Zbl. Bakt. 277, 28-33 (1992) © Gustav Fischer Verlag, StuttgartlNew York
Polymorphism of Outer Surface Proteins of Borrelia burgdorferi as a Tool for Classification O. PETER and A. G. BRETZ Clinical Microbiology, Institut Central des Hopitaux Valaisans, 1950 Sion, Switzerland
With 2 Figures· Received September 12, 1991 . Accepted January 15, 1992
Summary
A total of 23 isolates of Borrelia burgdorferi were characterized by SDS-PAGE and immunoblot analysis. One isolate came from the CSF of a Lyme neuro-borreliosis patient in Valais (Switzerland) and 22 were tick isolates (2 from I. dammini of Shelter Island, USA and 20 from I. ricinus of Valais, Switzerland). Based on the electrophoretic mobility of outer surface proteins (OspA and OspB), four groups of B. burgdorferi could be defined. Group I isolates possess an OspA of 31 KD and an OspB of 34 KD. The group II isolate showed an OspA of 32 KD and OspB of 35 KD. Group III isolates have a 33 KD OspA and group IV a 33.5 KD OspA. This classification was confirmed by the reactiviry of a monoclonal antibody (D6) to a 12 KD antigen that was recognized in group III only. A Lyme patient's serum showed a 2-band pattern (10 and 13 KD) for group I and a one-band pattern (12 KD) for the other 3 groups. Therefore OspA, OspB and other proteins of low molecular weight (10, 12, and 13 KD) seem to be important keys for the classification of B. burgdorferi isolates. This typing system correlates with genetic analysis.
Zusammenfassung Insgesamt 23 Isolate von B. burgdorferi wurden mittels SDS-PAGE und Immunoblot charakterisiert. Ein Isolat stammte aus dem Liquor eines Patienten mit Lyme-Borreliose des Nervensystems im Wallis (Schweiz) und 22 Isolate von Zecken (2 von I. dammini aus Shelter Island, USA und 20 von I. ricinus aus dem Wallis, Schweiz). Aufgrund der elektrophoretischen Mobilitar der aulSeren Oberflachenproteine (OspA und OspB) konnten vier Gruppen von B. burgdorferi definiert werden. Die Gruppe I wies ein OspA von 31 KD und ein OspB von 34 KD auf. Das Isolat der Gruppe II zeigte ein OspA von 32 KD und ein OspB von 35 KD, die Isolate der Gruppe III ein OspA von 33 KD und die der Gruppe IV ein OspA von 33.5 KD. Diese Klassifikation wurde durch die Reaktionsfahigkeit eines monoklonalen Antikorpers (D6) gegeniiber einem 12 KD-Antigen, das nur in der Gruppe III erkannt wurde, bestatigr, Das Serum eines Patienten mit Lyme-Borreliose zeigte ein Muster aus 2 Banden (10 und 13 KD) gegen Gruppe I und ein Muster mit einer Bande (12 KD) gegen die anderen drei Gruppen. Somit scheinen OspA, OspB und andere niedermolekulare Proteine (10,12 und 13 KD) wichtige Schlussel fur die Klassifikation von B. burgdorferi darzustellen. Dieses Typisierungsschema korreliert mit der genetischen Analyse.
Osp as a Tool for the Classification of B. burgdorferi
29
Introduction Since its discovery (10) Borrelia burgdorferi has been isolated from ticks, various species of mammals, a bird and humans o f the northern hemisphere. Some species of ticks of the genus Ixodes were found to be the main vect ors of this bacterium which causes a spiro chetosis first ca lled Lyme art h ritis, then Lyme disease, and now, Lyme borreliosis. In about 60 % o f human cases the disease is characterized by erythema (chronicum) migrans, a distinctive skin lesion that appears a few da ys to a few weeks after tick bite. This early ph ase of the disea se ma y be succeeded by ch ro nic systemic illnes s, invo lving the nervou s system, th e heart, the jo int s, the mu scles and the skin (15 ). In Europe, late neurological manifestati on s and skin lesions are more frequent than art hritis whereas in th e United States the rever se is true. In Europe, B. burgdorferi is maintained and tr an smitted by the sheep tick, 1. ricinus, of wh ich up to 55 % have been found infected in Switzerland (2, 11 ). Am ong European isolates o f B. burgdorferi, there appears to be a high degr ee of antigenic variability whereas American isolates are more homogeneous (6, 17). A system for preliminary typing of B. burgdorjeri, using monoclonal antibodies, has been proposed by Barbour (7). More extensive analyses of European and American iso lates were performed by Barbour and Schrumpf (8) and by Wilske et al. (19) . However , non e of the se classi fications fully agreed with classification ba sed on genetic analysis, such as rRNA restriction p attern (12) . Th erefore, we proposed a simplified typing system based up on th e electrophoretic mobility of O spA and O spB and upon the reactivity of a 12 KO ant igen with polyclonal and mon oclon al antibod ies.
