Date of Completion

9-30-2016

Embargo Period

9-29-2016

Advisors

M. Kyle Hadden, Dennis Wright, Adam Zweifach, Jonathan Klassen, Andrew Wiemer

Field of Study

Pharmaceutical Science

Degree

Master of Science

Open Access

Open Access

Abstract

Natural products and natural product derivatives comprise some of the most successful drugs to date. Many natural products derive from the secondary metabolite profiles of high GC content actinomycete bacteria of the genus Streptomyces, which are sometimes associated with eukaryotic hosts. Identification of biosynthetic gene sequences for natural product secondary metabolites in Streptomyces can assist in isolation of the identified natural product. Moreover, the biosynthetic pathways of drug-like secondary metabolites derived from Streptomyces bacteria often provide insight into the available metabolites that can be produced. As a result, identification of bacteria capable of producing antibacterial products, including Streptomyces, can often lead to identification of biologically relevant secondary metabolites.

It has been hypothesized that symbiotic relationships between invertebrate hosts and bacteria have evolved to protect host organisms from pathogenic invasion. Many invertebrates contain only an innate immune system incapable of adaptation, suggesting one hypothesis that this primitive immunity may be supplemented through the presence of symbiotic bacteria. To identify immune system augmenting methods, recent research has identified new techniques that investigate adaptive immune system enhancement through protein kinase C (PKC) activation and calcium channel influx in addition to suppression.

The research described here investigated a novel strain of antibiotic-producing Streptomyces, isolated from the invasive tunicate Styela clava collected from the University of Connecticut at Avery Point (Groton, CT). During the investigation of the secondary metabolite production, tumor promoting and PKC activating metabolite teleocidin A-1 was identified via a novel assay screen. The assay screen was designed to investigate small molecules that can enhance lytic granule exocytosis from cytotoxic T-lymphocytes either by stimulating calcium influx or activation of PKC. Identification of teleocidin A-1 inspired further investigation as to whether the bacterial secondary metabolite is developed to influence specific pathways of the host tunicate immune system. This hypothesis was investigated through attempts to localize the bacteria producing teleocidin A-1 to specific areas within the tunicate, including the gut and outer tunic. It was hypothesized that bacterial localization in or around various organs would lead to influence by secondary metabolites produced by bacteria, including teleocidin A-1. Thus, localization of bacteria by the host may indicate an influence by secondary metabolites on various organs and organ systems. Whole genome sequencing was employed to identify biosynthetic gene clusters that might play a role in antibiotic natural product biosynthesis as well as biosynthesis of teleocidin. After sequencing the bacterial genome, DNA samples isolated from various tunicate tissues were examined for bacterial DNA involved in the biosynthesis of teleocidin and for the presence of other antibiotic-producing Streptomyces bacteria. Results from localization experiments indicate that bacteria associated with antibiotic and natural product production are localized within the tunicate body, but not within the outer tunic membrane.

Major Advisor

Marcy J. Balunas

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