Table 1.
Strains and Plasmids used in this study.
Fig 1.
pNB90 backbone for the genomic library screen.
Random size-selected genomic fragments were cloned 5’ to ‘phoA to create a library that could be screened to detect sequences capable of mediating secretion of PhoA. pBBR1 ori = origin of replication; nptII = neomycin phosphotransferase II conferring kanamycin resistance; ‘phoA = E. coli alkaline phosphatase gene with no native signal sequence.
Table 2.
Proteins identified from a genetic screen for secreted Asaia proteins.
Fig 2.
PhoA-Asaia protein fusion plasmids expressed in Asaia.
PhoA = alkaline phosphatase with no signal sequence. The other lanes have Asaia sequences fused to ‘phoA. Tsr = TonB-dependent siderophore receptor. Aap = amino acid permease. Tdr = TonB-dependent receptor plug. Gdh = glucose dehydrogenase. Ybp = YVTN beta-propeller repeat protein. Csy = cellulose synthase. Amu = aminomutase. Ptp = peptide transport permease. The top ELISA used an anti-PhoA-HRP antibody to detect the presence of the alkaline phosphatase protein. The bottom plate assay used a PNPP substrate, which turns yellow when cleaved by active PhoA.
Fig 3.
Secreted protein relative abundance and activity from PhoA-Asaia protein fusion plasmids expressed in Asaia.
PhoA = alkaline phosphatase. Tsr = TonB-dependent siderophore receptor. Aap = amino acid permease. Tdr = TonB-dependent receptor plug. Gdh = glucose dehydrogenase. Ybp = YVTN beta-propeller repeat protein. Csy = cellulose synthase. Amu = aminomutase. Ptp = peptide transport permease. Both the ELISA and PNPP plates were repeated five times, and quantified using a plate reader. Signal intensity was read at 450 nm for the ELISA and at 400 nm for the PNPP assay. Although there was a similar amount of protein secreted by Tsr compared to Ybp, PhoA activity was more than five times as strong when secreted by Tsr. Each plate assay was performed 5 times and the means compared by an unpaired t-test using GraphPad Prism software, version 5.0.
Fig 4.
Asaia antiplasmodial expression plasmid.
PnptII = constitutive promoter from nptII; RBS = ribosome binding site; signal = siderophore receptor or YVTN sequence from genomic clones; effector = antiplasmodial effector gene; ‘phoA = phoA without the native signal sequence.
Fig 5.
Western blot of Asaia culture supernatants detecting proteins secreted using the siderophore receptor fusion protein.
Proteins were detected using a rabbit polyclonal anti-PhoA primary antibody and goat anti-rabbit HRP secondary. In each lane, red boxes highlight the predicted protein size of the full fusion proteins (higher molecular weight) and the predicted size of the effector molecules plus PhoA lacking the siderophore receptor protein (lower molecular weight).
Fig 6.
Disruption of P. berghei development by anti-Pbs21 scFv-Shiva1 immunotoxin and scorpine constructs.
Three-day old A. stephensi mosquitoes were fed paratransgenic strains of Asaia expressing either a fusion constuct combining the siderophore receptor protein and PhoA (= control), or a fusion construct combining the siderophore receptor protein, an effector protein, and PhoA. These mosquitoes were then fed on an infective mouse and the parasite was allowed to develop for 14 days. The mosquitoes were then dissected, and oocysts on the midgut were counted for each individual. Each dot on the chart represents a single midgut count. The median number of oocysts for each data set is marked with a horizontal line. Inhibition = inhibition of oocyst formation relative to the control; Mean = mean oocyst number per midgut; Median = median oocyst number per midgut; N = number of mosquitoes analyzed; Prevalence = percentage of mosquitoes carrying at least one oocyst; Range = range of oocyst numbers per midgut; Tbp = transmission-blocking potential: 100 − {(prevalence of mosquitoes fed with recombinant P. agglomerans)/[prevalence of control mosquitoes] × 100}. The scorpine construct produced a significant inhibition of 80.1% calculated by the median oocyst number.