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Low-passage-associated proteins of Borrelia burgdorferi B31: characterization and molecular cloning of OspD, a surface-exposed, plasmid-encoded lipoprotein.

ABSTRACT: Borrelia burgdorferi, the causative agent of Lyme disease, loses its ability to infect and cause disease in mammalian hosts after repeated in vitro passage. To identify proteins preferentially expressed by the low-passage strain and thus representing potential virulence factors, the polypeptide profiles of virulent, low-passage and nonvirulent, high-passage forms of B. burgdorferi B31 were compared by nonequilibrium pH gradient two-dimensional gel electrophoresis. Four low-passage-associated proteins with relative molecular masses (M(r)s) of 35,000, 28,000, 24,000, and 20,000 were identified. Of these, the 28- and 35-kDa polypeptides were not expressed in detectable quantities in the high-passage B31 strain, whereas the 24- and 20-kDa proteins were present in reduced quantities. All four of these proteins were lipoproteins, as determined by labelling with [3H]palmitate. The abundant 28-kDa component, called outer surface protein D (OspD), is surface exposed on the basis of its proteolysis during treatment of intact organisms with proteinase K. The ospD gene is located on a 38-kb linear plasmid present in seven of nine low-passage strains of B. burgdorferi examined but absent in most high-passage, nonvirulent strains tested. Molecular cloning and sequence analysis of the ospD gene locus revealed an open reading frame encoding a 28,436-Da polypeptide with a putative signal peptidase II leader sequence. An unusual feature of the region upstream of the gene was the presence of seven contiguous, direct repeats of a 17-bp sequence that includes consensus -35 and -10 transcription initiation signals; however, only one transcription initiation site was active as determined by primer extension analysis. Further study of these and other polypeptides associated with low-passage strains may lead to identification of B. burgdorferi gene products required for infection and pathogenesis in mammalian hosts.

SUBMITTER: Norris SJ

PROVIDER: S-EPMC258216 | biostudies-other | 1992 Nov

REPOSITORIES: biostudies-other

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Low-passage-associated proteins of Borrelia burgdorferi B31: characterization and molecular cloning of OspD, a surface-exposed, plasmid-encoded lipoprotein.

Norris S J SJ Carter C J CJ Howell J K JK Barbour A G AG

Infection and immunity 19921101 11

Borrelia burgdorferi, the causative agent of Lyme disease, loses its ability to infect and cause disease in mammalian hosts after repeated in vitro passage. To identify proteins preferentially expressed by the low-passage strain and thus representing potential virulence factors, the polypeptide profiles of virulent, low-passage and nonvirulent, high-passage forms of B. burgdorferi B31 were compared by nonequilibrium pH gradient two-dimensional gel electrophoresis. Four low-passage-associated pro ...[more]

PMID: 1398980

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Project description:Previously we have found that sera from immunocompetent mice infected either naturally by ticks or experimentally with low numbers of Borrelia burgdorferi ZS7 bacteria lack OspA- and OspB-specific antibodies but confer optimal protection on severe combined immunodeficiency mice against challenge with spirochetes (U.E. Schaible, L. Gern, R. Wallich, M. D. Kramer, M. Prester, and M. M. Simon, Immunol. Lett. 36:219-226, 1993). We have now used the latter immune sera to identify new spirochetal structures with relevance for protection from an expression library of the virulent European strain B. burgdorferi ZS7. Here we report the cloning and characterization of a novel lipoprotein, designated pG, the gene for which is located on a 48-kb linear plasmid. Sequence analysis of the pG gene revealed an open reading frame encoding a putative lipoprotein of 196 amino acids with a calculated molecular mass of 22 kDa and a consensus cleavage sequence (Leu-X-Y-Z-Cys) recognized by signal peptidase II. Restriction fragment length polymorphism analyses of pG derived from independent B. burgdorferi isolates from different geographic areas revealed that the gene is species specific, with, however, extensive genotypic heterogeneity. Comparison of the protein sequence of pG with those of other known B. burgdorferi outer surface lipoproteins (OspA to OspF and P27) demonstrated that pG is most related to OspF. Furthermore, the upstream region of pG exhibited extensive sequence homology (> 94%) with the ospEF promoter region. Mouse immune sera to recombinant pG did not recognize a corresponding molecule in lysates of in vitro-propagated ZS7 spirochetes. However, experimental or natural infection of mice with ZS7 resulted in the induction of antibodies with reactivity for pG and the potential to delay the development of clinical arthritis. Together with the finding that sera from Lyme disease patients also contain antibodies to pG, our data suggest that the pG gene is preferentially expressed in the mammal environment.

Dataset Information

Low-passage-associated proteins of Borrelia burgdorferi B31: characterization and molecular cloning of OspD, a surface-exposed, plasmid-encoded lipoprotein.

Publications

Low-passage-associated proteins of Borrelia burgdorferi B31: characterization and molecular cloning of OspD, a surface-exposed, plasmid-encoded lipoprotein.

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