Assessing the Contribution of an HtrA Family Serine Protease During Borrelia turicatae Mammalian Infection.

Tick-borne relapsing fever (TBRF), characterized by recurring febrile episodes, is globally distributed and among the most common bacterial infections in some African countries. Despite the public health concern that this disease represents, little is known regarding the virulence determinants required by TBRF Borrelia during infection. Because the chromosomes of TBRF Borrelia show extensive colinearity with those of Lyme disease (LD) Borrelia, the exceptions represent unique genes encoding proteins that are potentially essential to the disparate enzootic cycles of these two groups of spirochetes. One such exception is a gene encoding an HtrA family protease, BtpA, that is present in TBRF Borrelia, but not in LD spirochetes. Previous work suggested that btpA orthologs may be important for resistance to stresses faced during mammalian infection. Herein, proteomic analyses of the TBRF spirochete, Borrelia turicatae, demonstrated that BtpA, as well as proteins encoded by adjacent genes in the B. turicatae genome, were produced in response to culture at mammalian body temperature, suggesting a role in mammalian infection. Further, transcriptional analyses revealed that btpA was expressed with the genes immediately upstream and downstream as part of an operon. To directly assess if btpA is involved in resistance to environmental stresses, btpA deletion mutants were generated. btpA mutants demonstrated no growth defect in response to heat shock, but were more sensitive to oxidative stress produced by t-butyl peroxide compared to wild-type B. turicatae. Finally, btpA mutants were fully infectious in a murine relapsing fever (RF) infection model. These results indicate that BtpA is either not required for mammalian infection, or that compensatory mechanisms exist in TBRF spirochetes to combat environmental stresses encountered during mammalian infection in the absence of BtpA.

Generation of Conditional Mutants in Borrelia burgdorferi.

Mutational studies aimed at characterizing the function(s) of bacterial genes required for growth or viability are constrained by the inability to generate deletion strains lacking the gene of interest. To circumvent this limitation, it is possible to generate conditional mutants in which a copy of the gene of interest is introduced into the bacteria to compensate for the loss of the native allele. Expression of the non-native copy of the target gene is typically under control of an inducible promoter, which allows for controllable and regulated production of the gene of interest. Conditional mutants are also broadly useful for phenotypic analyses of genes that require a tightly regulated and artificially inducible copy of the target gene. Herein, we describe the methods used to generate and confirm conditional mutant clones in Borrelia burgdorferi utilizing the Borrelia-adapted lac operator/repressor system.