The role of pH-regulated genes in Borrelia burgdorferi pathogenesis

Lyme disease, caused by the spirochete bacterium Borrelia burgdorferi, is the most common arthropod-borne infectious disease in the USA. The life cycle of B. burgdorferi is complex requiring both a tick vector and a vertebrate host. Several recent whole genome transcriptional profile analyses of B. burgdorferi propagated under tick or mammalian host-specific conditions (pH, temperature) revealed significant up-regulation of several linear plasmid 54 (lp54) encoded open reading frames (ORFs). The extent to which this differential gene expression contributes to pathogenesis is yet to be determined, and hence our long-term goal was to analyze the role of the temperature- and pH-regulated lp54-encoded genes of B. burgdorferi in the infectivity of the mammalian host.

We have inactivated individually or in combination three genes (bba64, bba65 and bba66) that are significantly upregulated in B. burgdorferi under mammalian host specific conditions. We have determined the phenotype of each of the mutants using the murine model of Lyme disease. There were no significant differences in the ability of all the mutants to infect C3H/HeN mice compared to their isogenic control strains. These observations suggest that B. burgdorferi can establish infection in the absence of expression of BBA64, BBA65 and BBA66 in the murine model of Lyme disease, and that B. burgdorferi might utilize multiple, functionally related determinants to establish infection.

To further investigate differentially expressed genes on lp54 that are part of a "mini-transcriptome" and not amenable to insertional inactivation or targeted deletions, we have modified a genetic tool to facilitate manipulation of the B. burgdorferi genome. We have inserted two Flp-recombinase target sites (FRTs) at different places on lp54, and delivered Flp-recombinase on an inducible shuttle vector. The orientation of the two FRT sites will allow for deletion of the intervening DNA between bba55 and bba73, where the two FRT sites are inserted, in the presence of Flp-recombinase enzyme. This strategy will provide significant advantages in manipulating the genome of B. burgdorferi.