Res. Microbiol. 1992, 143, 75-.79
~) INSTITUTPASTEUR/ELsEVn.R Paris 1992
Mycobacterium paratuberculosis binds fibronectin P. Valentin-Weigand (*) and K.M. Moriarty
Department of Veterinary Pathology and Public Health, Massey University, Palmerston North (New Zealand) SUMMAnY Fibroneetin, an adhesive glycoprotein which is present in plasma and on many host cell surfaces of many host organisms, binds to certain bacterial pathogens. This study demonstrates the ability of Mycobacterium paratuberculosis (M.ptb) to interact with ~251-1abelledfibronectin purified from bovine and ovine plasma. T w o M,ptb strains were tested : a clinical isolate and a commercially available vaccine strain. Both strains showed significant fibronectin-binding activities of 22 and 4 1 % , respectively, whereas nonpathogenic M.phlei had almost no affinity for fibronectin. Binding activities were similar for ovine and bovine fibronectin. We found that fibronectin binding by M.ptb was (1) time-dependent, reaching saturation within 90 min, (21 specific, since it was inhibited by an excess of unlabelled fibronectin but not by albumin, (3) saturable, with an apparent dissociation constant of 1.25 x 10 -9 M and a maxima! number of 1,600 binding sites per bacterium, and (4) sensitive to detergents, proteases and heat treatments, indicating the protein nature of the responsible binding component(s). Scatchard plot analysis gave a straight line suggesting the presence of a single type of fibronectin receptor on M.ptb.
Key-words: Fibronectin, Mycobacteriumparatubercu/osis, Mycob3cteriumph/ei, BCG, Johne's disease; Adherence, Phagocytosis, Host defences.
INTRODUCTION
Mycobacterium paratuberculosis (M.ptb) is the causative agent o f J o h n e ' s disease, a chronic eateritis in ruminants occurring worldwide. At present, there exists no specific therapy against J o h n e ' s disease, and effective control i~ still hir.dered by the lack of appropriate diagnn~tic methods and protective vaccines (Chiodini et al., 1984). Thus, there has been increasing interest in identifying factors which are involved in the interaction o f M.ptb with the host immune system in order to further understand the basic
mechanisms of mycobacterial pathogenicity (Thoen and Baum, 1988). Adherence and phagocytosis are m a j o r mechanisms in the process of bacterial infection. The multi functional adhesive glycoprotein fibronectin has been shown to play an important role in these mechanisms. It is now well established that se!uble and cell-bound fibronectin can mediate the adherence of a n u m b e r of pathogens to host cells by interacting with specific binding sites on surfaces o f both bacteria and host cells (Vercellotti et al., 1984; Simpson et al., 1987). Furthermore, fibronectin might be of
Submitted January 28, 1991, accepted July 5, 1991. (*) Correspondingauthor's presentaddress: AG Mikrobiologieder GBF, Bio;:entrumder TechnischenUniversidit,Konstantin-UhdeStr.5, Postfach 171. D-3300Braunschweig(Germany).
P. VALENTIN-WEIGAND AND K.M. M O R I A R T Y
importance in non-opsonic phagoeytosis and wound infection (Kluftinger et al., 1989; Chhatwal et al., 1990). Recently, Abou-Zeid et al. (1988a) reported on specific binding of M. tuberculosis and M bovis to immobilized fibronectin and discussed a potential role of this interaction in the pathogenicity of mycobacterioses. They observed that fibronectin was b o u n d by some of the secreted mycobacterial antigens, which have gained recent interest because of their immunological significance (Abou-Zeid et al., 1988b). These findings p r o m p t e d us to undertake the present study, in which we report, for the first time, on the binding of soluble fibronectin by M . p t b and describe some of the characteristics of this interaction.
