Anna Chen, Colby CollegeFollow

Date of Award

2023

Document Type

Honors Thesis (Open Access)

Department

Colby College. Biology Dept.

Advisor(s)

Suegene Noh

Second Advisor

Lynn Hannum

Third Advisor

Ron Peck

Abstract

Paraburkholderia are amoeba resistant, gram negative bacteria that form facultative symbiotic relationships with D. discoideum, a soil dwelling amoeba host. Three species of Paraburkholderia, P. agricolaris, P. hayleyella, and P. bonniea are able to persistently infect nonnative, or symbiont free, D. discoideum. These three species share a type III secretion system (T3SS) and type VI secretion system (T6SS) that is absent in other close relatives We hypothesized that the ability to persistently infect D. discoideum may be partially attributed to the T3SS and T6SS shared across the three species of Paraburkholderia.

The goal was to test the phenotypic effect of a P. bonniea tssH ATPase gene knockout within the shared T6SS complex. We hypothesized that the ability of the mutant ∆tssH to infect and influence host fitness compared to the wild type would be significantly reduced. To compare the wildtype versus mutant variants of P. bonniea, we performed two assays. In each, D. discoideum hosts were paired with wildtype P. bonniea bb859 and bb433 strains, along with their respective mutant strains. These assays assessed differences between mutant and wildtype strains in changes in host fitness as infection prevalence increases and how host fitness was affected. We also generated and analyzed RNA sequencing data to understand the molecular impact of T6SS perturbation in this amoeba-bacteria symbiosis.

Although some phenotypic differences were observed between ∆tssH variants with the tssH-ATPase gene knockout within the T6SS and wildtype P. bonniea infected D. discoideum hosts, their impact on how host fitness changed as infection prevalence increased was not different between the ∆tssH mutant and wildtype. This observation was also reflected in our RNA-sequencing data, where there were no statistically-significant differentially expressed genes between them. However, significant differences were observed between the variants during horizontal transmission, where infection was spread from host to host. Thus, the T6SS may be closely involved with virulence after the bacteria has entered the cell, rather than immediately upon encountering the host.

Keywords

symbiosis, type 6 secretion system, horizontal transmission, host fitness, RNA-sequencing, ATPase

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