Antibody-resistant mutants of Borrelia burgdorferi: in vitro selection and characterization.

A Sădziene,D M Hogan,A G Barbour,P A Thompson,P A Rosa

doi:10.1084/jem.176.3.799

Abstract

We used polyclonal antisera and monoclonal antibodies (mAbs) to inhibit the growth of clonal populations of two strains of Borrelia burgdorferi, the Lyme disease agent, and thereby select for antibody-resistant mutants. mAbs were directed at the outer membrane proteins, OspA or OspB. Mutants resistant to the growth-inhibiting properties of the antibodies were present in the populations at frequencies ranging from 10(-5) to 10(-2). The several escape variants that were examined were of four classes. Class I mutants were resistant to all mAbs; they lacked OspA and OspB and the linear plasmid that encodes them. Two other proteins were expressed in larger amounts in class I mutants; mAbs to these proteins inhibited the mutant but not the wild-type cells. Class II mutants were resistant to some but not all mAbs; they had truncated OspA and/or OspB proteins. Class III mutants were resistant only to the selecting mAb; they had full-length Osp proteins that were not bound by the selecting antibody in Western blots. In two class III mutants resistant to different anti-OspA mAbs, missense mutations were demonstrated in the ospA genes. Class IV mutants were likewise resistant only to selecting antibody, but in this case the selecting antibody still bound in Western blots.

Highlights

Borrelias were grown in broth BSK II medium and harvested by methods previously described [41, 42]
In some experiments borrelias were grown as isolated in solid medium (42a)
Borrelias were added to BSK II medium with 1% low-geUing temperature agarose (SeaPlaque; FMC Corporation, Rockland, ME) kept liquid at 37~ The cell suspension was poured as a thin layer over solid BSK II medium containing 1.5% agarose in small polystyrene petri dishes [30]