MATERI~LS AND METHODS A clinical isolate of M.ptb (identified by acid-fast staining, slow mycobactin-dependent growth and morpholo~ y), a commercially available vaccine strain (M.ptb strain 319F, Neoparasec, Rh6ne M6rieux Animal Health, Upper Hut, New Zealand) and a M.phlei culture were used. Organisms were cultured in liqu!a medium (modified Reid's synthetic medium; Watsoa, 1935) supplemented (for M.ptb) with 2 mg/l mycobactin J (Allied Lab., Ames, IO, USA) and harvested by centrifugation (10,000 g, 20 min). As controls, one culture each of Staphylococcus aureus (positive for fibronectin binding) and Streptococcus agalactiae (negative for fibronectin binding) were grown on Todd-Hewitt broth (Gibco, Uxbridge, England) for 18 h at 37°C and harvested by centrifuga~ion. For binding assays, bacteria were washed in 0.15 M phosphate-buffered saline pH 7.4 (PBS) containing 0.1% Tween-20 (PBST) to eliminate nonspecific binding and adjusted in the same buffer to a concentration of approx. 108 cells/ml by optical density. Fibronectin was purified from plasma of sheep and cattle by affinity chromatography on gelatin and heparin agarose as described by Miekka et aL (1982). Pur.fied fibronectin was labelled with J251 by the chloramin T method (Hunter and Greenwood, 1962). The specific actwities of ovine and bovine fibronectin were 1.03 aud 0.82 mCi/mg, respectively. For binding experiments, 506 ~! of the bacterial suspen-
M.ptb = Mycobucteriumparatuberculosis. PBS = phosphate-buffeledsaline.
sions were incubated with I00 ng of labelled fibroneetin for 90 rain at room temperature (unless stated otherwise). Binding reactions were stopped by washing bacteria with ice-cold PBST (12,000 g, 5 min) and the radioactivity of the sedimented bacteria was determined in a "r-counter (LKB Wallace 1261, North Ryde, NSW, Australia). All tests were done in duplicate. Results were expressed as % binding obtained by relating the radioactivity of the pellet to the total radioactivity used. Characterization of the binding reaction involved experiments in which the following parameters were modified: (1) the nnmbers of bacteria (6.25-100 x l& per assay), (2) the times of incubation (10-120 min) and (3) the amounts of labelled fibronectin (2-250 ng per assay) to as5ess saturability of the binding. The data were plotted by the method of Scatchard et al. (1949) and the dissociation constant and number of fibronectin molecules bound per bacterium calculated. Specificity of the binding was evaluated by inhi!~ition experiments with unlabelled fibronectin and albumin at 100 g.g per assay. Furthermore, prior to binding bacteria were treated with 1070Triton-Xl00 (v/v), heat (90 min at 80°C) and 100 g.g/ml trypsin (Calbiochem, Alexandria, NSW, Australia) as previously described for streptococci (Valentin-Weigand et aL, 1988).
RESULTS AND DISCUSSION In the present study, we showed that M . p t b is able to interact with purified fibronectin from ovine a n d bovine plasma. Both the clinical isolate and the vaccine strain o f M . p t b used in this study showed strong binding activities towards fibronectin (table I). Binding was reproducible in a series of experiments and appeared tc, be significant when compared to the fibronecdnbinding activity of S.a~'reus (table 1), a species well known for its strong affinity for fibronectin (Kuusela, 1978). Optimal binding was observed when 0.5 × 10s M . p t b were incubated with 100 ng of labelled fibronectin for 90 min (fig 1A and B). The binding of fibronectin was almost completely (78 °70) inhibited by an excess (100 g.g) of unlabelled fibronectin but not by albumin, indicating that binding might occur through specific binding sites.
PBST = PBS containing0.1 07oTriton-XI00.
M. PARATUBERCULOSIS BINDS FIBRONECTIN interestingly, saprophytic M.phlei had almost no affinity for fibronectin, similar to the group B streptococcal culture used as negative control in our experiments (table I). This is in contrast
Table i. Binding of ~2Sl-labelled fibronectin from ovine and bovine plasma by M.ptb, ,~¢I.phlei, S. aureus and S. agalactiae (group B streptococci). Mean °70 binding of fibronectin from Sheep Cattle
Species
M, ptb c*) M.ptb 319F t''~ M.phlei S.aureus S. agalactiae
22 41
23 43
5
3
28
30
4
5
Results o b t a i n e d f r o m d u p lic a te s . ( * ) C l i a i c a l (**) C o m m e r c i a l l y available vaccine strain.
