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The insect-vectored disease malaria is a major world health problem. New control strategies are needed to supplement the current use of insecticides and medications. A genetic approach can be used to inhibit development of malaria parasites (Plasmodium spp.) in the mosquito host. We hypothesized that Pantoea agglomerans, a bacterial symbiont of Anopheles mosquitoes, could be engineered to express and secrete anti-Plasmodium effector proteins, a strategy termed paratransgenesis. To this end, plasmids that include the pelB or hlyA secretion signals from the genes of related species (pectate lyase from Erwinia carotovora and hemolysin A from Escherichia coli, respectively) were created and tested for their efficacy in secreting known anti-Plasmodium effector proteins (SM1, anti-Pbs21, and PLA2) in P. agglomerans and E. coli. P. agglomerans successfully secreted HlyA fusions of anti-Pbs21 and PLA2, and these strains are under evaluation for anti-Plasmodium activity in infected mosquitoes. Varied expression and/or secretion of the effector proteins was observed, suggesting that the individual characteristics of a particular effector may require empirical testing of several secretion signals. Importantly, those strains that secreted efficiently grew as well as wild-type strains under laboratory conditions and, thus, may be expected to be competitive with the native microbiota in the environment of the mosquito midgut.

Secretion of Anti-Plasmodium Effector Proteins from a Natural Pantoea agglomerans Isolate by Using PelB and HlyA Secretion Signals