Dawn C. Bisi

Defense Date

6-29-2009

Graduation Date

Fall 2009

Availability

Immediate Access

Submission Type

dissertation

Degree Name

PhD

Department

Biological Sciences

Committee Chair

David J. Lampe

Committee Member

Mary Alleman

Committee Member

Joseph McCormick

Committee Member

Marcelo Jacobs-Lorena

Keywords

malaria, plasmodium, protein secretion, Pantoea agglomerans, bacterial symbiont

Abstract

The disease malaria originates from the transmission of the parasite Plasmodium to humans by female anopheline mosquitoes. Estimates put the number of deaths at 1-3 million people annually and this number will increase without the establishment of new control strategies. There is currently no vaccine and the effectiveness of insecticides and drugs are thwarted by the gain of resistances for both the insect and parasite. An alternative genetic engineering approach to combating malaria is presented here. The bacterial mosquito symbiont Pantoea agglomerans, which resides in the mosquito gut, was chosen to express anti-Plasmodium effector gene products that are known to inhibit Plasmodium development. A caveat is finding an appropriate protein secretion signal for export of the effectors from the cell. A two-pronged approach to finding a secretion signal involved identifying and testing a native secreted protein signal in P. agglomerans, and also testing heterologous secretion signals shown to work previously in related species. Proteomic analysis of native secreted proteins in spent growth medium followed by the identification of the corresponding genes revealed the best native candidate for trials was the secreted protein FliC/Flagellin. Various constructs involving the fliC 5' UTR and the fliC ORF were used for secretion of a test protein (an anti-BSA scFv) in P. agglomerans and found to not be a sufficient secretion signal. The heterologous PelB (from Erwinia carotovora), and OmpA, TolB, and HlyA (all from E. coli) signals were also used in secretion trials with the anti-BSA scFv. The PelB and HlyA signals were shown to secrete the scFv in P. agglomerans, however it was only active in the case of PelB-induced secretion. In addition, four anti-Plasmodium effector proteins (SM1, Anti-Pbs21, PLA2, and CEL-III) were available for testing in constructs containing the heterologous secretion signals. Varying success was observed with the different combinations of signals and effector genes. The OmpA and TolB signals were not functional in P. agglomerans. P. agglomerans was able to secrete Anti-Pbs21-HlyA and PLA2 H67N-HlyA fusions and these strains are now available for testing inside malaria-infected anophelines for the inhibition of Plasmodium development.

Format

PDF

Language

English

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