A METAGENOMIC STUDY OF THE TICK MIDGUT

A Metagenomic Study of the Tick Midgut Daniel Yuan, B.S. Supervisory Professor : Steven J. Norris, Ph.D. Southern tick–associated rash illness (STARI) or Master’s disease is a Lyme-like illness that occurs following bites by Amblyomma americanum, the lone-star tick. Clinical symptoms include a bull’s eye rash similar to the erythema migrans lesions of Lyme disease, as well as fever and joint pains. Lyme disease is caused by Borrelia burgdorferi and related spirochetes. However, B. burgdorferi has not been detected in STARI patients, or in ticks in the South Central U.S. The causative agent of STARI has not been identified, although it was once thought to be caused by another Borrelia species, Borrelia lonestari. Furthermore, while adult A. americanum have up to a 5.6% Borrelia lonestari infection rate, the prevalence of all Borrelia species in Texas ticks as a whole is not known. Previous studies indicate that 6%-30% of Northern Ixodes scapularis ticks are infected by Borrelia burgdorferi while only 10% of Northern A. americanum and I. scapularis ticks are infected by Borrelia species. The first specific aim of this project was to determine the bacterial community that inhabits the midgut of Texas and Northeastern ticks by using high throughput metagenomic sequencing to sequence bacterial 16S rDNA. Through the use of massively parallel 454 sequencing, we were able to individually sequence hundreds of thousands of 16S rDNA regions of the bacterial flora from 133 ticks from the New York, Missouri and Texas. The presence of previously confirmed endosymbionts, specifically the Rickettsia spp. and Coxiella spp., that are commonly found in ticks were confirmed, as well as some highly prevalent genera that were previously undocumented. Furthermore, multiple pathogenic genera sequences were often found in the same tick, suggesting the possibility of co-infection of multiple pathogenic species. The second specific aim was to use Borrelia specific primers to screen 344 individual ticks from Missouri, Texas and the Northeast to determine the prevalence of Borrelia species in ticks. To screen for Borrelia species, two housekeeping genes, uvrA and recG, were selected as well as the 16S-23S rDNA intergenic spacer. Ticks from Missouri, Texas and New York were screened. None of the Missouri or Texas ticks tested positive for Borrelia spp. The rate of I. scapularis infection by B.burgdorferi is dependent on tick feeding activity as well as reservoir availability. B. burgdorferi is endemic in the Northeast, sometimes reported as highly present in over 50% of all I. scapularis ticks. 11.6% of all New York ticks were positive for a species of Borrelia, however only 6.9% of all New York ticks were positive for B. burgdorferi. Despite being significantly lower than 50%, the results still fall in line with previous reports of about the prevalence of B. burgdorferi. 1.5% of all Texas ticks were positive for a Borrelia species, specifically B. lonestari. While this study was unable to identify the causative agent for STARI, 454 sequencing was able to provide a tremendous insight into the bacterial flora and possible pathogenic species of both the I. scapularis and the A. americanum tick.

Transcriptional regulation of the Borrelia burgdorferi antigenically variable VlsE surface protein.

The Lyme disease agent Borrelia burgdorferi can persistently infect humans and other animals despite host active immune responses. This is facilitated, in part, by the vls locus, a complex system consisting of the vlsE expression site and an adjacent set of 11 to 15 silent vls cassettes. Segments of nonexpressed cassettes recombine with the vlsE region during infection of mammalian hosts, resulting in combinatorial antigenic variation of the VlsE outer surface protein. We now demonstrate that synthesis of VlsE is regulated during the natural mammal-tick infectious cycle, being activated in mammals but repressed during tick colonization. Examination of cultured B. burgdorferi cells indicated that the spirochete controls vlsE transcription levels in response to environmental cues. Analysis of PvlsE::gfp fusions in B. burgdorferi indicated that VlsE production is controlled at the level of transcriptional initiation, and regions of 5' DNA involved in the regulation were identified. Electrophoretic mobility shift assays detected qualitative and quantitative changes in patterns of protein-DNA complexes formed between the vlsE promoter and cytoplasmic proteins, suggesting the involvement of DNA-binding proteins in the regulation of vlsE, with at least one protein acting as a transcriptional activator.