Borrelia burgdorferi is the causative agent of Lyme disease, the most common vector-borne illness in the Northern hemisphere. Low-passage-number infectious strains of B. burgdorferi exhibit extremely low transformation efficiencies-so low, in fact, as to hinder the genetic study of putative virulence factors. Two putative restriction-modification (R-M) systems, BBE02 contained on linear plasmid 25 (lp25) and BBQ67 contained on lp56, have been postulated to contribute to this poor transformability. Restriction barriers posed by other bacteria have been overcome by the in vitro methylation of DNA prior to transformation. To test whether a methylation-sensitive restriction system contributes to poor B. burgdorferi transformability, shuttle plasmids were treated with the CpG methylase M.SssI prior to the electroporation of a variety of strains harboring different putative R-M systems. We found that for B. burgdorferi strains that harbor lp56, in vitro methylation increased transformation by at least 1 order of magnitude. These results suggest that in vitro CpG methylation protects exogenous DNA from degradation by an lp56-contained R-M system, presumably BBQ67. The utility of in vitro methylation for the genetic manipulation of B. burgdorferi was exemplified by the ease of plasmid complementation of a B. burgdorferi B31 A3 BBK32 kanamycin-resistant (B31 A3 BBK32::Kan(r)) mutant, deficient in the expression of the fibronectin- and glycosaminoglycan (GAG)-binding adhesin BBK32. Consistent with the observation that several surface proteins may promote GAG binding, the B. burgdorferi B31 A3 BBK32::Kan(r) mutant demonstrated no defect in the ability to bind purified GAGs or GAGs expressed on the surfaces of cultured cells.

Borrelia burgdorferi, the agent of Lyme disease, causes a multisystemic illness that can affect the skin, heart, joints, and nervous system and is capable of attachment to diverse cell types. Among the host components recognized by this spirochete are fibronectin and glycosaminoglycans (GAGs). Three surface-localized GAG-binding bacterial ligands, Bgp, DbpA, and DbpB, have been previously identified, but recent studies suggested that at least one additional GAG-binding ligand is expressed on the spirochetal surface when the spirochete is adapted to the mammalian host environment. BBK32 is a surface lipoprotein that is produced during infection and that has been shown to bind to fibronectin. In this study, we show that, when BBK32 was produced from a shuttle vector in an otherwise nonadherent high-passage B. burgdorferi strain, the protein localized on the bacterial surface and conferred attachment to fibronectin and to mammalian cell monolayers. In addition, the high-passage strain producing BBK32 bound to purified preparations of the GAGs dermatan sulfate and heparin, as well as to these GAGs on the surfaces of cultured mammalian cells. Recombinant BBK32 recognized purified heparin, indicating that the bacterial attachment to GAGs was due to direct binding by BBK32. This GAG-binding activity of BBK32 is apparently independent of fibronectin recognition, because exogenous heparin had no effect on BBK32-mediated bacterial binding to fibronectin.

Host cell binding is an essential step in colonization by many bacterial pathogens, and the Lyme disease agent, Borrelia burgdorferi, which colonizes multiple tissues, is capable of attachment to diverse cell types. Glycosaminoglycans (GAGs) are ubiquitously expressed on mammalian cells and are recognized by multiple B. burgdorferi surface proteins. We previously showed that B. burgdorferi strains differ in the particular spectrum of GAGs that they recognize, leading to differences in the cultured mammalian cell types that they efficiently bind. The molecular basis of these binding specificities remains undefined, due to the difficulty of analyzing multiple, potentially redundant cell attachment pathways and to the paucity of genetic tools for this pathogen. Complementation of a high-passage non-adherent B. burgdorferi strain reveals that the expression of DbpA, DbpB, or BBK32, is sufficient to confer efficient spirochete attachment to 293 epithelial cells. Epithelial cell attachment by DbpA and B was mediated by dermatan sulfate, while BBK32 recognized dermatan and heparan sulfate. The GAG binding properties of bacteria expressing DbpB or DbpA were distinguishable in that DbpB, but not DbpA, promoted spirochetal attachment to C6 glial cells. Furthermore, DbpA alleles from diverse Lyme disease spirochetes exhibit allelic variation with respect to binding decorin, dermatan sulfate, and epithelial cells. Targeted disruption of bbk32 resulted in decreased spirochete binding to fibronectin, GAGs, and mammalian cells. Thus, DbpA, DbpB, and BBK32 may play central but distinct roles in cell type-specific binding by Lyme disease spirochetes. This study illustrates that transformation of high-passage B. burgdorferi strains and targeted gene disruption provide a comprehensive genetic approach to analyze virulence-associated phenotypes conferred by multiple bacterial factors.

Host cell binding is an essential step in colonization by many bacterial pathogens, and the Lyme disease agent, Borrelia burgdorferi, which colonizes multiple tissues, is capable of attachment to diverse cell types. Glycosaminoglycans (GAGs) are ubiquitously expressed on mammalian cells and are recognized by multiple B. burgdorferi surface proteins. We previously showed that B. burgdorferi strains differ in the particular spectrum of GAGs that they recognize, leading to differences in the cultured mammalian cell types that they efficiently bind. The molecular basis of these binding specificities remains undefined, due to the difficulty of analyzing multiple, potentially redundant cell attachment pathways and to the paucity of genetic tools for this pathogen. In the current study, we show that the expression of decorin-binding protein (Dbp) A and/or DbpB, two B. burgdorferi surface proteins that bind GAGs, is sufficient to convert a high-passage nonadherent B. burgdorferi strain into one that efficiently binds 293 epithelial cells. Epithelial cell attachment was mediated by dermatan sulfate, and, consistent with this GAG-binding specificity, these recombinant strains did not bind EA-Hy926 endothelial cells. The GAG-binding properties of bacteria expressing DbpB or DbpA were distinguishable, and DbpB but not DbpA promoted spirochetal attachment to C6 glial cells. Thus, DbpA and DbpB may each play central but distinct roles in cell type-specific binding by Lyme disease spirochetes. This study illustrates that transformation of high-passage B. burgdorferi strains may provide a relatively simple genetic approach to analyze virulence-associated phenotypes conferred by multiple bacterial factors.