BB0844, an RpoS-regulated protein, is dispensable for Borrelia burgdorferi infectivity and maintenance in the mouse-tick infectious cycle

Abstract

The genome of Borrelia burgdorferi, the causative agent of Lyme disease, is comprised of a large linear chromosome and numerous smaller linear and circular plasmids. B. burgdorferi exhibits substantial genomic variation, and previous studies revealed genotype-specific variation at the right chromosomal telomere. A correlation has also been established between genotype and invasiveness. The correlation between chromosome length and genotype and between genotype and invasiveness suggested that a gene(s) at the right chromosome telomere may be required for virulence. Of particular interest was bb0844, an RpoS-regulated gene at the right telomere, the expression of which is induced when the spirochete undergoes adaptation to the mammalian host. The structure of the right chromosomal telomere was examined in 53 B. burgdorferi clinical isolates of various genotypes. Four distinct patterns were observed for bb0844: (i) chromosomal localization, (ii) plasmid localization, (iii) presence on both chromosome and plasmid, and (iv) complete absence. These patterns correlated with the B. burgdorferi genotype. On the basis of available sequence data, we propose a mechanism for the genomic rearrangements that accounts for the variability in bb0844 genomic localization. To further explore the role of BB0844 in the spirochete life cycle, a bb0844 deletion mutant was constructed by allelic exchange, and the viability of wild-type and bb0844 deletion mutants was examined in an experimental mouse-tick infection model. The bb0844 mutant was fully infectious in C3H/HeJ mice by either needle inoculation or tick transmission with B. burgdorferi-infected Ixodes scapularis larvae. Naïve larval ticks acquired both wild-type and mutant spirochetes with equal efficiency from B. burgdorferi-infected mice. The results demonstrate that BB0844 is not required for spirochete viability, pathogenicity, or maintenance in the tick vector or the mammalian host. At present, a defined role for BB0844 in B. burgdorferi cannot be ascertained.

FIG. 1.

Construction and characterization of a bb0844 deletion mutant. (A) Strategy for partial deletion of bb0844. (B) PCR analysis of the wild type and two independent deletion mutant clones (B1 and A2). The combination of primers used for each PCR is shown at the top, and the positions of the primers are shown in panel A. The migration positions of a 1-kb DNA ladder are shown on the right.

FIG. 2.

(A) Real -time RT-PCR analysis of bb0844 and ospC expression in flat (unfed) nymphs, fed nymphs, and fed larvae. Ticks were infected as larvae by infestation of mice that had been syringe inoculated with B. burgdorferi strain 297. Expression of bb0844 was significantly induced upon nymphal feeding (P = 0.0005). (B) Real-time RT-PCR analysis of bb0844 in fed nymphal ticks infected as larvae by immersion with either wild-type (WT) or rpoS mutant (ΔrpoS) B. burgdorferi strain 297. Expression of bb0844 was significantly reduced in the mutant (P < 0.0001). The values represent the average flaB-normalized copy number and standard error of the mean (SEM) for each gene.

FIG. 3.

Proposed evolution of the right chromosomal end of B. burgdorferi and the bb0844 genomic location. The schematic diagram shows the final product following ResT-mediated reversal of telomere resolution based on the mechanisms proposed by Chaconas and coworkers (13, 14, 30). The rectangles denote the presence of the chromosome and plasmid in a single cell. The numbers refer to alternative outcomes discussed in the text.