Chemiluminescent analysis of Borrelia burgdorferi penicillin-binding proteins using ampicillin conjugated to digoxigenin

Michael V. Norgard, Scott I. Baker, Justin D. Radolf

Research output: Contribution to journalArticle

3 Scopus citations

Abstract

Knowledge of the penicillin-binding proteins (PBPs) of Borrelia burgdorferi is important for understanding both the targets of β-lactams used therapeutically for Lyme borreliosis and the complex membrane biology of the distinctive spirochetal pathogen which causes Lyme disease. In this study, the PBPs of a number of B. burgdorferi strains and variants were examined using a rapid and sensitive chemiluminescent assay which employs ampicillin conjugated to digoxigenin (dig-amp). The minimum inhibitory concentration of dig-amp for B. burgdorferi high-passage strain B31 (0.012 μg/ml) was essentially no different from that of free ampicillin (0.025 μg/ml). Dig-amp bound specifically to B. burgdorferi B31 PBPs with molecular masses of 92, 80, 65, 46, 40, 34, 31, 29, 22, 20 and 13 kDa; the 31 kDa and 34 kDa PBPs were proven to be OspA and OspB, respectively. All of the borrelial PBPs were present in the cytoplasmic membrane fraction of B. burgdorferi, findings consistent with their activities as PBPs but inconsistent with OspA and OspB as surface-exposed outer membrane lipoproteins. Furthermore, among the PBP profiles of other high- and low-passage variants of B. burgdorferi strains Sh-2-82, HB19, and N40, which differed somewhat from one another, OspD (28 kDa) but not OspC (22-25 kDa) also was strongly implicated as a PBP; however, OspC possessed a gel mobility easily misconstrued as that of a 26 kDa PBP often expressed reciprocally with OspB. The ramifications of classifying OspA, OspB, and OspD as PBPs are discussed. While the current inability to genetically manipulate B. burgdorferi hinders determining which of the borrelial PBPs are essential for spirochetal viability (i.e., are the lethal targets of β-lactams), a priori knowledge of the borrelial PBPs will facilitate the production and purification of recombinant derivatives whose activities can be assessed further in vitro.

Original languageEnglish (US)
Pages (from-to)257-272
Number of pages16
JournalMicrobial Pathogenesis
Volume19
Issue number4
DOIs
StatePublished - Oct 1995

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Keywords

  • Borrelia
  • Chemiluminescence
  • Digoxigenin
  • Penicillin-binding proteins
  • β-lactams

ASJC Scopus subject areas

  • Microbiology
  • Infectious Diseases

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