Characterization of the protein recognized by the monoclonal antibody D6 specific for Borrelia

garinii isolates1O. Péter*, 1 J-C. Wyss, 1A.G.Bretz, 1L. Toutoungi, 2A. Scherl, 1X. Zhang, 2J.-C. Sanchez, 3R. Sahli

1 Institut Central des Hôpitaux Valaisans, Microbiologie (ICHV), Service of Infectious Diseases, 1950 Sion, Switzerland2 Biomedical Proteomics Research Group, Department of Bioinformatics and Structural Biology, Geneva University, 1200 Geneva3 Centre Hospitalier Universitaire Vaudois (CHUV), Institute of Microbiologie, 1011 Lausanne

Background : In Europe, Borrelia burgdorferi sensu lato isolates belong to 4 major species: B. burgdorferi sensu stricto, B. afzelii, B. garinii and B. valaisiana.The objective of this study was to characterize low molecular weight proteins of B. burgdorferi sensu lato. Our main focus was a protein around 12 kDa, that is reactive with D6, a monoclonal antibody specific for B. garinii isolates.

Methods :Proteins of a B. garinii isolate (VS 102) were prepared as described schematically below.

The Bb477, Bb061, Bb390 open reading frames of 28 isolates (5 B. burgdorferi sensu stricto, 5 B. afzelii, 13 B. garinii and 5 B. valaisiana) was analysed by PCR and DNA sequencing using the BigDye chemistry. Sequence alignments were done with clustalw included in the Vector NTI advance package (Invitrogen). Genes were cloned and expressed in E. coli PQR9. Putative epitope sequence was synthezised and mab D6 was saturated and to observe absence of reactivity.

Tandem mass spectrometry analysis

Design of primers for PCR

Mascot Search

Search in Genbank

Amplification by PCR of 13-28 Borrelia isolates

Sequencing of amplicons with ABI 310 instrument

Results :Partial sequences of 3 proteins of Borrelia burgdorferi were obtained from the tandem mass spectrometry analysis: Bb477 (30S ribosomal protein S10, rpsJ), BB061 (thioredoxine A, TrxA), Bb390 (50s ribosomal protein L7/L12, rpIL). All 3 complete genes were amplified by PCR from 13 to 28 isolates. DNA sequences were translated and aligned.

B31

VS123

VS215

A26s

ACA1

VS461

935T

387

NT29

VS102

UK

VS116

AG1

B

A

G

V

Recombinants rpIL and TrxA, dilutions (1)1/2, (2)1/20, (3)1/200, (4)1/2000

C+ 1 2 3 4 1 2 3 4 ……..rpIL…………….. TrxA

Expression of rpIL and TrxA, in E. coli PQR9

Immuno-absorption of mab D6 with synthetic peptide (TrxA 4-20)

Conclusion 1:After purification of low molecular mass proteins of B. garinii, partial sequences of 3 proteins of Borrelia burgdorferi were obtained from the mass spectrometry analysis: Bb390 (50s ribosomal protein L7/L12, rpIL), Bb477 (30S ribosomal protein S10, rpsJ), BB061 (thioredoxine A, TrxA). All 3 complete genes were amplified by PCR from 13 to 28 Borrelia isolates belonging to B. burgdorferi, B. afzelii, B. garinii and B. valaisiana.

Conclusion 2:TrxA and rpIL were expressed in E. coli PQR9 and mab D6 showed weak reactivity to recombinant TrxA protein and no reactivity to recombinant rpIL. Based on sequence alignements, epitope was suspected to be in position 7-12 of TrxA

Conclusion 3:A synthetic peptide was ordered and was used for immuno-absorption. It confirmed that epitope of mab D6 corresponds to KEDFVA sequence of Thioredoxyne A protein (amino acids 7-12).

rpsJ

TrxA

rpIL

B

A

G

V

B31VS123

VS215

VS461

ACA1

A26s

935T

387

20047

A19s

A77c

FAR01

G25

M63

VS BM

VS BP

HP3

NT19

VS102

UK

VS116

AG1

C+ C+

B

A

G

V

B31

VS123

VS215

Geho

IP1

VS461

ACA1

A26s

A38s

Bo23

935T

387

20047

A19s

A77c

FAR01

G25

M63

VS BM

VS BP

HP3

NT29

VS102VS116

UK

AG1

F10.08.94

FRANK