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.

DYSREGULATION OF MEOX2 FOLLOWING WT1 MUTATION IN KIDNEY DEVELOPMENT AND WILMS TUMORIGENESIS

Wilms tumor (WT) is a childhood tumor of the kidney and a productive model for understanding the role of genetic alteration and interactions in tumorigenesis. The Wilms tumor gene 1 (WT1) is a transcriptional factor and one of the few genes known to have genetic alterations in WT and has been shown be inactivated in 20% of WTs. However, the mechanisms of how WT1 mutations lead to Wilms tumorigenesis and its influence on downstream genes are unknown. Since it has been established that WT1 is a transcriptional regulator, it has been hypothesized that the loss of WT1 leads to the dysregulation of downstream genes, in turn result in the formation of WTs. To identify the dysregulated downstream genes following WT1 mutations, an Affymetrix GeneChip Human Genome Array was previously conducted to assess the differentially expressed genes in the WT1-wildtype human and WT1-mutant human WTs. Approximately 700 genes were identified as being significantly dysregulated. These genes were further prioritized based on their statistical significance, fold change, chromosomal region, spatial pattern of gene expression and known or putative cellular functions. Mesenchyme homeobox 2 (MEOX2) was one of the most significantly upregulated genes in WT1-mutant WT. MEOX2 is known to play a role in cell proliferation, apoptosis, and differentiation. In addition to its biological roles, it is expressed during early kidney development in the condensed mesenchyme similar to WT1. Furthermore, the use of the Match® web-based tool from the BIOBASE Biological Data base identified a significant predicted WT1 binding site within the first intron of MEOX2. The similarity in spatial gene expression in the developing kidney and the significant predicted WT1 binding site found in the first intron of MEOX2 lead to the development of my hypothesis that MEOX2 is upregulated via a WT1-dependent manner. Here as a part of my master’s work, I have validated the Affymetrix GeneChip Human Genome Array data using an independent set of Wilms tumors. MEOX2 remained upregulated in the mutant WT1 Wilms tumor by 41-fold. Wt1 and Meox2 gene expression were assessed in murine newborn kidney; both Wt1 and Meox2 were expressed in the condensed, undifferentiated metanephric mesenchyme. I have shown that the in vivo ablation of Wt1 during embryonic development at embryonic day (E) 13.5 resulted in the slight increase of Meox2 gene expression by two fold. In order to functionally demonstrate the effect of the loss of Wt1 on Meox2 gene expression in undifferentiated metanephric mesenchyme, I have generated a kidney mesenchymal cell line to genetically ablate Wt1 in vitro by adenoviral infection. The ablation of Wt1 in the kidney mesenchymal cell line resulted in the upregulation of Meox2 by 61-fold. Moreover, the upregulation of Meox2 resulted in the significant induction of p21 and Itgb5. In addition to the dysregulation of these genes the ablation of Wt1 in the kidney mesenchymal cells resulted in decrease in cell growth and loss of cellular adherence. However, it is uncertain whether the upregulation of Meox2 caused this particular cellular phenotype. Overall, I have demonstrated that the upregulation of Meox2 is Wt1-dependent during early kidney development.