TY - JOUR
T1 - Chemiluminescent analysis of Borrelia burgdorferi penicillin-binding proteins using ampicillin conjugated to digoxigenin
AU - Norgard, Michael V.
AU - Baker, Scott I.
AU - Radolf, Justin D.
N1 - Funding Information:
The authors thank Linda M. Weigel for performing dig-amp experiments, Alan Barbour and Stephen Barthold for providing strains of B. burgdorferi, Steven Norris for supplying OspD antiserum, Bryan Riley for assistance with cultivation and cloning of borreliae, isolation of escape mutants, and animal infection experiments, Martin Goldberg and Leslie Arndt for outstanding technical assistance, and Darrin Akins for helpful discussions. This work was partially supported by Public Health Service Grant AI-29735 from the National Institute of Allergy and Infectious Diseases, Grant 1-0940 from the Robert A. Welch Foundation, and Grant-in-Aid 91015470 from the American Heart Association. JDR was the recipient of an Established Investigatorship Award from the American Heart Association.
PY - 1995/10
Y1 - 1995/10
N2 - 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.
AB - 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.
KW - Borrelia
KW - Chemiluminescence
KW - Digoxigenin
KW - Penicillin-binding proteins
KW - β-lactams
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UR - http://www.scopus.com/inward/citedby.url?scp=0028790189&partnerID=8YFLogxK
U2 - 10.1016/S0882-4010(95)90308-9
DO - 10.1016/S0882-4010(95)90308-9
M3 - Article
C2 - 8825913
AN - SCOPUS:0028790189
SN - 0882-4010
VL - 19
SP - 257
EP - 272
JO - Microbial Pathogenesis
JF - Microbial Pathogenesis
IS - 4
ER -