Defense Date

4-13-2022

Graduation Date

Summer 8-13-2022

Availability

Immediate Access

Submission Type

dissertation

Degree Name

PhD

Department

Biological Sciences

School

Bayer School of Natural and Environmental Sciences

Committee Chair

David Lampe

Committee Member

Jana Patton-Vogt

Committee Member

Kyle Selcer

Committee Member

Robert Shanks

Keywords

Paratransgenesis, mosquito, microbiome, malaria, protein export, Asaia

Abstract

Mosquitoes transmit many pathogens that cause human disease. One such disease, malaria, is caused by parasites in the genus Plasmodium, infecting over 200 million people and killing over 600,000 per year. Current strategies to control vector-transmitted diseases are increasingly undermined by mosquito and pathogen resistance. Research has turned to additional and novel methods of control, such as altering the microbiota of the vectors. In this method, called paratransgenesis, symbiotic bacteria are genetically modified to affect the mosquito’s phenotype by engineering them to deliver antiplasmodial molecules into the midgut to kill parasites. These molecules must be released by the bacteria into the midgut of the mosquito host to effectively interfere with parasite development. This research focuses on one paratransgenesis candidate, Asaia bogorensis, a bacterium colonizing target tissues of Anopheles sp. mosquitoes. Since common signal sequences do not function in Asaia, the first step was to evaluate native Asaia N-terminal signal sequences predicted from bioinformatics for their ability to mediate increased levels of effector molecules outside the cell. Six signals resulted in significant levels of the reporter protein alkaline phosphatase released from the Asaia bacterium. Three signals were successfully used to drive the release of the antimicrobial peptide, scorpine, and two of these strains effectively interfered with the development of Plasmodium within the mosquito midgut.

When these strains were assessed for their maximum growth rate and their ability to colonize the mosquito midgut, we saw high fitness costs associated with the production of the recombinant protein, which suggests that simply increasing the amount of effector molecules in the midgut is insufficient to create superior paratransgenic bacterial strains. Symbiont fitness must be considered as well since these transgenic strains must be able to compete with the natural microorganisms found within the mosquito host. Five different partner proteins were fused to scorpine and evaluated for effects on the fitness of the transgenic bacteria’s maximum growth rate, ability to compete against wild-type Asaia, and the ability to colonize the mosquito midgut. Overall, three of the new partner proteins resulted in significant levels of protein released from the Asaia bacterium while also reducing the prevalence of mosquitoes infected with P. berghei, two of which performed as well as the previously tested Asaia strain that used the alkaline phosphatase partner in the fitness analyses. It may be that there is a maximum level of fitness and parasite inhibition that can be achieved with scorpine being driven constitutively, and that use of a Plasmodium specific effector molecule in place of scorpine would help to mitigate the stress on the symbionts. This research provides more groundwork for the future use of paratransgenic Asaia strains to combat malaria in the wild. While further modifications will be required to make this bacterium ready for field use, the ability of these transgenic strains to reduce the Plasmodium parasite within the mosquito host demonstrates the effectiveness of using paratransgenesis to control vector-borne disease.

Language

English

Additional Citations

Grogan C, Bennett M, Moore S, and Lampe D (2021). Novel Asaia bogorensis Signal Sequences for Plasmodium Inhibition in Anopheles stephensi. Front. Microbiol. 12:633667. doi: 10.3389/fmicb.2021.633667.

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