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Molecular cloning and characterization of nlpH, encoding a novel, surface-exposed, polymorphic, plasmid-encoded 33-kilodalton lipoprotein of Borrelia afzelii.

ABSTRACT: Borrelia burgdorferi sensu lato organisms, comprising B. burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii, are tick-borne pathogens causing Lyme borreliosis in humans. To identify putative virulence determinants, a B. afzelii DNA library was screened for Congo red dye binding, a property associated with virulence in pathogenic bacteria. One clone was found to carry a 663-nucleotide-long open reading frame encoding a Congo red dye-binding protein with a calculated molecular mass of 25,660 Da. The amino acid sequence deduced from its nucleotide sequence was found to include a consensus bacterial lipidation site present at residues 15 to 18 (Leu-Ser-Gly-Cys). The lipoprotein nature was demonstrated by incorporation of radioactive palmitate; hence, this protein has been termed NlpH, for new lipoprotein H. NlpH is located on the surface of B. afzelii, and the nlpH gene is found on a circular plasmid. The nlpH gene is also found in B. burgdorferi sensu stricto and B. garinii. Immediately upstream of nlpH is located a smaller reading frame encoding a polypeptide containing the casein kinase II phosphorylation recognition sequence, (Ser/Thr)-X-Y-(Glu/Asp), repeated 10 times.

SUBMITTER: Theisen M

PROVIDER: S-EPMC178528 | biostudies-other | 1996 Nov

REPOSITORIES: biostudies-other

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Molecular cloning and characterization of nlpH, encoding a novel, surface-exposed, polymorphic, plasmid-encoded 33-kilodalton lipoprotein of Borrelia afzelii.

Theisen M M

Journal of bacteriology 19961101 22

Borrelia burgdorferi sensu lato organisms, comprising B. burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii, are tick-borne pathogens causing Lyme borreliosis in humans. To identify putative virulence determinants, a B. afzelii DNA library was screened for Congo red dye binding, a property associated with virulence in pathogenic bacteria. One clone was found to carry a 663-nucleotide-long open reading frame encoding a Congo red dye-binding protein with a calculated molecular mass ...[more]

PMID: 8932298

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Project description:Leptospira is the etiologic agent of leptospirosis, a bacterial zoonosis distributed worldwide. Leptospiral lipopolysaccharide is a protective immunogen, but the extensive serological diversity of leptospires has inspired a search for conserved outer membrane proteins (OMPs) that may stimulate heterologous immunity. Previously, a global analysis of leptospiral OMPs (P. A. Cullen, S. J. Cordwell, D. M. Bulach, D. A. Haake, and B. Adler, Infect. Immun. 70:2311-2318, 2002) identified pL21, a novel 21-kDa protein that is the second most abundant constituent of the Leptospira interrogans serovar Lai outer membrane proteome. In this study, we identified the gene encoding pL21 and found it to encode a putative lipoprotein; accordingly, the protein was renamed LipL21. Southern hybridization analysis revealed the presence of lipL21 in all of the pathogenic species but in none of the saprophytic species examined. Alignment of the LipL21 sequence from six strains of Leptospira revealed 96 to 100% identity. When specific polyclonal antisera to recombinant LipL21 were used, LipL21 was isolated together with other known leptospiral OMPs by both Triton X-114 extraction and sucrose density gradient membrane fractionation. All nine strains of pathogenic leptospires investigated by Western blotting, whether culture attenuated or virulent, were found to express LipL21. In contrast, the expression of LipL21 or an antigenically related protein could not be detected in nonpathogenic L. biflexa. Infected hamster sera and two of eight human leptospirosis sera tested were found to react with recombinant LipL21. Native LipL21 was found to incorporate tritiated palmitic acid, consistent with the prediction of a lipoprotein signal peptidase cleavage site. Biotinylation of the leptospiral surface resulted in selective labeling of LipL21 and the previously known OMPs LipL32 and LipL41. These findings show that LipL21 is a surface-exposed, abundant outer membrane lipoprotein that is expressed during infection and conserved among pathogenic Leptospira species.

Dataset Information

Molecular cloning and characterization of nlpH, encoding a novel, surface-exposed, polymorphic, plasmid-encoded 33-kilodalton lipoprotein of Borrelia afzelii.

Publications

Molecular cloning and characterization of nlpH, encoding a novel, surface-exposed, polymorphic, plasmid-encoded 33-kilodalton lipoprotein of Borrelia afzelii.

Similar Datasets

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