species) and reproduction of the disease. Vet. Med. Small Anim. Clin., 67: 61-64. Hitchcock, P.J. and Braun, T.M., 1983. Morphological heterogeneity among salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J. Bacteriol., 154: 269-277. Hudson, M.J., Alexander, T.J.L. and Lysons, R.J., 1976. Diagnosis of swine dysentery: Spirochaetes which may be confused with Treponema hyodysenwriae, Vet. Rec., 99: 498-500. Hughes, R., Olancer, M.V. and Williams, C.B., 1975. Swine dysentery: pathogenicity of Treponema hyodysenteriae. Am. J. Vet. Res., 36:971-977. Joens, L.A. and Marquez, R.B., 1986. Molecular characterisation of proteins from porcine spirochaetes. Infect. Immun., 54: 893-896. Joens, L.A., Whipp, S.C., Glock, R.D. and Nuessen, M.E., 1983. Serotype-specific protection against infection in ligated colonic loops of pigs recovered from swine dysentery. Infect. Immun., 39: 460-462. Kinyon, J.M., Harris, D.L. and Glock, R.D., 1977. Enteropathogenicity of various isolates of Treponema hyodysenteriae. Infect. Immunol., 15: 638-646. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227: 680-685. Mapother, M.E. and Joens, L.A., 1985. New serotypes of Treponema hyodysenteriae. J. Clin. Microbiol., 22: 161-164. Markwell, M.A.K., Naas, S.M., Biaber, L.L. and Tolbert, N.W., 1978. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal. Biochem., 87: 206-210. Nakae, T., 1976. Identification of the outer membrane proteins of Escherichia coli that produces transmembrane channels in reconstituted vesicle membranes. Biochem. Biophys. Res. Commun., 71: 877-884. Paakkanen, J., Gotschlich, E.C. and Makela, P.H., 1979. Protein K: a new major outer membrane protein found in encapsulated Escherichia coll. J. Bacteriol., 139: 835-841. Smibert, R.M., 1984. Anaerobic Spirochaetes. In: E.M. Lennette, A. Balows, W.J. Housler, Jr. and H.J. Shadonyl (Editors), Manual of Clinical Microbiology 4th Edn. American Society for Microbiology, Washington, DC, pp. 490-494. Taylor, D.J., 1970. An agent possibly associated with swine dysentery. Res. Vet. Sci., 12:177179. Towbin, M., Staehelin, T. and Gordon, J., 1979. Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedures and applications. Proc. Nat. Acad. Sci. U.S.A., 76: 4350-4354.
29
Biochemical and immunochemical characterisation of strains of Treponema
hyodysenteriae Stuart C. Smith 1, Felicity Roddick 1, Soong Ling 1, Norman L. Gerraty 2 and Peter J. Coloe ~ IBiotechnology Unit, Department of Applied Biology, Royal Melbourne Institute of Technology, GPO Box 2476V, Melbourne, Vic. 3001 (Australia) 2E.R. Squibb & Sons Pty. Ltd., Box 39, Noble Park, Vic. 3174 (Australia) (Accepted 4 December 1989 )
ABSTRACT Smith, S.C., Roddick, F., Ling, S., Gerraty, N.L. and Coloe, P.J., 1990. Biochemical and immunochemical characterisation of strains of Treponema hyodysenteriae. Vet. Microbiol., 24:29-41. The protein composition of 18 clinical isolates of Treponema hyodysenteriae from pigs with swine dysentery in Australia were compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis. Coomassie Blue stained SDS-PAGE-profiles of whole cell and outer membrane (OM) proteins demonstrated the same gel pattern among the T. hyodysenteriae isolates, particularly the OM proteins in the molecular mass (Mr) range of 30 kDa to 40 kDa. The T. hyodysenteriae isolates were categorised into two distinct groups (A and B ) based on the strain-variability in the 37 kDa OM protein. Immunoblotting of whole cell proteins after SDS-PAGE using serum from rabbits and pigs immunised with known T. hyodysenteriae serotypes revealed a number of common immunoreactive bands in all isolates. LPS typing of the T. hyodysenteriae isolates by immunoblotting with the rabbit antiserum revealed one additional serotype emphasising the LPS heterogeneity among the strains isolated from geographic locations in Australia, Great Britain and the U.S.A. Immunoblotting of the OM preparations revealed several common immunoreactive polypeptides corresponding to Mr values of 34 kDa to 30 kDa among the T. hyodysenteriae and T. innocens isolates but a distinct 39 kDa found only in the T. hyodysenteriae isolates. Trypsin proteolysis of intact T. hyodysenteriae cells caused selective loss of these and other major abundant proteins identifying the location of the 39 kDa, 36 kDa, 34 kDa and 30 kDa proteins on the cell surface and suggesting a possible role of these proteins in the pathogenesis of swine dysentery.