3o
isolate.
A
0
L~I 40
60
80 100 120
NUMBER OF BACTERIA (x 106l
51 . . . . . . . . 0 20 40 6l] 80 leo 1~0 140 INCUitATION TIME (min)
to the findings o f Ratliff et al. (1988) who observed the binding of M, phlei to immobilized fibronectin. However, these discrepancies could be due to the different bindings o f soluble and immobilized fibronectin, as has been observed with other bacteria (Kuusela et aL, 1985). We found that fibronectin binding by h4.ptb was saturable with an apparent dissociation constant o f 1.25 × !0 -9 M and with a number o f approx. 1,600 binding sites per bacterium (fig. 1 C). Scatchard analysis o f tt~e dose response experiments gave a single line ir,dicating the presence of a single class of receptors on M.ptb (fig. IC, inset). Fibronectin binding was relatively sensitive (70 °7o reduction) to treatment with detergent Triton-X and moderately affected by treatment with heat and trypsin (fig. 2). These characteristics show some similarities with the fibrov_ectin binding o f BCG (bacillus Calmette-Gu&in) and b. aureus (Ratiiff et al., 1988; Aslanzadeh et al., 1989; Ryden et aL, 1983). In contrast to BCG, the M.ptb strains that we studied appeared to have a significantly lower number of fibronectin-binding site-s (1,600 vs. 8,000 per bacterium) (Aslanzadeh et al., 1989). However, more detailed studies are needed to determine the exact nature of the fibronectin-binding component(s) of M.ptb.
30! 20
0
20
40 69 80 I O0 FIBRO/|ECTIN ADDED Ing)
[]
120
Fig. 1. Characteristics of the interaction of M.ptb (clinical isolate) with soluble ~-'51-1abelledfibronectin. Bindingexperimentswereperformed with variousconcentrationsof bacteria (A) and increasingincubation times (B); in figure C the saturabitity of fibronectin binding is shown as the result of a dose-responseexperimentand Scatchard analysis (inset) of the binding reaction. Results express mean bindings obtained from duplicate samples.
c Fig. 2. Fibronectinbindingby ~l.ptb (clinical isolate)after treatment ~,ith buffer (control, solia bar), 1% v/v TritonXI00 (open bar). heat (90 rain at 80°C, cross-hatchedbar) or 100 ~.g/ml trypsin (hatched bar). Results expressmean bindingsobtained from duplicate samples.
78
P. V A L E N T I N - W E I G A N D A N D K . M . M O R I A R T Y
Studies o f bacterial i n t e r a c t i o n s with fibronectin z,n d their p o t e n t i a l role in t h e i n f e c t i o n process h a v e been f r e q u e n t l y p u b l i s h e d o v e r t h e last decade. H o w e v e r , f i b r o n e c t i n b i n d i n g b y m y c o b a c t e r i a is a relatively n e w a n d very interesting o b s e r v a t i o n since f i b r o n e c t i n r e c e p t o r s have been f o u n d o n m a c r o p h a g e s a n d l y m p h o c y tes ( M o l n a r et al., 1987 ; Cardarelli a n d P i e r s c h b a c h e r , 1987), b o t h o f w h i c h play a central role in t h e h o s t i m m u n e r e s p o n s e to m y c o b a c t e r i a l i n f e c t i o n . F u r t h e r m o r e , s t u d i e s b y R a t l i f f et aL (1987) s u g g e s t e d t h a t t h e a n t i t u m o r a l activity m e d i a t e d by B C G t r e a t m e n t requires t h e a t t a c h m e n t o f t h e b a c t e r i a to f i b r o n e c t i n w i t h i n t h e bladder. Detailed c h a r a c t e r i z a t i o n o f t h e m y c o bacteria/fibronectin interaction could, thus c o n t r i b u t e to a better u n d e r s t a n d i n g o f t h e p a t h o g e n i c p r o c e s s e s involved in m y c o b a c t e r i o ses. In o u r l a b o r a t o r y , s t u d i e s a r e c u r r e n t l y in p r o g r e s s to a n a l y s e in detail f i b r o n e c t i n b i n d i n g by M . p t b , to i d e n t i f y t h e b i n d i n g sites a n d to e v a l u a t e t h e p o s s i b l e role o f f i b r o n e c t i n in M . p t b / h o s t cell i n t e r a c t i o n s .
