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Antibody-resistant mutants of Borrelia burgdorferi: in vitro selection and characterization
A Sădziene1, P A Rosa, P A Thompson
1Department of Microbiology, University of Texas Health Science Center, San Antonio 78284.
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.
Insights
Researchers identified antibody-resistant Borrelia burgdorferi (Lyme disease agent) mutants using monoclonal antibodies (mAbs) targeting outer membrane proteins OspA and OspB. These mutants exhibit varied resistance mechanisms, including loss or alteration of target proteins.
Area of Science:
- Microbiology
- Immunology
- Bacteriology
Background:
- Borrelia burgdorferi is the causative agent of Lyme disease.
- Outer membrane proteins OspA and OspB are key targets for antibody-mediated inhibition of bacterial growth.
Purpose of the Study:
- To investigate the mechanisms of antibody resistance in Borrelia burgdorferi.
- To select and characterize antibody-resistant mutants of Borrelia burgdorferi using monoclonal antibodies (mAbs).
Main Methods:
- Inhibition of clonal populations of Borrelia burgdorferi using polyclonal antisera and monoclonal antibodies (mAbs).
- Selection of antibody-resistant mutants.
- Characterization of mutant strains using Western blots and analysis of outer membrane proteins (OspA, OspB) and encoding plasmids.
Main Results:
- Antibody-resistant mutants were found at frequencies of 10(-5) to 10(-2).
- Four classes of escape variants were identified: Class I (lacked OspA/OspB and encoding plasmid), Class II (truncated OspA/OspB), Class III (full-length Osp proteins with mutations preventing antibody binding), and Class IV (antibody binding intact but resistance observed).
Conclusions:
- Borrelia burgdorferi can develop diverse resistance mechanisms against antibody-mediated growth inhibition.
- Mutations in ospA genes and alterations in outer membrane protein expression contribute to antibody resistance in Lyme disease bacteria.