INTRODUCTION
Treponema hyodysenteriae, a large Gram-negative spirochaete, is the causative agent of swine dysentery (SD), a disease of pigs characterised by in0378-1135/90/$03.50
© 1990 Elsevier Science Publishers B.V.
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S.C. SMITH ET AL.
flammation, oedema and haemorrhage in the large intestine and colon (Taylor, 1970; Harris et al., 1972; Hughes et al., 1975; Baum and Joens, 1979). The disease is highly contagious for breeding and post-weaned pigs in large herds yet little is known of the mechanism of pathogenesis or the host immunological response to the T. hyodysenteriae infection. There are a number of spirochaetes that are often detected as "normal" gastrointestinal flora of the porcine intestinal tract. T. innocens appears to be closely related to T. hyodysenteriae on growth and biochemical characteristics (Hudson et al., 1976 ) but it has not been shown to induce the same pathological lesions as T. hyodysenteriae (Kinyon et al., 1977). Likewise avirulent isolates of T. hyodysenteriae have been found that do not induce swine dysentery (P.J. Coloe, unpublished observations, 1988 ). Pathogenic isolates of T. hyodysenteriae have been classified serologically, on the basis of outer envelope lipopolysaccharide (LPS) (Baum and Joens, 1979; Mapother and Joens, 1985). This LPS may play an important role in the pathogenesis of T. hyodysenteriae in swine dysentery since protection against infection with T. hyodysenteriae appears serotype specific (Joens et al., 1983 ). Moreover, T. innocens may also be differentiated from the pathogenic T. hyodysenteriae by hemolysin production (Kinyon et al., 1977 ). The factors responsible for virulence and protection have not been identified. In many Gram-negative bacterial infections the outer membrane (OM) plays a critical role in the interaction between the pathogen and host (Di Rienzo et al., 1978). The OM components contribute importantly to host cell attachment, initiation of infection, resistance to phagocytosis and virulence. Recent studies using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting have identified the protein composition of a number of T. hyodysenteriae isolates from locations in the United States and Great Britain with respect to T. innocens (Joens and Marquez, 1966; Chatfield et al., 1988 ). To better define the extent of heterogeneity of T. hyodysenteriae from geographical locations the present study was undertaken to examine by SDSPAGE the polypeptide profiles of whole cell and OM enriched fractions of clinical isolates from Australia with reference to known T. hyodysenteriae strains, to determine by immunoblotting the LPS-serotypes of isolates from Australia; and to evaluate the surface distribution and immunogenicity of the OM involved in the immunological response to infection by clinical isolates from Australia. MATERIALS AND METHODS
Bacterial strains and growth conditions The T. hyodysenteriae strains used in this study were all isolated from pigs suffering from natural outbreaks of swine dysentery and were identified as T.