Acknowledgements P.V. was supported by a fellowship from Deutsche Forschungs-gemeinschaft, Bonn, Germany.
Fixation de Mycobacterium paratuberculosis sur la fibronectine Une liaison sp6cifique est d6montr6e entre Mycobacterium paratuberculosis et la fibronectine plasmatique ovine et bovine. La fixation de la fibronectine radiomarqu6e est fonction du temps, elle est observ6e ~t saturation et elle est sp6cifique (non observ6e avec de l'albumine). La sensibilit6 de la fixation au Triton-X100, ~ la chaleur et ~ l'action de la trypsine sugg6re que les 616ments de cette liaison avec les cel!ules de M. paratuberculosis sont de nature prot6inique, et les caract6ristiques de la fixation paraissent analogues h ce qui s'observe avec le BCG et Staphylococcus aureus. Mots-cl~s : Fibronectine, Mycobacterium paratuberculosis, Mycobacterium phlei, BCG, Maladie de Johne; Adh6rence, Phagocytose, D6fenses de l'h6te.
References Abou-Zeid C., Ratliff T.L., Wiker, H.G., Harboe, M., Bennedsen, J. & Rook, G.A.W. (1988a), Characterization of fibronectin-binding antigens released by Mycobacterium tuberculosis and Mycobacterium bovis BCG. Infect. Immun., 56, 3046-3051. Abou-Zeid, C., Smith, 1., Grange, J.M., Ratliff, T.L., Steele, J. & Rook, G.A.W. (1988b), The secreted antigens of Myeobacterium tuberculosis and their relationship to those recognized by the available antibodies. J. gen. Microbiol., 134, 531-538. Aslanzadeh, J., Brown, E.J., Quillin, S.P., Ritchey, J.K. & Ratliff, T.L. (1989), Characterization of soluble fibronectin binding to Bacille Calmette-Gu&in. J. gen. MicrobioL, 135. 2735-2741. Cardarelli, P.M. & Pierschbacher, M.D. 0987), Identification of fibronectin receptors of T lymphocytes. J. Cell BioL, 105, 499-506. Chhatwal, G.S., Valentin-Weigand, P. & Timmis, K.N. (1990), Bacterial infection of wounds: fibronectinmediated adherence of group A and C streptococci to fibrin thrombi in vitro. Infect. lmmun., 58, 3015-3019. Chiodini, R.J., van Kruiningen, H.J. & Merkal, R.S. (1984), Ruminant paratuberculosis (Johne's disease). The current status and future prospects. Cornell Vet., 74, 218-262. Huater, W.H. & Greenwood, F. (1962), Preparation of iodine-I 3 l-labelled human growth hormone of high specific activity. Nature (Lond.), 194, 495-496. Kluftinger, J.L., Kelly, N.M., Jost, B.H. & Hancock, E.W. (1989), Fibronectin as an enhancer of nonopsonic phagocytosis of Pseudomonas aeruginosa by macrophages. Infect. Immun., 57, 2782-2785. Kuusela, P. (1978), Fibronectin binds to Staphylococcus aureus. Nature (Lond.), 276, 718-720. Miekka, S.J., Ingham, K.C. & Menache, D. (1982), Rapid methods for isolation of human plasma fibronectin. Thromb. Res., 27, 1-14. Molnar, J., Hoekstra, S., Ku, C.S.