CHARACTERIZING VIRULENCE AND CYTOTOXICITY IN BORRELIA BURGDORFERI

Abstract

Lyme disease is one of the most common vector-borne illness affecting millions of people worldwide. It is caused by the bacteria Borrelia burgdorferi or related spirochetes, which are transmitted to mammals by infected Ixodes sp. ticks. Although antibiotic treatment is typically curative, a small subset of patients experiences non-specific symptoms several months after treatment, which may include pain, fatigue, and cognitive deficits, collectively termed post-treatment Lyme disease syndrome, for which no further therapy is currently available. In addition, new isolates or species of pathogens causing Lyme disease were recently discovered that display enhanced virulence, inducing even more severe infections. These observations underscore the need for a deeper understanding of spirochete biology, host-pathogen interactions, and pathogenesis, as well as the development of novel, comprehensive intervention strategies against B. burgdorferi. Virulence of a pathogen, which is a measure of the pathogen’s ability to infect and cause disease in a host, is a complex interplay of host and pathogen components, with the microbial factors involved in colonizing the host, avoiding immune response and acquiring nutrition while the host factors interact with the microbial virulence factors to trigger immune responses. To study infection biology and virulence of a pathogen, it is important to determine the host factors responsible for the same.To this end, we have attempted a collaborative effort to identify potential host factors that are involved in pathogen interactions and/or disease pathogenesis, by screening a library of human interactome exoproteins with several pathogens, including diverse spirochete strains and isolates that cause Lyme disease. Screening of the initial interactome with spirochetes identified a potential host target, called lipocalin-2 (LCN2), which appears to be a promising factor for further study. We sought to test the hypothesis that LCN2 could be a novel host molecule involved in host-pathogen interactions and that influences B. burgdorferi virulence. Immunization studies with this protein revealed a possible effect of LCN2 in local immune response at the tick bite site, as we recorded a decrease in tick weight and a decrease in B. burgdorferi burden at the tick bite site. However, our in vitro protein-protein interaction studies, including Far Western Blot analysis and pull-down assays, showed no direct interaction between LCN2 and B. burgdorferi. Finally, using LCN2 knockout mice, we show that the deficiency of LCN2 also had negligible effect on B. burgdorferi burden. Taking together, our study suggests that host LCN2 may have an indirect or redundant effect on B. burgdorferi infection and the pathogenesis of Lyme disease. In addition to exploring novel host targets that could be involved in pathogen interactions, as detailed above, we also explored the identification of cytotoxic compounds as antimicrobials against B. burgdorferi. Our collaborators at the National Center for Advancing Translational Sciences (NCATS) performed a high-throughput drug screening assay using multiple libraries of diverse small molecules, including existing antibiotics and antimicrobials, which identified selected compounds that have a potent cytotoxic effect on B. burgdorferi. We further performed in vitro screening of these compounds and selected one topical antibiotic (designated as N-Abx-101), which displayed a robust antimicrobial effect against spirochetes. N-Abx-101 was selected from the library of antimicrobial compounds for its cytotoxic effects against B. burgdorferi and we determined that at higher concentrations, this antibiotic was cytotoxic to B. burgdorferi, in vitro. Currently, we lack a topical antibiotic that can be used against B. burgdorferi; therefore, we sought to explore whether N-Abx-101 could be used as prophylactic treatment or a preventive option against B. burgdorferi. Our initial in vivo studies show that N-Abx-101 treatment on mice can impair B. burgdorferi survival in the host, especially at the vector bite site. Further experiments were done with modified parameters, which showed a preventive use of N-Abx-101, as final dose applied on mice at least 5 hours before tick feeding, after 7 days of treatment, can result in complete eradication of B. burgdorferi in at least 50% of the challenged animals. Our next assessment of N-Abx-101 as a therapeutic agent, when used as a treatment within 10-14 hours after tick repletion twice daily for a total of 7 days, also results in a reduction in the antibody titer in the serum against B. burgdorferi, although we recorded the presence of spirochetes in the host. Therefore, while our initial studies suggest that N-Abx-101, a candidate topical antibiotic, could be developed as a new arsenal against B. burgdorferi, additional studies need to be performed to identify optimal conditions for more effective use of the antibiotic against Lyme disease.