IMMUNOREACTIVE PROTEINS OF TREPONEMA HYOD YSENTERIAE
31
hyodysenteriae on the basis of their biochemical reactions and hemolytic activity on horse blood agar plates. The T. hyodysenteriae strains 70A, 5380, 924, 933, 947, 1155 and 4943, 508, 3636, 1415, were isolated from naturally occurring SD in Victoria. Strains 2612 and 2482 were from New South Wales, strain WA 2528 from Western Australia, and strains L7273, Q5363, Q5392, Q4662 and Q4374 from Queensland. Strains $78 and $82 were donated by R.J. Lysons, Institute for Research on Animal Diseases, Compton, Great Britain. The reference strains 31287, 31212 and 27164 were obtained as stock cultures from the American Type Culture Collection (ATCC), U.S.A. Serotype isolates B78, B204, B 169 and A 1, which were representatives of serotype I, II, III and IV, respectively were donated by D. Hampson, Murdoch University, Perth, Western Australia. T. innocens strains 9509 and 9510, 9690, 8841 and 8441 were isolated from pigs and identified on the basis of biochemical tests (Smibert, 1985 ). T. innocens strains B256 was donated by D. Hampson. A selection of bacterial isolates held in the Royal Melbourne Institute of Technology laboratory culture collection including Escherichia coli strain JP777, Salmonella typhimurium strain V27a, Yersinia enterocolitica strain 430-1, Campylobacter jejuni strains F1 and F14, and Campylobacterfetus subsp, fetus strain 45 were used as representatives of other Gram-negative bacteria. All isolates were stored at - 7 0 ° C . When required, the frozen isolates were thawed at 37 °C and cultivated on 5% horse blood agar at 37 °C in air or at 37°C or 42°C in an anaerobic jar in an atmosphere of H2/CO2 1 : 1. Cells of each culture were removed from the blood agar plates with sterile phosphate-buffered saline (PBS; 0.15 M sodium chloride, 10 mM sodium phosphate, pH 7.4 ), washed four times by repeated centrifugation (8000 ×g, 10 min, 4°C) and resuspension in 0.1 M Tris-HC1 (pH 8.3). If the cell mass was not used immediately it was stored at - 2 0 ° C until extractions were performed. Preparation of whole-cell and outer membrane extracts and peptidoglycan associated proteins Whole T. hyodysenteriae cells were dissolved in sodium dodecyl sulfate (SDS) buffer ( 125 mM Tris-HC1 (pH 6.9), 2% (w/v) SDS, 10% (w/v) glycerol, 5 ¢tM phenylmethyl sulphonyl fluoride (PMSF)) and heated in a waterbath for 5 min at 100 ° C. Outer membranes of T. hyodysenteriae, T. innocens and other Gram-negative bacteria were isolated from harvested cells after cell disruption, removal of whole-cell debris by centrifugation and differential solubilisation of the cell envelope with sodium N-lauryl sarcosinate (Sarkosyl; Sigma Chemical Co. ) according to the method of Achtman et al. ( 1983 ). Enriched OM and Sarkosyl-soluble inner membranes (IM) were separated by centrifugation ( 100 000 × g, 1 h, 4 ° C) and the OM suspended in SDS buffer prior to storage at - 2 0 ° C . Peptidoglycan-associated proteins were prepared by preferential solubilisation of the cell envelope in 2% (w/v) SDS at 37°C for 30 min (Nakae, 1976).
32
s.c, SMITHETAL.
The insoluble peptidoglycan and associated proteins (including porins) were separated from the soluble material by centrifugation ( 100 000 × g, 1 h, 4 °C ) and the peptidoglycan-associated proteins suspended in SDS buffer prior to storage at - 2 0 ° C. The protein content of the peptidoglycan-associated proteins and the outer membrane and whole-cell extracts was determined by a modification of the Lowry method (Markwell et al., 1978 ).
SDS-PAGE Samples ( 10-100 pg) were solubilised by heating at 100 ° C for 3 min in an equal volume of SDS sample buffer (SDS buffer containing 10% (w/v) 2-mercaptoethanol, (Sigma Chemical Co. ), and 0.01% (w/v) bromophenol blue (Sigma)). SDS-PAGE was performed as described by Laemmli (1970) with 12% (w/v) resolving gels and 4.75% (w/v) stacking gels. Gels were stained with either Coomassie Brilliant blue to visualise proteins or silver nitrate to visualise LPSs (Hitchcock and Brown, 1983) and the molecular masses (Mr) of the proteins determined using low Mr standards (Pharmacia LKB, Uppsala, Sweden).