-L. & Van Alten, P. (1987), Evidence for the recycling ~t~ture of the fibronectin receptor of macroph=ges. Z Cell. PhysioL, !3!, 374-383. Ratliff, T.L., Palmer, J.O., McGarr, J.A. & Brown, E.J. (1987), Intravesical Bacille Calmette.Gu6rin (BCG) therapy for murine bladder tumors : initiation of the response by fibronectin-mediated attachment of BCG. Cancer Res., 47, 1762-1766. Ratliff, T.L., McGarr, J.A., Abou-Zeid, C., Rook, G.A.W., Stanford, J.L., Aslanzadeh, J. & Brown, E.J. (1988), Attachment of mycobacteria to fibronectin-coated surfaces. J. gen. Microbiol., 134, 1307-1313. Ryd6n, C., Rubiu, K., Speziale, P., H66k, M., Lindberg, M. & Wadstr6m, T. (1983), Fibronectin receptors from Staphylococcus aureus. J. biol. Chem., 258, 3396-3401. Scatchard, G. (1949), The attractions of proteins for small molecules and ions. Ann. N.Y. Acad. ScL, 51, 660-672. Simpson, W.A., Courtney, H.S. & Ofek, I. (1987), Interactions of fibronectin with streptococci : the role of fibronectin as a receptor for Streptococcuspyogenes. Rev. infect. Dis., 9, $351-$359.
M. PARATUBERCULOS1S BINDS F I B R O N E C T I N Thoen, C.O. & Baum, K. (1988). Current knowledge on paratuberculosis. J. Amer. vet. Ass., 190, 676-680. Valentin-Weigand, P., Gruhlich-Henn, J., Chhatwal, G.S., Miiller-Berghaus, G., Blobel, H. & Preissner, K.T. (1988), Mediation of adherence of streptococci to human endothelial cells by complement S protein (vitronectin). lt~fect, lmmun., 56, 2851-2855.
Vercellotti, G.M., Lus~enhop, D., Petcrson, P.K., Furcht, L.T., McCarthy, J.B., Jacob, H.S. & Moldow, C.F. (1984), Bacterial adherence to fibronectin and endothelial cells: a possible mechanism for bacterial tissue tropism. J. Lab. Clin. Med., 103, 34-43. Watson, E.A. (1935), Tuberculin, Johnin and mallein derived from ii~,it-~ro~ein ~nedia. Canad. J. pubL Hlth, 26, 268-275.
~) INSTITUTPASTEUR/ELsEVn.R Paris 1992
Mycobacterium paratuberculosis binds fibronectin P. Valentin-Weigand (*) and K.M. Moriarty
Department of Veterinary Pathology and Public Health, Massey University, Palmerston North (New Zealand) SUMMAnY Fibroneetin, an adhesive glycoprotein which is present in plasma and on many host cell surfaces of many host organisms, binds to certain bacterial pathogens. This study demonstrates the ability of Mycobacterium paratuberculosis (M.ptb) to interact with ~251-1abelledfibronectin purified from bovine and ovine plasma. T w o M,ptb strains were tested : a clinical isolate and a commercially available vaccine strain. Both strains showed significant fibronectin-binding activities of 22 and 4 1 % , respectively, whereas nonpathogenic M.phlei had almost no affinity for fibronectin. Binding activities were similar for ovine and bovine fibronectin. We found that fibronectin binding by M.ptb was (1) time-dependent, reaching saturation within 90 min, (21 specific, since it was inhibited by an excess of unlabelled fibronectin but not by albumin, (3) saturable, with an apparent dissociation constant of 1.25 x 10 -9 M and a maxima! number of 1,600 binding sites per bacterium, and (4) sensitive to detergents, proteases and heat treatments, indicating the protein nature of the responsible binding component(s). Scatchard plot analysis gave a straight line suggesting the presence of a single type of fibronectin receptor on M.ptb.
Key-words: Fibronectin, Mycobacteriumparatubercu/osis, Mycob3cteriumph/ei, BCG, Johne's disease; Adherence, Phagocytosis, Host defences.
