Graduation Date
Fall 2017
Document Type
Thesis
Program
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
Keywords
Rickettsia, HPLC, Folate, Gene knockout
Subject Categories
Biology
Abstract
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.
Citation Style
APA
Recommended Citation
Chen, Junyan, "HPLC-UV Quantitation of folate synthesized by Rickettsia endosymbiot ixodes pacificus (REIP)" (2017). Cal Poly Humboldt theses and projects. 94.
https://digitalcommons.humboldt.edu/etd/94