Transfer and immunodetection of cellular and outer membrane proteins and LPS T. hyodysenteriae and T. innocens whole cell and OM proteins were electrophoretically transferred from one-dimensional SDS-polyacrylamide gels to nitrocellulose membranes (pore size, 0.2/~m) (Bio-Rad Laboratories) according to the Western blotting method of Towbin et al. ( 1979 ). For optimal detection of antigen-specific antibodies, blocking of non-specific binding sites and washing of zetaprobe membranes was performed in 5% (w/v) skim milk in TST buffer (10 mM Tris/HC1, 10 mM-NaC1 0.1% (w/v), Tween 20 pH 7.4 ), and all antibodies were diluted in TST buffer. Antibody adsorption was performed for 2 h using hyperimmune or pre-immune antisera from pigs or rabbits at a 1:200 dilution. Antibody binding was detected with either [ 35S ] protein A (0.1/tCi/ml, Amersham) and subsequent autoradiography, or horse-radish peroxidase-conjugated anti-rabbit or anti-porcine immunoglobulin ( 1 : 400, Cappel Laboratories) using 4-chloro- 1-naphthol (Sigma Chemical Co., St. Louis, MO) as a chromogenic substrate. Serotyping of T. hyodysenteriae LPS from 20 clinical isolates was determined by comparing control whole cell extracts (containing LPS ) with pronase treated extracts after SDS-PAGE and electrophoretic transfer to nitrocellulose. LPS was detected immunologically by using hyperimmune rabbit serum against defined serotypes I (B78), II (B204), III (B169), IV (A1) and Australian isolates 5380 and 70A at 1:200 dilution and antibody detection as above.
IMMUNOREACTIVE PROTEINS OF TREPONEMA HYODYSENTERIAE
33
Preparation of antisera Antiserum against whole cells of T. hyodysenteriae strains 5380, 70A, and known serotypes B78, B204, B 169 and A 1 were produced in rabbits or in pigs by intramuscular injection of whole T. hyodysenteriae cells ( 108 cells per ml) emulsified in an equal volume of Freund's incomplete adjuvant (CSL, Parkville). Booster injections were administered intramuscularly at 2, 4 and 8 weeks. Serum was collected prior to immunisation and 2 weeks after the last injection, and titrated by immunoblot analysis.
Protease treatment T. hyodysenteriae cells were harvested and washed as described above, and resuspended in PBS buffer at a concentration of 1 × 109 organisms per ml. To 1 ml of cell suspension was added either 50/~g of trypsin or 200 #g of proteinase K (Boehringer, Mannheim). A cell suspension without protease treatment was included as control. After incubation for 30 min at room temperature, cells were washed twice in PBS containing 5/~M PMSF (Sigma) and the pellets suspended in SDS sample buffer and subjected to SDS-PAGE. RESULTS
Comparison ofT. hyodysenteriae strains by SDS-PAGE SDS-PAGE profiles of polypeptides of whole-cell lysates from 18 isolates of T. hyodysenteriae from pigs with clinical SD in Australia, two isolates from the United Kingdom, three ATCC type strains and four serotype strains of T. hyodysenteriae were grown at 37 °C and compared as an initial step towards identifying potential immunogenic treponenal antigens. The majority of T. hyodysenteriae strains, including the serotype strains, revealed similar protein banding profiles to that of type strain ATCC 31287 (Fig. 1, lane 9) with respect to the major abundant proteins in the Mr range of 30 kDa to 40 kDa and at least 30 additional less intense Coomassie blue stained proteins. A typical profile is demonstrated in Fig. 1. The 36 kDa proteins were detected as closely migrating doublets in most isolates. Additional diffuse bands present in most strains in the Mr region 16-25 kDa were identified as complexes of LPS on the basis of silver staining and nitrocellulose binding characteristics (data not shown). Type strain ATCC 27164 (Fig. 1, lane 8) and Great Britain strain $78 (Fig. 1, lane 10) and $82 (data not shown) demonstrated identical polypeptide profiles to strains ATCC 31287 except that they did not express the 39 kDa protein doublet. After numerous passages on growth medium different preparations of whole-cell lysates from the same T. hyodysenteriae strains ( 5380 70A, 31287, 31212 ) showed identical polypeptide profiles on SDS-PAGE demonstrating that the stable protein composition of the isolates did not vary (data not shown). Furthermore the protein profile of strain 5380 (Fig. 1, lane 2) re-
34 ii~Da
S.C. SMITH ¸
i
!!ii!~ ! !~iiii!i i!!!il~¸ ~iiii
i!!!~!~~iii~,~,~, ~i¸~!~ii~,iii ii~i,~
!
i!
