Masters Thesis

Characterization of de novo folate biosynthesis of rickettsia endosymbiont Ixodes pacificus in Ixodes scapularis ISE6 tick cell line

Bacterial endosymbionts often contribute to the nutritional fitness of their host, as seen with Buchnera endosymbionts in aphids and Wolbachia endosymbionts in Cimex lectularius. Conventionally, amino acids and vitamins provided by bacterial endosymbionts maintain host survival and fitness. Previous research in our lab has detected Rickettsia endosymbiont in Ixodes pacificus that is 100% transovarially and transstadially transmitted. MUMmer genome alignments of whole genome sequence of I. scapularis (the closest relative to I. pacificus) and metabolic pathway reconstructions indicated that the Rickettsia endosymbiont has the genetic capacity to produce folate de novo. In this study, rickettsial isolate was obtained from ovaries of engorged I. pacificus. The isolate was named Rickettsia endosymbiont I. pacificus (REIP) and was cultured in vitro using IRE11 and ISE6 tick embryonic cell lines. Furthermore, REIP isolate was characterized by the transmission electron microscopy and the gene sequencing. Multilocus sequence typing (MLST) of PCR-products targeting rickettsial 16S rRNA, gltA, and ompA genes revealed 99% identity of 16S rRNA between the REIP isolate and other Rickettsia species. The gltA sequences revealed 99% identity between REIP isolate and Rickettsia monacensis. The ompA sequences revealed 99% identity between REIP isolate and R. buchneri. Also, multi-spacer typing (MST) of PCR-products targeting rickettsial dksA-xerC, mppA-purC and rpmE-tRNAfMet intergenic spacers result in one genotype, and 99% identity between REIP isolate and R. monacensis. Phylogenetic analysis of the six genes demonstrated that the REIP isolate fulfills the criteria to be classified as representative of a novel Rickettsia species closely related to ‘Rickettsia monacensis’ and ‘Rickettsia buchneri’. We named REIP species as ‘Rickettsia humboldti’ strain IPO1 (Ixodes pacificus ovary tick1). Furthermore, RT-qPCR assay detected the expression of genes in the folate biosynthesis pathway (folA, folE, and folKP) in REIP- infected ISE6 cells. In changing the growth temperature of REIP-infected ISE6 cells, which simulated the ticks life cycle, revealed that folate genes are not differentially expressed (P≥0.05) between 25°C and 36°C. This study provided a piece of evidence about the nutritional interactions between R. humboldti and I. pacificus ticks.