Investigating putative antimicrobial product biosynthesis in a tick endosymbiont. - Project Summary The blacklegged tick lxodes scapularis is the major vector of tick-borne disease agents in North America, transmitting seven known human pathogens. However, despite its capability as a vector and its geographical overlap with other ticks that host pathogenic spotted fever group rickettsiae (SFGR), /. scapularis transmits no disease-causing Rickettsia species, and instead is widely infected by the endosymbiont Rickettsia tamurae subspecies buchneri (Hardt et al. 2020; hereafter R. buchnen). Presence of this symbiont has been proposed as the primary reason that/. scapularis is rarely infected by nor transmits pathogenic members of the SFGR. Intriguingly, the R. buchneri genome encodes putative antibiotic synthesis operons and a toxin/antidote module, which may contribute to the symbiont's role in preventing rickettsial superinfection of its tick host. However, beyond bioinformatic analyses, no investigation of the antibiotic activity of the products of these genes has been attempted. In tick cell culture the presence of R. buchneri has been shown to prevent infection with the human pathogen Rickettsia parkeri, and reduce infection by Anaplasma phagocytophilum and Rickettsia monacensis. Besides the effect of the encoded genes for antibacterial antagonism, we also hypothesize that the endosymbiont plays a role in modulating the immune response of its tick host to further reduce infection by tick-borne pathogens. Both these mechanisms driven by R. buchneri may play important roles in determining the tick's vector competence for human-pathogenic bacteria and shaping tick borne disease epidemiology. Furthermore, identification of antimicrobial products from R. buchneri may result in important leads for new drug development for the treatment of human infections. To improve understanding of tick-symbiont interactions, research will focus on the following Aims: Aim 1: Investigate regulation of the activity of antibiotic synthesis gene clusters and toxin/antidote genes in R. buchneri under conditions of challenge with tick-borne pathogens in vitro, and assessment of tick cell responses. Using qRT-PCR, the expression of R. buchneri's putative antibacterial antagonism genes will be analyzed in tick cells when the symbiont is present only by itself or when tick cells are additionally challenged with pathogenic R. parkeri. The expression of genes from multiple immune pathways of the tick host will also be examined through RNAseq and single-cell genomics. Aim 2. Discover the antibiotic natural products encoded in Rb genome. Homologous and heterologous expression strategies will be employed to identify and produce the target natural products for characterization. This research will clarify to what extent R. buchneri's antibacterial machinery is employed during tick infection, whether the symbiont is involved in shaping /. scapularis immunity, and will seek to identify and isolate the natural products synthesized by the symbiont.