~¸¸
~
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i!i~i~ii~
,,i,~ ~'~i~!'¸...... ¸ ~, ii ...... ~,~
~
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ET AL.
~,
i,,,~, ¸t~!~ ~,,~,~,,~,~! ,ii
Fig. 1. S D S - P A G E profiles o f w h o l e cell lysates o f isolates a n d reference s t r a i n s o f T. hyodysenteriae. L a n e 1, 70A; lane 2, 5380; lane 3, L7273; lane 4, Q 5 3 6 3 ; lane 5, 2612; lane 6 , 5 0 8 ; lane 7, 3630; lane 8, A T C C 27164; lane 9, A T C C 31287; lane 10, $78; lane 11, 9690; lane 12, 8841: lane 13, 8441.
mained stable after in vivo challenge and subsequent reisolation from several pigs. When T. hyodysenteriae strains 5380 and 70A were grown at 42 °C there were no major differences in the abundant proteins in the Coomassie blue stained protein profiles compared with the whole cell-lysates from cells grown at 37°C (data not shown).
Detection of immunogenic proteins and LPS heterogeneity of T. hyodysenteriae strains Using the Western immunoblotting procedure (Towbin et al., 1979 ) rabbit h y p e r i m m u n e serum raised against whole T. hyodysenteriae 5380 cells recognised at least 20 immunoreactive polypeptides with apparent molecular masses in the 20 kDa to 70 kDa Mr range (Fig. 2). No proteins were recognised by the pre-immune control serum. Rabbit h y p e r i m m u n e serum raised against T. hyodysenteriae cells from serotypes 1 to 4 and strain 70A revealed similar immunoblot profiles for each of the isolates with strong immunoreactivity with proteins in the Mf range 30 kDa to 47 kDa. The majority of immunoreactive proteins appear conserved in all isolates tested but some differences were detected in the immunoreactive proteins recognised in the T. innocens isolates, this is consistent with the data of Chatfield et al. ( 1988 ). Immunoblotting of the whole cell lysates with h y p e r i m m u n e porcine serum revealed the same bands were recognised by pigs as rabbits.
IMMUNOREACTIVE PROTEINS OF TREPONEMA HYODYSENTER1AE
35
Fig. 2. Western immunoblot analyses of T. hyodysenteriae whole cell solubilised proteins using specific rabbit serum. Cell lysates were prepared as described in the text, separated by SDSPAGE and transferred to nitrocellulose for Western blot analyses. Antigens were detected using pre-immune control serum (lane 1 ) or polyvalent rabbit anti-T, hyodysenteriae (isolate 5380) serum (lanes 2 to 14) diluted 1:200. Lanes 1 and 2, 5380; lane 3, L7273; lane 4, Q5363; lane 5, Q5392; lane 6, Q4662; lane 7, Q9374; lane 8, $825; lane 9, $78.4; lane 10, 508; lane 11, 3636; lane 12, 1415; lane 13, 2612; lane 14, 2482. Bound antibody was detected using [ 35S] Protein A and subsequent autoradiography. Arrows indicate the position of LPS and the 39 kDa and 36 kDa polypeptides.
