Genome-wide screen identifies novel genes required for Borrelia burgdorferi survival in its Ixodes tick vector

Borrelia burgdorferi, the causative agent of Lyme disease in humans, is maintained in a complex biphasic life cycle, which alternates between tick and vertebrate hosts. To successfully survive and complete its enzootic cycle, B. burgdorferi adapts to diverse hosts by regulating genes required for survival in specific environments. Here we describe the first ever use of transposon insertion sequencing (Tn-seq) to identify genes required for B. burgdorferi survival in its tick host. We found that insertions into 46 genes resulted in a complete loss of recovery of mutants from larval Ixodes ticks. Insertions in an additional 56 genes resulted in a >90% decrease in fitness. The screen identified both previously known and new genes important for larval tick survival. Almost half of the genes required for survival in the tick encode proteins of unknown function, while a significant portion (over 20%) encode membrane-associated proteins or lipoproteins. We validated the results of the screen for five Tn mutants by performing individual competition assays using mutant and complemented strains. To better understand the role of one of these genes in tick survival, we conducted mechanistic studies of bb0017, a gene previously shown to be required for resistance against oxidative stress. In this study we show that BB0017 affects the regulation of key borrelial virulence determinants. The application of Tn-seq to in vivo screening of B. burgdorferi in its natural vector is a powerful tool that can be used to address many different aspects of the host pathogen interaction.
Author summary Borrelia burgdorferi, the causative agent of Lyme disease, must adjust to environmental changes as it moves between its tick and vertebrate hosts. We performed a screen of a B. burgdorferi transposon library using massively parallel sequencing (Tn-seq) to identify fitness defects involved in survival in its tick host. This screen accurately identified genes known to cause decreased fitness for tick survival and identified new genes involved in B. burgdorferi survival in ticks. All of the genes tested individually confirmed the Tn-seq results. One of the genes identified encodes a protein whose function was previously unknown that appears to be involved in regulating expression of proteins known to be involved in environmental adaptation. Tn-seq is a powerful tool for understanding vector-pathogen interactions and may reveal new opportunities for interrupting the infectious cycle of vector-borne diseases.