Material and Methods
Strains and culture conditions. A total of 23 isolates of B. burgdorferi was used in this stud y (Table 1). Twenty origina ted from I. ricinus from Valais, Switzerland (11) and one from the cerebro spinal fluid of a Lyme borr eliosis pat ient from Valais. In addition, the reference strain of B. burgdorferi (B31) from I. dammini and one isolate from the same tick species from Shelter Island , New York , (provided by W. Burgdorfer) were included. All the isolate s were grow n in BSK II medium (5 ) and incubated at 34 °C. With th e exception of B31, all the isolates had been passaged less than 10 times. Preparation of the antigens. The borreli ae were collected dur ing their logarithmi c growth pha se, by centrifugation at 9000 g for 20 min. The y were washed twice in PBS-Mg-CIz5mM (pH 7.2) and centr ifuged at 10000 g for 5 min. Th e pellet was resuspended in distilled water and protein concentration was adju sted to 1 mg/ml, using the method of Biuret. The material was kept frozen until used. Monoclonal and polyclonal antibodies. Monoclonal antib odies were prepared by using the invitrotech kit (Bioinvent, Lund). Spleen cells from three BALB/c mice were excised and prepared with supplied medium according to the manu factur er's pro cedure. In vitro immuniz ation was mad e by adding 20 ~t1 of SOS lysate of B. burgdorferi stra in VS 102 (1 mg/ ml ). Hybridoma s were produ ced by fusion with PAl myeloma cells (kindly supplied by Or. D. Lauancby, CHUV, Lausanne, Switzerland). Th e mon oclonal antibody (0 6) coming from one of the hybridomas was used in this study. Polyclon al anti bodies came fro m a Lyme patient' s serum reactive with ant igens of 10 to 13 KD. Electrophoresis and immunoblot. The cell suspensions of B. burgdorferi (1 mg/ml) were dissolved in the sample buffer with 0.6 % SDS (final concentration) and 50 mM dithiothreitol as a reducing agent. The samples were boiled for 5 min before und ergoing elec-
30
O. Peter and A. G. Bretz
Table 1. Source of B. burgdorferi isolates Origin biologic
geographic
I. dammini
l.tlammini
USA, NY, Shelter Island CH, VS, Sion 1 CH, VS, Martigny CH, VS, Martign y CH, VS, Martigny CH, VS, Rarogne CH , VS, Rarogne USA, NY, Shelter Island
VS 461
I. ricinus
CH, VS, Vouvry
VS 100 VS 102 VS 185 VS 286 VS 290 VS 3 VS 307 VS 464 VS 468 VS 488 VS 492 VS 5 18 VS BP
I. ricinus I. ricinus I. ricinus 1. ricinus I. ricinus I. ricinus I. ricinus I. ricinus I. ricinus I. ricinus I. ricinus I. ricinus human CSF
CH, VS, Rarogne CH, VS, Rarogne CH, VS, Sierre CH, VS, Rarogne CH, VS, Rarogn e CH, VS, Sion CH, VS, Rarogne CH, VS, Vouvry CH, VS, Vouvry CH, VS, Monthey CH, VS, Monrh ey CH, VS, Raro gne CH, VS, Viege
VS 116
I. ricinus
CH, VS, Raro gne
Isolate VS 2 VS 130 VS 134 VS 215 VS 2 19 VS 293 VS 393 B 31 2
I
2
I. ricinus I. ricinus
I. ricinus I. ricinus I. ricinus 1. ricinus
CH (Switzerland), VS (Canton of Valais), locality. Source: Will y Burgdorier, Rocky Mountain Laboratory, H amilton , MT 59840 , USA.