Differences between the T. hyodysenteriae clinical isolates were observed in the immunoreactivity of the antigen in the 16 kDa to 20 kDa region to the strain-specific porcine or rabbit hyperimmune serums raised against whole T. hyodysenteriae cells. This antigen was determined to be LPS complex by using silver nitrate staining (Hitchcock and Brown, 1983) and by examining control whole cell lysates with pronase-treated cell lysates prior to SDS-PAGE and immunoblotting. Of 18 field isolates of T. hyodysenteriae probed with rabbit hyperimmune serum raised against the known serotypes of T. hyodysenteriae, only six strains (508, 3636, 2612, 924, 933 and 4943 ) had LPS immunoreactive with antiserotype II (strain B204) serum and five strains ($82, $78, 1415, WA2528 and L7273 ) immunoreactive with anti-serotype I (strain B78 ) serum. Rabbit anti-serotype III (strain B 169 ) serum did not recognise the LPS complex from any of the Australian isolates tested. The majority of Australian field isolates examined, reacted strongly with hyperimmune serum raised against T. hy-
36
s.c. SMITH ET AL.
odysenteriae strains 5380 and 70A and to a lesser extent with serum raised against serotype IV (strains A~ ). This diversity suggests further heterogeneity in the LPS complexes within serotype 4 in the field isolates from Australia. OM protein profiles ofT. hyodysenteriae Cell envelope fractions from the field isolates and type strains of T. hyodysenteriae and two isolates of T. innocens were extracted with Sarkosyl and the Sarkosyl-insoluble, OM protein-enriched preparations resolved by SDSPAGE (Fig. 3 ). The major OM proteins present in T. hyodysenteriae type and field strains corresponded to the major abundant proteins migrating in the M r range 30 kDa to 40 kDa in the cellular protein preparations (refer to Fig. 1 ). The OM protein profiles revealed all isolates expressed common 36 kDa, 34 kDa, 33 kDa, 31.5 kDa and 30 kDa OM proteins when grown at 37°C (Fig. 3, lanes 1 to 6 ) or 42 ° C (data not shown) but of the 18 clinical isolates tested 11 expressed a major 37 kDa OMP and 7 expressed a major 37.5 kDa OMP (see Fig. 3, lanes 1 and 2, respectively). These isolates have been designated groups A and B, respectively based upon the variable migration on kDa
Fig. 3. SDS-PAGE profiles ofSarkosyl-insoluble OM preparations. Ten pg of OM proteins from T. hyodysenteriae isolates. Lane 1, 70A; lane 2, ATCC 31287; lane 3, ATCC 27164; lane 4, Q5363; lane 5, 508; lane 6, 5380; lane 7, T. innocens 9690; lane 8, T. innocens 8841; lane 9, T. innocens 8441; lane 10, T. innocens 9509; lane 11, T. innocens 9510; lane 12, 5380 whole cell lysate. Asterisks indicate the position of the 37 kDa and 37.5 kDa polypeptides.
37
IMMUNOREACTIVEPROTEINSOF TREPONEMAHYODYSENTERIAE
SDS-PAGE of the 37 kDa protein. The OM protein-enriched composition of T. hyodysenteriae was readily distinguished from that of T. innocens (Fig. 3, lanes 7 to 11 ) and the OMP-enriched preparations from E. coli, S. typhimurium, Y. enterocolitica, C. jejuni and C. fetus subsp, fetus.
Detection of immunoreactive OM proteins ofT. hyodysenteriae When Sarkosyl-insoluble OM preparations from seven T. hyodysenteriae (four group A and two group B) isolates and two T. innocens isolates were separated by SDS-PAGE and immunoblotted and probed with porcine antiT. hyodysenteriae strain 5380 hyperimmune serum, the pattern of reactivity was similar in almost all isolates except for a small degree of strain-variation in the proteins in the 38 kDa to 40 kDa molecular mass range (Fig. 4). Included for comparative purposes was the pattern of reactivity with isolate 5380 whole-cell lysate (Fig. 4, lanes 1 and 5 ). The OM proteins from groups A and B and the T. innocens isolates were reactive with antibodies present in A
B
kDa 94
.
43
--
34
--
20.1
~
J
.......
