LONG-TERM IN-VITRO CULTIVATION OF BORRELIA-BURGDORFERI SENSU-LATO STRAINS - INFLUENCE ON PLASMID PATTERNS, GENOME STABILITY AND EXPRESSION OF PROTEINS

Low (7th) and high (298th/304th) in vitro passages (cultivated over a period of 3 years) of two human Borrelia burgdorferi sensu late strain s, PKo (B. afzelii) and PBi (B. garinii) were compared by pulse-field gel electrophoresis, Southern blot, sequencing of the ospA gene, SDS-P AGE and Western blot. Digestion of genomic DNA with ApaI, BssHII, KspI , MluI, SmaI and XhoI did not reveal any differences between low and h igh passages. The loss of two linear plasmids with sizes of 6 and 31 k bp was detected in strain PKo between passages 34-50 and 101-304, resp ectively, whereas the ospA-carrying plasmid remained unchanged. In con trast, analysis of linear plasmid profiles obtained from low and high passages of B. garinii strain PBi showed no differences Sequence analy sis of the ospA gene demonstrated no difference in the strain PBi and one nucleotide exchange in the strain PKo when low and high passages w ere compared. The observed transition (G-A) in the third codon positio n did not alter the amino acid sequence. However, the rate of expressi on of the outer surface proteins OspA, OspB and OspC of strain PKo dur ing low and high stages of cultivation varied significantly. In summar y, our data suggest that the B. burgdorferi sensu late genome is stabl e during long-term in vitro cultivation.