Novel Asaia bogorensis signal sequences for Plasmodium inhibition in Anopheles stephensi

Mosquitoes vector many pathogens that cause human disease, such as malaria
that is caused by parasites in the genus Plasmodium. Current strategies to
control vector-transmitted diseases are hindered by mosquito and pathogen
resistance, so research has turned to altering the microbiota of the
vectors. In this strategy, called paratransgenesis, symbiotic bacteria are
genetically modified to affect the mosquito’s phenotype by engineering
them to deliver antiplasmodial effector molecules into the midgut to kill
parasites. One paratransgenesis candidate is Asaia bogorensis, a
Gram-negative, rod-shaped bacterium colonizing the midgut, ovaries, and
salivary glands of Anopheles sp. mosquitoes. However, common secretion
signals from E. coli and closely-related species do not function in Asaia.
Here, we report evaluation of 20 native Asaia N-terminal signal sequences
predicted from bioinformatics for their ability to mediate increased
levels of antiplasmodial effector molecules directed to the periplasm and
ultimately outside the cell. We tested the hypothesis that by increasing
the amount of antiplasmodials released from the cell we would also
increase parasite killing power. We scanned the Asaia bogorensis SF2.1
genome to identify signal sequences from extra-cytoplasmic proteins and
fused these to the reporter protein alkaline phosphatase. Six signals
resulted in significant levels of protein released from the Asaia
bacterium. Three signals were successfully used to drive the release of
the antimicrobial peptide, scorpine.  Further testing in
mosquitoes demonstrated that these three Asaia strains were able to
suppress the number of oocysts formed after a blood meal containing P.
berghei to a significantly greater degree than wild-type Asaia, although
prevalence was not decreased beyond levels obtained with a previously
isolated siderophore receptor signal sequence. We interpret these results
to indicate that there is a maximum level of suppression that can be
achieved when the effectors are constitutively driven due to stress on the
symbionts. This suggests that simply increasing the amount of
antiplasmodial effector molecules in the midgut is insufficient to create
superior paratransgenic bacterial strains and that symbiont fitness must
be considered as well.