troph oresis (constant current 45 rnA) on 12.5% polyacrylamide gel overlaid with a 4 % stacking gel, according to the method of Barbour (3). The standard molecular weights (MW) of BioRad (low range protein molecular weight standards) were used as reference for the calculation of relative molecular weights. The transfer to PVDF membrane (Immobilon, Millipore, Zurich, Switzerland ) was performed according to Towbin et al. (16). After transfer, the membrane was cur to separate the MW and the strip was stained wit h Coom assie blue. The membrane was then cut slightly above the 14.4 KD. The upper pa rt of the membrane was stained with Coo massie blue and the lower part was saturated with 3% gelatine in a Tris buffer, pH . 7.5 at 37 °C, for 1 h. It was washed three times for 5 min each in Tris-Tween 20 (0.05% ) buffer and incuba ted for 2 h at room temperature with a human serum diluted 1 : 200 or with one of our monoclonal antibody (D6) diluted (1 : 100 in the same buffer containing 1% gelatin. After washing, antibody-ant igen reactions were demon strated by a second goat anti-hum an IgG or goat anti-mouse IgM antib ody conjugated to alkaline phosphatase, followed by three washes and the addition of BCIPINBT substrate (5bromo-4-chloro -3-indoyl-p-toluidine phosphate / p-nitro blue tetrazoliumchloride).
Osp as a Tool for the Classification of B. burgdorferi
31
Results Based on the electrophoretic mob ility of Os pA, and OspB if present , four groups of borreliae could be defined. Gro up I was represented by the strain B31. Although profiles identi cal to B31 could not be observed, they possessed an OspA of 31 KD and an O spB of 34 KD (Fig. 1). Six of the l. ricinus isolates (VS 130, VS 134, VS 2 15, VS 219 , VS 293, VS 393 ) and the I. dammini isolat e VS 2 belonged to this group. Gro up II showed an OspA of 32 KO and an OspB of 35 KO. Only one tick isolate (VS 461 ) was found in this group (Fig. 1). Group III wa s the largest group with 12 isolates from I . ricinus and the patient isolate (VS HP), exemplified by the strain VS 102. Th ey had an OspA of 33 KD and no appa rent OspB (Fig. 1). Th e fourth gro up had only one representat ive strain VS 116 (l. ricinus isolat e) which possessed an OspA of 33 .5 KD witho ut app ar ent O spB (Fig. 1). Compariso n of the electroph oretic mob ility of OspA and OspB in the isolate VS 2 with 5 and 97 passages showe d identical pattern s.
lAW
vs vs vs 2
130
134
vs
vs
vs
vs
215
219
293
393
-
-
Grl
B 31
vs
ca,
VS vs vs vs vs 102 100 185 28lI 290
_: -: + + + +
:Gtl:
vs vs vs vs vs vs vs vs vs 3
307 46'
+ + + + Grill
C48
Polymorphism of Outer Surface Proteins of Borrelia burgdorferi as a Tool for Classification O. PETER and A. G. BRETZ Clinical Microbiology, Institut Central des Hopitaux Valaisans, 1950 Sion, Switzerland
With 2 Figures· Received September 12, 1991 . Accepted January 15, 1992
Summary
A total of 23 isolates of Borrelia burgdorferi were characterized by SDS-PAGE and immunoblot analysis. One isolate came from the CSF of a Lyme neuro-borreliosis patient in Valais (Switzerland) and 22 were tick isolates (2 from I. dammini of Shelter Island, USA and 20 from I. ricinus of Valais, Switzerland). Based on the electrophoretic mobility of outer surface proteins (OspA and OspB), four groups of B. burgdorferi could be defined. Group I isolates possess an OspA of 31 KD and an OspB of 34 KD. The group II isolate showed an OspA of 32 KD and OspB of 35 KD. Group III isolates have a 33 KD OspA and group IV a 33.5 KD OspA. This classification was confirmed by the reactiviry of a monoclonal antibody (D6) to a 12 KD antigen that was recognized in group III only. A Lyme patient's serum showed a 2-band pattern (10 and 13 KD) for group I and a one-band pattern (12 KD) for the other 3 groups. Therefore OspA, OspB and other proteins of low molecular weight (10, 12, and 13 KD) seem to be important keys for the classification of B. burgdorferi isolates. This typing system correlates with genetic analysis.