-,m
..........
t0
1t
__
1
2
3
4
5
6
7
8
9
12
13
Fig. 4. Western blot profiles of OM proteins of T. hyodysenteriae and T. innocens isolates using porcine anti-T, hyodysenteriae serum. OM proteins were prepared by Sarkosyl solubilisation. Panel A, immune blot with pre-immune control sera; panel B, immune blot with hyper-immune sera. Serum was diluted 1 : 100. Lanes 1 and 5, T. hyodysenteriae 5380 cell extract; lanes 2 and 6, 5380 OMP; lanes 3 and 7, ATCC 31287 OMP; lanes 4 and 8, 70A OMP; lane 9, Q5363 OMP; lane 10, 508 OMP; lane 11, ATCC 27164 OMP; lane 12, T. innocens 8841 OMP; lane 13, T. in nocens 9510 OMP. Antibody binding was detected by incubation with [35S] Protein A and subsequent autoradiography.
38
S.C. SMITH ET AL.
the hyperimmune sera but not with prechallenge control serum (Fig. 4, lanes 1 to 4). In all T. hyodysenteriae isolates, strongly immunoreactive common OM proteins were observed at 34 kDa and 30 kDa. The 34 kDa and 30 kDa bands were also reactive in the T. innocens isolates (Fig. 4, lanes 12 and 13 ). The major 40 kDa in the T. innocens isolate 8841 (Fig. 4, lane 12), the 38 kDa and 38.5 kDa protein bands in the T. innocens isolate 9510 (Fig. 4, lane 13) and the specific 39 kDa band conserved in T. hyodysenteriae groups A and B isolates (Fig. 4, lanes 6 to 11 ) were also immunoreactive. A similar antibody response was observed when OM proteins were probed with rabbit anti- T. hyodysenteriae hyperimmune serum. Immunoblotting of the OM proteins from other Gram-negative organisms with the same porcine anti-T, hyodysenteriae hyperimmune serum revealed no detectable antibody crossreactivity.
Surface location of protease-sensitive OM proteins To further characterise the T. hyodysenteriae antigens important in the immunological response, the OM proteins recognised in the Western blots were A
B
kDa 94
-
67
_
3
.-
2:0.1
--
Fig. 5. Effect of protease digestion on T. hyodysenteriae isolate 5380 whole cells. Intact cells were incubated with 50 #g/ml of trypsin (lane A) or with PBS buffer (lane B). After 30 min, reactions were stopped by the addition of PMSF and cells solubilised in SDS sample buffer prior to SDS-PAGE. Arrows indicate the position of the Coomassie blue-stained proteins sensitive to proteolysis.
IMMUNOREACTIVE PROTEINS OF TREPONEMA HYODYSENTERIAE
39
correlated with proteins located on the surface of T. hyodysenteriae. Intact T. hyodysenteriae strain 5380 whole cells were treated with trypsin and the proteolysis stopped with PMSF. Both protease-treated and control T. hyodysenteriae 5380 cells were solubilised in SDS sample buffer and subjected to SDSPAGE (Fig. 5 ). Trypsin proteolysis caused selective loss of the protein bands at Mr values of 39 kDa, 34 kDa (doublet), and 30 kDa indicating that these proteins extend out through the OM to the treponemal cell surface. The same pattern of proteolysis was observed when proteinase K treatment of the T. hyodysen teriae 5380 cells was used rather than trypsin (data not shown ). The 37.5 kDa protein band was shown to be a peptidoglycan-associated protein based upon the resistance of this outer membrane protein to solubilisation by 2% (w/v) SDS at 37 °C (Nakae, 1976 ). Solubilisation at 37 °C resulted in the absence of the 37 kDa protein from the outer membrane profile from T. hyodysenteriae strain 5380 and the appearance of apparent higher molecular weight proteins which may represent oligomeric forms of this protein due to the anomalous migration of such complexes on SDS-PAGE (data not shown ). DISCUSSION In the present study we have examined by SDS-PAGE the whole cell proteins of 18 strains of T. hyodysenteriae isolated from pigs from several locations in Australia and compared those with isolates from the United States and Great Britain. The high degree of uniformity in the whole cell polypeptide profiles of the T. hyodysenteriae isolates is in agreement with observations made by another research group (Chatfield et al., 1988) and demonstrates the usefulness of SDS-PAGE for differentiation of T. hyodysenteriae from the commensal spirochaete T. innocens. OM proteins from the 18 field T. hyodysenteriae isolates demonstrated minor differences in the OM proteins especially the strain variable 37 kDa OM protein suggesting the subdivision of the T. hyodysenteriae isolates into two groups, A and B. E. coli OM protein profiles have been used as epidemiologic markers in the study of disease (Achtman et al., 1983; Barenkamp et al., 1981 ) and OM protein patterns can be used to characterise T. hyodysenteriae strains. However, there was no clear correlation between groups A and B OM patterns and LPS serotype, indicating that the two features do not appear closely related. These resuits suggest further evaluation of the OM proteins is required to determine the role of these OM proteins and how they interact with LPS in pathogenicity. SDS-PAGE and immunoblotting demonstrated a heterogeneous response to LPS but a conserved protein response, with three strongly immunoreactive proteins of 39 kDa, 34 kDa and 30 kDa being recognised by anti-T, hyodysenteriae sera, irrespective of serotype. Several of these OM proteins were crossreactive to antisera raised against T. hyodysenteriae and T. innocens (data not shown) suggesting that the 39 kDa OM protein appears specific to T. hyodys-
40
S.C. SMITH ET AL.
