Defense Date

7-24-2018

Graduation Date

Fall 12-21-2018

Availability

One-year Embargo

Submission Type

dissertation

Degree Name

PhD

Department

Biological Sciences

Committee Chair

David Lampe

Committee Member

Robert Shanks

Committee Member

Michael Jenson-Seaman

Committee Member

John Stolz

Keywords

Paratransgenesis, mosquito, microbiome, malaria, transcriptional regulation, Asaia

Abstract

The control of vector-borne diseases such as malaria has been an extremely important research subject for hundreds of years. Because of the complex lifecycles of the pathogens that cause these diseases, finding a comprehensive treatment or preventative strategy has proven extremely difficult. Malaria alone is responsible for almost half a million deaths annually, most of them children under 5 years old. This disease is caused by parasitic protists in the genus Plasmodium that are transmitted to humans from Anopheles sp. mosquitoes. Most preventative strategies that are in use today revolve around controlling the vectors, including bed nets, insecticides, and larval habitat removal. Transgenic mosquito lines are also being produced that inhibit the mosquito from transmitting the pathogen; however, progress in this area is hindered by the number of species that vector Plasmodium as well as the ability of the transgenes to spread through wild populations. The way in which this study overcomes these hurdles is by using a technique called paratransgenesis.

Paratransgenesis is the process of genetically engineering symbionts to affect host phenotype; in this study, the natural midgut symbiont Asaia sp. SF2.1 was engineered to secrete antimalarial effectors causing the mosquito to be unable to transmit malaria. However, constitutive expression of these effectors causes a fitness disadvantage to the transgenic Asaia. Therefore, it is desirable to express these molecules only when Plasmodium is present in the mosquito midgut, namely when a mosquito takes an infected blood-meal. The first step in this process was to optimize this wild-type bacterium for use in the laboratory to find putative promoters that were induced under blood meal conditions. Three techniques were employed to find conditional promoters using the plasmid pGLR1: a promoter trap library, screening the promoter regions of conditional Asaia homologs, and RNA-seq analysis under the varying conditions. The latter two methods produced four blood meal induced (BMI) promoters that were then cloned into an antimalarial expression plasmid pCG18.

These conditional strains were evaluated for fitness against a constitutive control using maximum growth rate, ability to compete against wild-type Asaia, and the ability to colonize the mosquito midgut. Overall, the conditional strains outperformed the constitutive control and were tested for Plasmodium inhibition. All conditional constructs show significantly reduced infection rates of the mosquito from the wild-type Asaia, with three performing significantly better than the constitutive control. The ability of these conditional strains to not only reduce disease burden but compete effectively with wild-type bacteria demonstrates the effectiveness of using a paratransgenic approach to control vector-borne disease. These promoters will be used in further testing to insert antimalarial constructs into the Asaia genome and eventually be tested in the field.

Language

English

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