Publication Date

5-2013

Type of Culminating Activity

Thesis

Degree Title

Master of Science in Chemistry

Department

Chemistry

Major Advisor

Ken Cornell, Ph.D.

Abstract

The bacterial enzyme Methylthioadenosine/S-adenosylhomocysteine (MTA/SAH) nucleosidase (MTN) is involved in methionine and adenine salvage from the by-products of S-adenosylmethionine (SAM, AdoMet) dependent reactions. MTN plays a critical role in alleviating product inhibition of SAM dependent reactions, including methylation reactions and the synthesis of polyamines, vitamins, and autinducer signals. Due to its absence from humans and its importance to bacterial metabolism, MTN is a potential target for the development of novel antibiotics to treat microbial infections. In this study, a MTN gene knock-out (MTN KO) strain of the pathogen E. coli O157:H7 was created to study the impact of MTN activity on bacterial growth, virulence, and autoinducer-dependent events; and to model the effects that would be expected from complete pharmacologic interruption of enzyme activity. E. coli O157:H7 was chosen as the topic of study since it is a serious gram-negative pathogen responsible for severe diarrhea that can progress to cause hemolytic uremic syndrome (HUS), and factors influencing its virulence are well known.

The MTN KO strain showed delayed growth, minimal biofilm production, and decreased in vitro virulence when compared to the parental wild type (WT) strain. Notably, the MTN KO strain showed a reduced ability to adhere to cultured mammalian cells. An analysis of virulence factor expression showed that the MTN KO strain secreted less Shiga-toxin, type-III secretory proteins, and hemolysin activity than the parental WT strain. Culture supplementation with autoinducer-2 precursor, 4,5-dihydroxy-2,3-pentanedione (DPD) did not restore the growth or virulence of the MTN KO strain, suggesting that the virulence defect was not the result of a loss of autoinducer-2 signaling. However, culture supplementation with lipoate, thiamine and biotin partially reconstituted the growth and virulence phenotypes of the KO strain to WT levels, indicating that altered vitamin-dependent metabolic activity played a role in the defect. The lipoate- and thiamine-dependent enzymes in the pyruvate dehydrogenase complex were overexpressed in the MTN KO strain, but the enzyme complex showed lower specific activity than the WT strain, suggesting that the ability to synthesize these vitamins was compromised in the KO strain. Other metabolic enzymes (lactate dehydrogenase, alcohol dehydrogenase, glutamate dehydrogenase) were found to have specific activities equal to the WT strain, thus providing release points for excess metabolites in the KO strain.

This study provides support for MTN as a target for antibiotic treatment. Our results indicate that one mechanism by which MTN specific inhibitors could exert their antibiotic effect is by interrupting vitamin dependent processes, particularly in central carbon metabolism. While loss of MTN activity was not bactericidal in this case, the significant reduction in bacterial growth, biofilm formation and virulence suggests that the bacteria treated with MTN inhibitors could have increased susceptibility to traditional antibiotics and the host immune system responses. This supports the incorporation of MTN inhibitors into combination drug therapies with standard antibiotics. Finally, the creation and analysis of a MTN KO strain provides a valuable tool to explore potential mechanisms of antibiotic action that can be used in comparative studies to examine the antimicrobial activities of future MTN inhibitors.

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