INTRODUCTION
Mycobacterium paratuberculosis (M.ptb) is the causative agent o f J o h n e ' s disease, a chronic eateritis in ruminants occurring worldwide. At present, there exists no specific therapy against J o h n e ' s disease, and effective control i~ still hir.dered by the lack of appropriate diagnn~tic methods and protective vaccines (Chiodini et al., 1984). Thus, there has been increasing interest in identifying factors which are involved in the interaction o f M.ptb with the host immune system in order to further understand the basic
mechanisms of mycobacterial pathogenicity (Thoen and Baum, 1988). Adherence and phagocytosis are m a j o r mechanisms in the process of bacterial infection. The multi functional adhesive glycoprotein fibronectin has been shown to play an important role in these mechanisms. It is now well established that se!uble and cell-bound fibronectin can mediate the adherence of a n u m b e r of pathogens to host cells by interacting with specific binding sites on surfaces o f both bacteria and host cells (Vercellotti et al., 1984; Simpson et al., 1987). Furthermore, fibronectin might be of
Submitted January 28, 1991, accepted July 5, 1991. (*) Correspondingauthor's presentaddress: AG Mikrobiologieder GBF, Bio;:entrumder TechnischenUniversidit,Konstantin-UhdeStr.5, Postfach 171. D-3300Braunschweig(Germany).
P. VALENTIN-WEIGAND AND K.M. M O R I A R T Y
importance in non-opsonic phagoeytosis and wound infection (Kluftinger et al., 1989; Chhatwal et al., 1990). Recently, Abou-Zeid et al. (1988a) reported on specific binding of M. tuberculosis and M bovis to immobilized fibronectin and discussed a potential role of this interaction in the pathogenicity of mycobacterioses. They observed that fibronectin was b o u n d by some of the secreted mycobacterial antigens, which have gained recent interest because of their immunological significance (Abou-Zeid et al., 1988b). These findings p r o m p t e d us to undertake the present study, in which we report, for the first time, on the binding of soluble fibronectin by M . p t b and describe some of the characteristics of this interaction.
MATERI~LS AND METHODS A clinical isolate of M.ptb (identified by acid-fast staining, slow mycobactin-dependent growth and morpholo~ y), a commercially available vaccine strain (M.ptb strain 319F, Neoparasec, Rh6ne M6rieux Animal Health, Upper Hut, New Zealand) and a M.phlei culture were used. Organisms were cultured in liqu!a medium (modified Reid's synthetic medium; Watsoa, 1935) supplemented (for M.ptb) with 2 mg/l mycobactin J (Allied Lab., Ames, IO, USA) and harvested by centrifugation (10,000 g, 20 min). As controls, one culture each of Staphylococcus aureus (positive for fibronectin binding) and Streptococcus agalactiae (negative for fibronectin binding) were grown on Todd-Hewitt broth (Gibco, Uxbridge, England) for 18 h at 37°C and harvested by centrifuga~ion. For binding assays, bacteria were washed in 0.15 M phosphate-buffered saline pH 7.4 (PBS) containing 0.1% Tween-20 (PBST) to eliminate nonspecific binding and adjusted in the same buffer to a concentration of approx. 108 cells/ml by optical density. Fibronectin was purified from plasma of sheep and cattle by affinity chromatography on gelatin and heparin agarose as described by Miekka et aL (1982). Pur.fied fibronectin was labelled with J251 by the chloramin T method (Hunter and Greenwood, 1962). The specific actwities of ovine and bovine fibronectin were 1.03 aud 0.82 mCi/mg, respectively. For binding experiments, 506 ~! of the bacterial suspen-
M.ptb = Mycobucteriumparatuberculosis. PBS = phosphate-buffeledsaline.
