An evaluation of fusion partner proteins for paratransgenesis in Asaia bogorensis

Abstract

Mosquitoes transmit many pathogens responsible for human diseases, such as malaria which is caused by parasites in the genus Plasmodium. Current strategies to control vector-transmitted diseases are increasingly undermined by mosquito and pathogen resistance, so additional methods of control are required. Paratransgenesis is a method whereby symbiotic bacteria are genetically modified to affect the mosquito's phenotype by engineering them to deliver effector molecules into the midgut to kill parasites. One paratransgenesis candidate is Asaia bogorensis, a Gram-negative bacterium colonizing the midgut, ovaries, and salivary glands of Anopheles sp. mosquitoes. Previously, engineered Asaia strains using native signals to drive the release of the antimicrobial peptide, scorpine, fused to alkaline phosphatase were successful in significantly suppressing the number of oocysts formed after a blood meal containing P. berghei. However, these strains saw high fitness costs associated with the production of the recombinant protein. Here, we report evaluation of five different partner proteins fused to scorpine that were evaluated for effects on the growth and fitness of the transgenic bacteria. Three of the new partner proteins resulted in significant levels of protein released from the Asaia bacterium while also significantly reducing the prevalence of mosquitoes infected with P. berghei. Two partners performed as well as the previously tested Asaia strain that used alkaline phosphatase in the fitness analyses, but neither exceeded it. 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.

Fig 1. Antiplasmodial vector pHyp4s.Myc.

This plasmid was created for fusion partner protein insertion by replacing the alkaline phosphatase region of Hyp4s (PhoA) [31] with a Myc tag and restriction enzyme recognition sites. The partner protein genes were inserted between the AvrII and AscI restriction sites for in frame fusion with the preceding Myc tag. pBBR, origin of replication; Kan^R, kanamycin resistance; P_nptII, promoter; hypothetical protein 4 signal, signal sequence for extracellular release of protein; scorpine, antiplasmodial effector protein; linker, (GGGGS)₃ flexible linker. Created with BioRender.com.

Fig 2. Antiplasmodial effector abundance in the supernatants from Asaia.

(A) A representative ELISA that utilized anti-Myc primary and anti-mouse-HRP secondary antibodies to detect the presence of the recombinant protein in the supernatant fraction of transgenic Asaia strains. SF2.1, wild-type Asaia. (B) Quantification of supernatant fractions of the ELISA analysis. Relative levels (expressed as absorbance values measured at 450 nm) of substrate cleaved by the HRP-conjugate anti-mouse antibody across five separate trials. Statistical significance was determined using one-way ANOVA with Dunnett’s correction where significance is represented by *P < 0.05, **P < 0.01, and ***P < 0.001 with experimental replicates. SF2.1, wild-type Asaia. (C) Representative western blot of supernatants from antiplasmodial Asaia strains. SF2.1, wild-type Asaia; +, pIT2-scFv Myc positive control; MBP*, MBP diluted to a final OD₆₀₀ of 10.

Fig 3. Fitness assessments of antiplasmodial Asaia strains.

(A) Cell viability of Asaia strains on solid media. The Myc and MBP strains had the highest significant percentage of dead cells while SF2.1, TrxA and GST had the lowest percentage. (B) Growth curves and maximum growth rates (μmax) were calculated from individual isolates of each strain. Only MBP showed a significantly lower μmax than the PhoA strain. (C) Each strain was competed in mannitol media with wild-type Asaia SF2.1. Ratios of transgenic vs. whole culture CFUs are displayed. A 50:50 ratio indicates no loss of the paratransgenic strain during the course of the experiment. (D) Relative colonization of mosquito midguts by antiplasmodial Asaia strains. The His strain had the highest rate of colonization, followed by the GST strain; the PhoA strain had the lowest rate of colonization. Statistical significance for each experiment was determined using one-way ANOVA with Dunnett’s correction where significance is represented by *P < 0.05, **P < 0.01, and ***P < 0.001 with experimental replicates (A. n ≤ 18, B. n ≤ 10, C. n = 3). Only significant comparisons are shown.

Fig 4. Suppression of P. berghei development by paratransgenic Asaia strains.

In four separate trials, oocysts were counted in mosquitoes infected with Asaia strains that were fed on a P. berghei infected mouse. Each dot represents an individual midgut and the number of P. berghei oocysts it contained. Prevalence is the fraction of midguts with at least one oocyst. SF2.1 is the wild type Asaia strain (negative control), Hyp4 is the Asaia strain releasing only alkaline phosphatase (negative control), and PhoA is a previously developed scorpine antiplasmodial strain with alkaline phosphatase as the fusion partner protein (positive control). All antiplasmodial strains significantly reduced the median number of oocysts (horizontal bars) compared to the wild-type strain (quantile regression, P < 0.01), while only the Myc and MBP antiplasmodial strains had a significantly higher median number of oocysts compared to the PhoA strain (quantile regression, P < 0.02161). The prevalence of infection was also significantly different between the wild-type and all antiplasmodial strains except for the Myc strain (χ², 1 df). Only the Myc and His antiplasmodial strains had a significantly higher prevalence of infection compared to the PhoA strain (χ², 1 df). There was no significant difference between the wild-type and Hyp4 negative controls. P-values: *P < 0.05, **P < 0.01, ***P < 0.001.