Zusammenfassung Insgesamt 23 Isolate von B. burgdorferi wurden mittels SDS-PAGE und Immunoblot charakterisiert. Ein Isolat stammte aus dem Liquor eines Patienten mit Lyme-Borreliose des Nervensystems im Wallis (Schweiz) und 22 Isolate von Zecken (2 von I. dammini aus Shelter Island, USA und 20 von I. ricinus aus dem Wallis, Schweiz). Aufgrund der elektrophoretischen Mobilitar der aulSeren Oberflachenproteine (OspA und OspB) konnten vier Gruppen von B. burgdorferi definiert werden. Die Gruppe I wies ein OspA von 31 KD und ein OspB von 34 KD auf. Das Isolat der Gruppe II zeigte ein OspA von 32 KD und ein OspB von 35 KD, die Isolate der Gruppe III ein OspA von 33 KD und die der Gruppe IV ein OspA von 33.5 KD. Diese Klassifikation wurde durch die Reaktionsfahigkeit eines monoklonalen Antikorpers (D6) gegeniiber einem 12 KD-Antigen, das nur in der Gruppe III erkannt wurde, bestatigr, Das Serum eines Patienten mit Lyme-Borreliose zeigte ein Muster aus 2 Banden (10 und 13 KD) gegen Gruppe I und ein Muster mit einer Bande (12 KD) gegen die anderen drei Gruppen. Somit scheinen OspA, OspB und andere niedermolekulare Proteine (10,12 und 13 KD) wichtige Schlussel fur die Klassifikation von B. burgdorferi darzustellen. Dieses Typisierungsschema korreliert mit der genetischen Analyse.
Osp as a Tool for the Classification of B. burgdorferi
29
Introduction Since its discovery (10) Borrelia burgdorferi has been isolated from ticks, various species of mammals, a bird and humans o f the northern hemisphere. Some species of ticks of the genus Ixodes were found to be the main vect ors of this bacterium which causes a spiro chetosis first ca lled Lyme art h ritis, then Lyme disease, and now, Lyme borreliosis. In about 60 % o f human cases the disease is characterized by erythema (chronicum) migrans, a distinctive skin lesion that appears a few da ys to a few weeks after tick bite. This early ph ase of the disea se ma y be succeeded by ch ro nic systemic illnes s, invo lving the nervou s system, th e heart, the jo int s, the mu scles and the skin (15 ). In Europe, late neurological manifestati on s and skin lesions are more frequent than art hritis whereas in th e United States the rever se is true. In Europe, B. burgdorferi is maintained and tr an smitted by the sheep tick, 1. ricinus, of wh ich up to 55 % have been found infected in Switzerland (2, 11 ). Am ong European isolates o f B. burgdorferi, there appears to be a high degr ee of antigenic variability whereas American isolates are more homogeneous (6, 17). A system for preliminary typing of B. burgdorjeri, using monoclonal antibodies, has been proposed by Barbour (7). More extensive analyses of European and American iso lates were performed by Barbour and Schrumpf (8) and by Wilske et al. (19) . However , non e of the se classi fications fully agreed with classification ba sed on genetic analysis, such as rRNA restriction p attern (12) . Th erefore, we proposed a simplified typing system based up on th e electrophoretic mobility of O spA and O spB and upon the reactivity of a 12 KO ant igen with polyclonal and mon oclon al antibod ies.
Material and Methods
Strains and culture conditions. A total of 23 isolates of B. burgdorferi was used in this stud y (Table 1). Twenty origina ted from I. ricinus from Valais, Switzerland (11) and one from the cerebro spinal fluid of a Lyme borr eliosis pat ient from Valais. In addition, the reference strain of B. burgdorferi (B31) from I. dammini and one isolate from the same tick species from Shelter Island , New York , (provided by W. Burgdorfer) were included. All the isolate s were grow n in BSK II medium (5 ) and incubated at 34 °C. With th e exception of B31, all the isolates had been passaged less than 10 times. Preparation of the antigens. The borreli ae were collected dur ing their logarithmi c growth pha se, by centrifugation at 9000 g for 20 min. The y were washed twice in PBS-Mg-CIz5mM (pH 7.2) and centr ifuged at 10000 g for 5 min. Th e pellet was resuspended in distilled water and protein concentration was adju sted to 1 mg/ml, using the method of Biuret. The material was kept frozen until used. Monoclonal and polyclonal antibodies. Monoclonal antib odies were prepared by using the invitrotech kit (Bioinvent, Lund). Spleen cells from three BALB/c mice were excised and prepared with supplied medium