Dataset Information

Targeted mutation of the outer membrane protein P66 disrupts attachment of the Lyme disease agent, Borrelia burgdorferi, to integrin alphavbeta3.

ABSTRACT: Borrelia burgdorferi, the agent of Lyme disease, expresses several adhesion molecules that are probably required for initial establishment of infection in mammalian hosts, and for colonization of various tissues within the host. The B. burgdorferi outer membrane protein P66 was previously identified as a ligand for beta3-chain integrins by using a variety of biochemical approaches. Although the earlier data suggested that P66 is an adhesin that mediates B. burgdorferi attachment to beta3-chain integrins, lack of genetic systems in B. burgdorferi precluded definitive demonstration of a role for P66 in beta3 integrin attachment by intact borreliae. Recent advances in the genetic manipulation of B. burgdorferi have now made possible the targeted disruption of the p66 gene. Mutants in p66 show dramatically reduced attachment to integrin alphavbeta3. This is, to our knowledge, the first description of the targeted disruption of a candidate B. burgdorferi virulence factor with a known biochemical function that can be quantified, and demonstrates the importance of B. burgdorferi P66 in the attachment of this pathogenic spirochete to a human cell-surface receptor.

SUBMITTER: Coburn J

PROVIDER: S-EPMC165870 | biostudies-literature | 2003 Jun

REPOSITORIES: biostudies-literature

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Targeted mutation of the outer membrane protein P66 disrupts attachment of the Lyme disease agent, Borrelia burgdorferi, to integrin alphavbeta3.

Coburn Jenifer J Cugini Carla C

Proceedings of the National Academy of Sciences of the United States of America 20030514 12

Borrelia burgdorferi, the agent of Lyme disease, expresses several adhesion molecules that are probably required for initial establishment of infection in mammalian hosts, and for colonization of various tissues within the host. The B. burgdorferi outer membrane protein P66 was previously identified as a ligand for beta3-chain integrins by using a variety of biochemical approaches. Although the earlier data suggested that P66 is an adhesin that mediates B. burgdorferi attachment to beta3-chain i ...[more]

PMID: 12748384

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Project description:Bacteria from the genus Borrelia are known to harbor numerous linear and circular plasmids. We report here a comparative analysis of the nucleotide sequences of 236 plasmids present in fourteen independent isolates of the Lyme disease agent B. burgdorferi.We have sequenced the genomes of 14 B. burgdorferi sensu stricto isolates that carry a total of 236 plasmids. These individual isolates carry between seven and 23 plasmids. Their chromosomes, the cp26 and cp32 circular plasmids, as well as the lp54 linear plasmid, are quite evolutionarily stable; however, the remaining plasmids have undergone numerous non-homologous and often duplicative recombination events. We identify 32 different putative plasmid compatibility types among the 236 plasmids, of which 15 are (usually) circular and 17 are linear. Because of past rearrangements, any given gene, even though it might be universally present in these isolates, is often found on different linear plasmid compatibility types in different isolates. For example, the arp gene and the vls cassette region are present on plasmids of four and five different compatibility types, respectively, in different isolates. A majority of the plasmid types have more than one organizationally different subtype, and the number of such variants ranges from one to eight among the 18 linear plasmid types. In spite of this substantial organizational diversity, the plasmids are not so variable that every isolate has a novel version of every plasmid (i.e., there appears to be a limited number of extant plasmid subtypes).Although there have been many past recombination events, both homologous and nonhomologous, among the plasmids, particular organizational variants of these plasmids correlate with particular chromosomal genotypes, suggesting that there has not been rapid horizontal transfer of whole linear plasmids among B. burgdorferi lineages. We argue that plasmid rearrangements are essentially non-revertable and are present at a frequency of only about 0.65% that of single nucleotide changes, making rearrangement-derived novel junctions (mosaic boundaries) ideal phylogenetic markers in the study of B. burgdorferi population structure and plasmid evolution and exchange.

Project description:The Borrelia burgdorferi outer membrane (OM) contains numerous surface-exposed lipoproteins but a relatively low density of integral OM proteins (OMPs). Few membrane-spanning OMPs of B. burgdorferi have been definitively identified, and none are well characterized structurally. Here, we provide evidence that the borrelial OMP P66, a known adhesin with pore-forming activity, forms a ?-barrel in the B. burgdorferi OM. Multiple computer-based algorithms predict that P66 forms a ?-barrel with either 22 or 24 transmembrane domains. According to our predicted P66 topology, a lysine residue (K487) known to be sensitive to trypsin cleavage is located within a surface-exposed loop. When we aligned the mature P66 amino acid sequences from B. burgdorferi and B. garinii, we found that K487 was present only in the B. burgdorferi P66 protein sequence. When intact cells from each strain were treated with trypsin, only B. burgdorferi P66 was trypsin sensitive, indicating that K487 is surface exposed, as predicted. Consistent with this observation, when we inserted a c-Myc tag adjacent to K487 and utilized surface localization immunofluorescence, we detected the loop containing K487 on the surface of B. burgdorferi. P66 was examined by both Triton X-114 phase partitioning and circular dichroism, confirming that the protein is amphiphilic and contains extensive (48%) ?-sheets, respectively. Moreover, P66 also was able to incorporate into liposomes and form channels in large unilamellar vesicles. Finally, blue native PAGE (BN-PAGE) revealed that under nondenaturing conditions, P66 is found in large complexes of ?400 kDa and ?600 kDa. Outer surface lipoprotein A (OspA) and OspB both coimmunoprecipitate with P66, demonstrating that P66 associates with OspA and OspB in B. burgdorferi. The combined computer-based structural analyses and supporting physicochemical properties of P66 provide a working model to further examine the porin and integrin-binding activities of this OMP as they relate to B. burgdorferi physiology and Lyme disease pathogenesis.

Dataset Information

Targeted mutation of the outer membrane protein P66 disrupts attachment of the Lyme disease agent, Borrelia burgdorferi, to integrin alphavbeta3.

Publications

Targeted mutation of the outer membrane protein P66 disrupts attachment of the Lyme disease agent, Borrelia burgdorferi, to integrin alphavbeta3.

Similar Datasets

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