Degree Type

Dissertation

Date of Award

2013

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

Nicola Pohl

Second Advisor

Richard Zuerner

Abstract

ABSTRACT

Spirochaete is a diverse phylum of Gram-negative bacteria that is located under the order, Spirochaetales, which is divided into three families: Spirochaetacea, Brachyspiraceae, and Leptospiraeae. Research was focused on the latter, specifically, the genus, Leptospira. Leptospira consists of many saprophytic, intermediate pathogenic, and pathogenic species, with L. interrogans (transmitted via contaminated water) and L. borpetersenii (acquired via host to host transmission) causing the majority of leptospirosis cases. To gain insight into the pathogenesis of Leptospira, a putative thiopurine S-methyltransferase (LBJ0800) was exploited. Because this gene is not present in any of the sequenced spirochetes, Brachyspira, Borrelia, Treponema, except Leptospira, and even then, only in L. borgpetersenii, work was concentrated on this novel gene.

The open reading frame predicted to encode the putative thiopurine methyl S-methyltransferase (645 bp) was PCR amplified and directionally cloned into pET101 with a polyhistidine tag at the C terminus to provide plasmid, LBJ0800/pET101. To confirm the presence of a functional recombinant thiopurine S-methyltransferase, the expression construct was introduced into E. coli BL21 Star (DE3) cells, which were then grown in LB broth for IPTG-initiated induction of the protein. Cells were harvested by centrifugation, resuspended in Lysis Buffer and purified by a Ni-NTA metal-affinity column. Western blot analysis confirmed the purified, recombinant protein with a molecular mass of approximately 28 kDa.

In the second chapter, in vivo characterization of the putative thiopurine S-methyltransferase was analyzed. It was determined by ELISA experiments and immunohistochemistry studies that although LBJ0800 is an unique protein, it was not expressed during infection at detectable levels. In chapter 3, research efforts focused on characterizing the gene enzymatically. Demonstrating substrate specificity, LBJ0800 methylated 6-thioguanine, validating its putative function as a thiopurine S-methyltransferase.

DOI

https://doi.org/10.31274/etd-180810-3219

Copyright Owner

Joy Marie Jackson

Language

en

File Format

application/pdf

File Size

86 pages

Included in

Microbiology Commons

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