DNA array analysis to detect gene expression changes in Borrelia burgdorferi after treatment with a bactericidal antibody

Identification and characterization of genes that contribute to infection by Borrelia burgdorferi and of those, genes that are targets of host responses, is important for understanding the pathogenesis of Lyme disease. The focus of this dissertation project was to expand the understanding of the bactericidal mechanism of a complement independent antibody. The monoclonal antibody (mAb) CB2 recognizes a carboxy terminal, hydrophilic epitope of the outer surface protein B (OspB) of B. burgdorferi. Upon binding of CB2 to OspB, differentially expressed gene products may be responsible for, or be associated with, the death of the organism.;Whole genome DNA array analysis at various time points within one hour of incubation of B. burgdorferi with the antibody disclosed that most significant changes occurred at 25 min. Circular plasmid 32 (cp32)-encoded genes were active in this period of time, including blyA homologs, phage holin system genes. DNA array data and quantitative real-time PCR analysis showed three blyA homologs were up-regulated significantly. A question to test in future studies, then, is whether the killing mechanism of CB2 is through uncontrolled expression of the blyA and blyB phage holin system.;When the genome sequence of Borrelia burgdorferi became available, several genes surfaced as possible targets for an autolytic response as an effect of CB2 treatment. Specifically, BB0059 was further investigated due to its significant homology to a putative Treponema pallidum hemolysin, TP0649. Results showed that both BB0059 and TP0649 displayed no hemolytic activity and furthermore did not lyse epithelial cells, suggesting their characterization as putative hemolysins may be incorrect. BB0059 was genetically inactivated to create a mutant Borrelia strain, B31 BB0059-. The Borrelia mutant strain was phenotypically characterized using assays for susceptibility to CB2, plasmid content, RNA quality, cDNA PCR analysis, growth rates and hemolysis on soft blood agar plates. B31 BB0059- had a significant delay in growth that could be partially restored with magnesium supplementation to the medium. BB0059 therefore may function as a metal ion influx/efflux accessory protein.