sions were incubated with I00 ng of labelled fibroneetin for 90 rain at room temperature (unless stated otherwise). Binding reactions were stopped by washing bacteria with ice-cold PBST (12,000 g, 5 min) and the radioactivity of the sedimented bacteria was determined in a "r-counter (LKB Wallace 1261, North Ryde, NSW, Australia). All tests were done in duplicate. Results were expressed as % binding obtained by relating the radioactivity of the pellet to the total radioactivity used. Characterization of the binding reaction involved experiments in which the following parameters were modified: (1) the nnmbers of bacteria (6.25-100 x l& per assay), (2) the times of incubation (10-120 min) and (3) the amounts of labelled fibronectin (2-250 ng per assay) to as5ess saturability of the binding. The data were plotted by the method of Scatchard et al. (1949) and the dissociation constant and number of fibronectin molecules bound per bacterium calculated. Specificity of the binding was evaluated by inhi!~ition experiments with unlabelled fibronectin and albumin at 100 g.g per assay. Furthermore, prior to binding bacteria were treated with 1070Triton-Xl00 (v/v), heat (90 min at 80°C) and 100 g.g/ml trypsin (Calbiochem, Alexandria, NSW, Australia) as previously described for streptococci (Valentin-Weigand et aL, 1988).
RESULTS AND DISCUSSION In the present study, we showed that M . p t b is able to interact with purified fibronectin from ovine a n d bovine plasma. Both the clinical isolate and the vaccine strain o f M . p t b used in this study showed strong binding activities towards fibronectin (table I). Binding was reproducible in a series of experiments and appeared tc, be significant when compared to the fibronecdnbinding activity of S.a~'reus (table 1), a species well known for its strong affinity for fibronectin (Kuusela, 1978). Optimal binding was observed when 0.5 × 10s M . p t b were incubated with 100 ng of labelled fibronectin for 90 min (fig 1A and B). The binding of fibronectin was almost completely (78 °70) inhibited by an excess (100 g.g) of unlabelled fibronectin but not by albumin, indicating that binding might occur through specific binding sites.
PBST = PBS containing0.1 07oTriton-XI00.
M. PARATUBERCULOSIS BINDS FIBRONECTIN interestingly, saprophytic M.phlei had almost no affinity for fibronectin, similar to the group B streptococcal culture used as negative control in our experiments (table I). This is in contrast
Table i. Binding of ~2Sl-labelled fibronectin from ovine and bovine plasma by M.ptb, ,~¢I.phlei, S. aureus and S. agalactiae (group B streptococci). Mean °70 binding of fibronectin from Sheep Cattle
Species
M, ptb c*) M.ptb 319F t''~ M.phlei S.aureus S. agalactiae
22 41
23 43
5
3
28
30
4
5
Results o b t a i n e d f r o m d u p lic a te s . ( * ) C l i a i c a l (**) C o m m e r c i a l l y available vaccine strain.
3o
isolate.
A
0
L~I 40
60
80 100 120
NUMBER OF BACTERIA (x 106l
51 . . . . . . . . 0 20 40 6l] 80 leo 1~0 140 INCUitATION TIME (min)
to the findings o f Ratliff et al. (1988) who observed the binding of M, phlei to immobilized fibronectin. However, these discrepancies could be due to the different bindings o f soluble and immobilized fibronectin, as has been observed with other bacteria (Kuusela et aL, 1985). We found that fibronectin binding by h4.ptb was saturable with an apparent dissociation constant o f 1.25 × !0 -9 M and with a number o f approx. 1,600 binding sites per bacterium (fig. 1 C). Scatchard analysis o f tt~e dose response experiments gave a single line ir,dicating the presence of a single class of receptors on M.ptb (fig. IC, inset). Fibronectin binding was relatively sensitive (70 °7o reduction) to treatment with detergent Triton-X and moderately affected by treatment with heat and trypsin (fig. 2). These characteristics show some similarities with the fibrov_ectin binding o f BCG (bacillus Calmette-Gu&in) and b. aureus (Ratiiff et al., 1988; Aslanzadeh et al., 1989; Ryden et aL, 1983). In contrast to BCG, the M.ptb strains that we studied appeared to have a significantly lower number of fibronectin-binding site-s (1,600 vs. 8,000 per bacterium) (Aslanzadeh et al., 1989). However, more detailed studies are needed to determine the exact nature of the fibronectin-binding component(s) of M.ptb.