enteriae. Joens and Marquez (1986) and Chatfield et al. ( 1988 ) have shown other major polypeptide antigens apparently specific to T. hyodysenteriae Chatfield et al. (1988) has shown that the predominant polypeptides between 30 kDa and 36 kDa are associated with the cell envelope fraction. In this study we have shown, by using the sodium N-lauryl sarcosinate (Sarkosyl) method of Achtman et al. ( 1983 ) which selectively solubilises the inner membrane and enriches for the OM Proteins that these same cell-envelopeassociated polypeptides reside in the OM of T. hyodysenteriae and are situated on the cell surface, and sensitive to trypsin proteolysis in situ, indicating that they must have surface-exposed epitopes. These major immunoreactive polypeptides may be targets for a protective porcine immune response against SD. The strain variable 37 kDa OM protein in both group A and B T. hyodysenteriae isolates did not appear strongly immunoreactive due to the fact that it appears to be a peptidoglycan-associated protein (data not shown) and is not degraded by trypsin treatment. This protein may represent an integral membrane protein due to its selective release from the cell membrane by Triton X-114 (M. Alderton, personal communication), a detergent used to extract integral membrane proteins (Bordier, 1981 ). By analogy with other enteric porins the 37 kDa OM protein may serve an important role in the synthesis of capsular polysaccharides (Paakkanen et al., 1979; Achtman et al., 1983 ) but not in the sero-specificity of the T. hyodysenteriae LPS. When isolates from varied geographical locations in Australia were typed using hyperimmune rabbit serum against four known T. hyodysenteriae serotypes (I to IV) and two Australian isolates, at least one additional LPS serotype, specific to Australia, was observed, confirming and extending the evidence of LPS heterogeneity among the T. hyodysenteriae isolates examined (Baum and Joens, 1979; Mapother and Joens, 1985; Chatfield et al., 1988). This serotype did not react with hyperimmune rabbit serum raised against serotypes 1, 2 or 3 (Baum and Joens, 1979) and is different to serotypes 5, 6 or 7 since it did not cross-react with serotypes 1 and 2 antisera, a feature of these serotypes (Mapother and Joens, 1985 ). Hampson et al. (1989) has proposed a revised typing scheme for T. hyodysenteriae isolates placing isolates which share common LPS antigens into serogroups A to E with serotypes within each serogroup. Serogroups A to D correspond to the serotypes 1 to 4 in the typing scheme of Baum and Joens ( 1979 ) while serogroup E is a novel group. Hampson et al. ( 1989 ) has placed Western Australia isolates of T. hyodysenteriae into the serogroups A, B and E. Additional studies will be carried out to determine if the additional LPS serotype found herein may represent another serogroup. In this paper we have demonstrated that the major polypeptides of T. hyodysenteriae are strong immunogens and are conserved with the various serotypes whereas there is considerable diversity in the antigenicity of LPS between those same serotypes.
IMMUNOREACTIVE PROTEINS OF TREPONEMA HYOD YSENTER1AE
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