according to the manu factur er's pro cedure. In vitro immuniz ation was mad e by adding 20 ~t1 of SOS lysate of B. burgdorferi stra in VS 102 (1 mg/ ml ). Hybridoma s were produ ced by fusion with PAl myeloma cells (kindly supplied by Or. D. Lauancby, CHUV, Lausanne, Switzerland). Th e mon oclonal antibody (0 6) coming from one of the hybridomas was used in this study. Polyclon al anti bodies came fro m a Lyme patient' s serum reactive with ant igens of 10 to 13 KD. Electrophoresis and immunoblot. The cell suspensions of B. burgdorferi (1 mg/ml) were dissolved in the sample buffer with 0.6 % SDS (final concentration) and 50 mM dithiothreitol as a reducing agent. The samples were boiled for 5 min before und ergoing elec-
30
O. Peter and A. G. Bretz
Table 1. Source of B. burgdorferi isolates Origin biologic
geographic
I. dammini
l.tlammini
USA, NY, Shelter Island CH, VS, Sion 1 CH, VS, Martigny CH, VS, Martign y CH, VS, Martigny CH, VS, Rarogne CH , VS, Rarogne USA, NY, Shelter Island
VS 461
I. ricinus
CH, VS, Vouvry
VS 100 VS 102 VS 185 VS 286 VS 290 VS 3 VS 307 VS 464 VS 468 VS 488 VS 492 VS 5 18 VS BP
I. ricinus I. ricinus I. ricinus 1. ricinus I. ricinus I. ricinus I. ricinus I. ricinus I. ricinus I. ricinus I. ricinus I. ricinus human CSF
CH, VS, Rarogne CH, VS, Rarogne CH, VS, Sierre CH, VS, Rarogne CH, VS, Rarogn e CH, VS, Sion CH, VS, Rarogne CH, VS, Vouvry CH, VS, Vouvry CH, VS, Monthey CH, VS, Monrh ey CH, VS, Raro gne CH, VS, Viege
VS 116
I. ricinus
CH, VS, Raro gne
Isolate VS 2 VS 130 VS 134 VS 215 VS 2 19 VS 293 VS 393 B 31 2
I
2
I. ricinus I. ricinus
I. ricinus I. ricinus I. ricinus 1. ricinus
CH (Switzerland), VS (Canton of Valais), locality. Source: Will y Burgdorier, Rocky Mountain Laboratory, H amilton , MT 59840 , USA.
troph oresis (constant current 45 rnA) on 12.5% polyacrylamide gel overlaid with a 4 % stacking gel, according to the method of Barbour (3). The standard molecular weights (MW) of BioRad (low range protein molecular weight standards) were used as reference for the calculation of relative molecular weights. The transfer to PVDF membrane (Immobilon, Millipore, Zurich, Switzerland ) was performed according to Towbin et al. (16). After transfer, the membrane was cur to separate the MW and the strip was stained wit h Coom assie blue. The membrane was then cut slightly above the 14.4 KD. The upper pa rt of the membrane was stained with Coo massie blue and the lower part was saturated with 3% gelatine in a Tris buffer, pH . 7.5 at 37 °C, for 1 h. It was washed three times for 5 min each in Tris-Tween 20 (0.05% ) buffer and incuba ted for 2 h at room temperature with a human serum diluted 1 : 200 or with one of our monoclonal antibody (D6) diluted (1 : 100 in the same buffer containing 1% gelatin. After washing, antibody-ant igen reactions were demon strated by a second goat anti-hum an IgG or goat anti-mouse IgM antib ody conjugated to alkaline phosphatase, followed by three washes and the addition of BCIPINBT substrate (5bromo-4-chloro -3-indoyl-p-toluidine phosphate / p-nitro blue tetrazoliumchloride).
Osp as a Tool for the Classification of B. burgdorferi
31
Results Based on the electrophoretic mob ility of Os pA, and OspB if present , four groups of borreliae could be defined. Gro up I was represented by the strain B31. Although profiles identi cal to B31 could not be observed, they possessed an OspA of 31 KD and an O spB of 34 KD (Fig. 1). Six of the l. ricinus isolates (VS 130, VS 134, VS 2 15, VS 219 , VS 293, VS 393 ) and the I. dammini isolat e VS 2 belonged to this group. Gro up II showed an OspA of 32 KO and an OspB of 35 KO. Only one tick isolate (VS 461 ) was found in this group (Fig. 1). Group III wa s the largest group with 12 isolates from I . ricinus and the patient isolate (VS HP), exemplified by the strain VS 102. Th ey had an OspA of 33 KD and no appa rent OspB (Fig. 1). Th e fourth gro up had only one representat ive strain VS 116 (l. ricinus isolat e) which possessed an OspA of 33 .5 KD witho ut app ar ent O spB (Fig. 1). Compariso n of the electroph oretic mob ility of OspA and OspB in the isolate VS 2 with 5 and 97 passages showe d identical pattern s.
lAW
vs vs vs 2
130
134
vs
vs
vs
vs
215
219
293
393
-
-
Grl
B 31
vs
ca,
VS vs vs vs vs 102 100 185 28lI 290
_: -: + + + +
:Gtl:
vs vs vs vs vs vs vs vs vs 3
307 46'
+ + + + Grill
C48