30! 20
0
20
40 69 80 I O0 FIBRO/|ECTIN ADDED Ing)
[]
120
Fig. 1. Characteristics of the interaction of M.ptb (clinical isolate) with soluble ~-'51-1abelledfibronectin. Bindingexperimentswereperformed with variousconcentrationsof bacteria (A) and increasingincubation times (B); in figure C the saturabitity of fibronectin binding is shown as the result of a dose-responseexperimentand Scatchard analysis (inset) of the binding reaction. Results express mean bindings obtained from duplicate samples.
c Fig. 2. Fibronectinbindingby ~l.ptb (clinical isolate)after treatment ~,ith buffer (control, solia bar), 1% v/v TritonXI00 (open bar). heat (90 rain at 80°C, cross-hatchedbar) or 100 ~.g/ml trypsin (hatched bar). Results expressmean bindingsobtained from duplicate samples.
78
P. V A L E N T I N - W E I G A N D A N D K . M . M O R I A R T Y
Studies o f bacterial i n t e r a c t i o n s with fibronectin z,n d their p o t e n t i a l role in t h e i n f e c t i o n process h a v e been f r e q u e n t l y p u b l i s h e d o v e r t h e last decade. H o w e v e r , f i b r o n e c t i n b i n d i n g b y m y c o b a c t e r i a is a relatively n e w a n d very interesting o b s e r v a t i o n since f i b r o n e c t i n r e c e p t o r s have been f o u n d o n m a c r o p h a g e s a n d l y m p h o c y tes ( M o l n a r et al., 1987 ; Cardarelli a n d P i e r s c h b a c h e r , 1987), b o t h o f w h i c h play a central role in t h e h o s t i m m u n e r e s p o n s e to m y c o b a c t e r i a l i n f e c t i o n . F u r t h e r m o r e , s t u d i e s b y R a t l i f f et aL (1987) s u g g e s t e d t h a t t h e a n t i t u m o r a l activity m e d i a t e d by B C G t r e a t m e n t requires t h e a t t a c h m e n t o f t h e b a c t e r i a to f i b r o n e c t i n w i t h i n t h e bladder. Detailed c h a r a c t e r i z a t i o n o f t h e m y c o bacteria/fibronectin interaction could, thus c o n t r i b u t e to a better u n d e r s t a n d i n g o f t h e p a t h o g e n i c p r o c e s s e s involved in m y c o b a c t e r i o ses. In o u r l a b o r a t o r y , s t u d i e s a r e c u r r e n t l y in p r o g r e s s to a n a l y s e in detail f i b r o n e c t i n b i n d i n g by M . p t b , to i d e n t i f y t h e b i n d i n g sites a n d to e v a l u a t e t h e p o s s i b l e role o f f i b r o n e c t i n in M . p t b / h o s t cell i n t e r a c t i o n s .
Acknowledgements P.V. was supported by a fellowship from Deutsche Forschungs-gemeinschaft, Bonn, Germany.
Fixation de Mycobacterium paratuberculosis sur la fibronectine Une liaison sp6cifique est d6montr6e entre Mycobacterium paratuberculosis et la fibronectine plasmatique ovine et bovine. La fixation de la fibronectine radiomarqu6e est fonction du temps, elle est observ6e ~t saturation et elle est sp6cifique (non observ6e avec de l'albumine). La sensibilit6 de la fixation au Triton-X100, ~ la chaleur et ~ l'action de la trypsine sugg6re que les 616ments de cette liaison avec les cel!ules de M. paratuberculosis sont de nature prot6inique, et les caract6ristiques de la fixation paraissent analogues h ce qui s'observe avec le BCG et Staphylococcus aureus. Mots-cl~s : Fibronectine, Mycobacterium paratuberculosis, Mycobacterium phlei, BCG, Maladie de Johne; Adh6rence, Phagocytose, D6fenses de l'h6te.
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