The role of Borrelia peptidoglycan-interacting protein (BpiP) in the patho-physiology of Borrelia burgorferi

Lyme disease is the most common arthropod-borne disease in the US with more than 300,000 cases reported to CDC in 2013. The agent of Lyme disease is Borrelia burgdorferi, a pathogenic spirochete, which is transmitted to humans following the bite of infected Ixodes scapularis ticks. Bioinformatic analysis of the amino acid sequence of BB_0167 locus, further referred to as Borrelia peptidoglycan-interacting protein, bpiP, revealed significant similarity to OmpA-like proteins and contains the conserved c-terminal MHELSEKRARAIGNYL motif used by this family of proteins to interact with the peptidoglycan (NX 2LSX2RAX2VX3L)(1). Recombinant BpiP was purified in E. coli and used to generate polyclonal antibodies that detected a 45kDa protein, consistent with the predicted size of BpiP. bpiP was deleted from B. burgdorferi using homologous recombination. Preliminary studies in the C3H/H3N mouse model of Lyme disease indicate that this gene is not necessary to establish infection. Knockouts such as the previously mentioned bpiP are essential for understanding the role of proteins in the physiology and infectivity of the causative agent, B. burgdorferi. Mutated strains are obtained by using various resistance cassettes such as resistance to kanamycin, streptomycin, and gentamicin as counter-selectable markers. Confirmation of the genetic manipulation is then carried our by PCR using primers which target the gene itself, the antibiotic resistance markers, and regions flanking the genetic manipulation. After confirmation by PCR positive colonies are then subjected to immunoblot analysis and finally southern blot. This screening process will be significantly expedited and provide greater confidence in potential mutants used for the more time consuming downstream confirmations by creating polyclonal antibodies to the proteins which confer the antibiotic resistance. This will lead to an ease in generating and confirming mutations in B. burgdorferi.