Graduation Date

Fall 2017

Document Type



Master of Science degree with a major in Biology

Committee Chair Name

Jianmin Zhong

Committee Chair Affiliation

HSU Faculty or Staff

Second Committee Member Name

David S. Baston

Second Committee Member Affiliation

HSU Faculty or Staff

Third Committee Member Name

Jenny Cappuccio

Third Committee Member Affiliation

HSU Faculty or Staff

Fourth Committee Member Name

Jacob Varkey

Fourth Committee Member Affiliation

HSU Faculty or Staff

Subject Categories



Ticks are the most important vector of many infectious diseases in the United States. Understanding the nature of the relationship between Rickettsia endosymbiont Ixodes pacificus (REIP) and Exudes pacificus will help develop strategies for the control of tick-borne diseases, such as Lyme disease, and Rocky Mountain spotted fever. Folate, also known as vitamin B9, is a necessary vitamin for tick survival, and plays a central role in one-carbon metabolism in cells. Folate exist as a large family of structurally related forms that transfer one-carbon groups among biomolecules that are important to cell growth, differentiation, and survival. In Dr. Zheng’s lab, REIP were cultured in Ixodes scapularis embryonic tick cell line ISE6. Previous research has shown that REIP in Ixodes pacificus carries all five de novo folate biosynthesis genes. Folate biosynthesis mRNAs were detected and all recombinant rickettsial folate proteins were overexpressed. To determine whether REIP synthesize folate, we sought to measure the folate concentration in REIP using HPLC-UV quantification with a Diamond HydrideTM liquid chromatography column. 5-methyltetrahydrofolate (5-MTHF), the active circulating form of folate in bacteria was detected. The averaged folate level in the REIP-infected ISE6 pellet is 0.2514 mg/L/106 cells, which is 5-fold higher than the pellet sample from uninfected ISE6 pellet sample. For the REIP-infected ISE6 supernatant sample, the measured concentration is 0.0029 mg/L/106 cells. Statistical analysis using t-test determined there is a statistical significance (p=0.05) in the folate concentrations between REIP-ISE6 pellets and ISE6 pellets; REIP-ISE6 pellet and REIP-ISE6 supernatant. However, more analysis is required to confirm the identity of the 5-MTHF peak before we draw the conclusion that REIP can synthesize folate. Additionally, we sought to knockout the folA gene in REIP to confirm folate is synthesized by REIP. We used the TargeTron system to construct the knockout, however, no successful mutant Rickettsia were obtained so far. Furthermore, because REIP classification was not determined yet, we sought to construct a phylogenetic tree of the species. Surprisingly, we identified that REIP was a new subspecies with the following classification Rickettsia monacensis subsp. pacifica subsp nov. To our knowledge, this is the first report of the present of R. monacensis in I. pacificus in North America.

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