Blood meal-induced inhibition of vector-borne disease by transgenic microbiota

Abstract

Vector-borne diseases are a substantial portion of the global disease burden; one of the deadliest of these is malaria. Vector control strategies have been hindered by mosquito and pathogen resistances, and population alteration approaches using transgenic mosquitos still have many hurdles to overcome before they can be implemented in the field. Here we report a paratransgenic control strategy in which the microbiota of Anopheles stephensi was engineered to produce an antiplasmodial effector causing the mosquito to become refractory to Plasmodium berghei. The midgut symbiont Asaia was used to conditionally express the antiplasmodial protein scorpine only when a blood meal was present. These blood meal inducible Asaia strains significantly inhibit pathogen infection, and display improved fitness compared to strains that constitutively express the antiplasmodial effector. This strategy may allow the antiplasmodial bacterial strains to survive and be transmitted through mosquito populations, creating an easily implemented and enduring vector control strategy.

Fig. 1

Putative BMI promoters significantly increase fluorescence of four Asaia conditional strains when exposed to blood. There was a significant difference in mean fluorescence for four BMI isolates in minimal media compared to media supplemented with blood (Welch’s t-test, n = 10 for each strain and condition). No significant difference between the different media was seen for both the positive (ANB50) and negative (AGLR1) controls, as well as the SodB and Ferr strains. Fluorescence readings were taken during log phase and normalized to the OD₆₀₀ of each culture. Height of bars are the mean value for that treatment. Error bars represent standard error of the mean (s.e.m.) (n = 10). Individual data points are superimposed on the bar charts. Statistical significance was determined using Welch’s two-tailed t-test where significance is represented by *P < 0.05, **P < 0.01, and ***P < 0.001. Only significant differences are labeled

Fig. 2

BMI Asaia strains retain GFP induction in vivo in response to a mosquito blood meal. Bacterial cultures of each strain were fed to mosquitoes and differences in fluorescence were analyzed by eye between the blood fed and sugar fed individuals of each strain. AGLR1, which contains no promoter, is the negative control and ANB50 is a constitutively fluorescent positive control. The strains outlined in gray, AGLR1.Hem, HF, HlyA and HlyC, show conditional fluorescence when exposed to a blood meal vs sugar meal indicating these promoters are induced by the blood meal in the mosquito. Images are representative of all observed midguts and all midguts were observed at the same magnification. The size bars are each 100 µM

Fig. 3

Antiplasmodial vector pCG18.glr1. BMI promoters were added at the multiple cloning site to drive the antiplasmodial effector scorpine in the promoterless pCG18.glr1. This plasmid was generated from the fusion of the promoter region of pGLR1 into the constitutive antiplasmodial plasmid pCG18 that contains a secreted protein fusion of scorpine and alkaline phosphatase. T1 transcriptional terminator from E. coli rrnB, Rep pBBR1 origin of replication, KanR kanamycin resistance, mcs multiple cloning site, TonB SS Asaia TonB dependent signal sequence for secretion, phoA E. coli alkaline phosphatase

Fig. 4

Scorpine is conditionally expressed in all BMI isolates when blood is present. Expression of scorpine was evaluated from colonies collected from minimal media agar plates (M) and chocolate agar plates that contain lysed blood (B) in a western analysis using an anti-alkaline phosphatase antibody to detect the fusion constructs. Two bands were detected by the antibody. The larger band (ca. 61.1 kD) correlates with the size of the secreted protein that contains the secretion signal while the smaller band (ca. 55.4 kD) correlates to the size of the protein construct lacking the secretion signal. All conditional constructs show protein production only when blood is supplemented in the media, whereas the constitutive strain (ACG18) produces the fusion protein on both media. SF2.1 = wild type Asaia; ACG18 = constitutive expression of scorpine by Asaia; all others are strains where scorpine is driven by a BMI promoter

Fig. 5

BMI strains demonstrate increased fitness compared to a constitutively-expressing strain. a Growth curves and maximum growth rates were calculated from 10 individual isolates of each strain grown over log phase of the bacteria using RStudio; b Each strain was competed in minimal media with wild-type Asaia SF2.1 in an initial 50/50 co-culture during log phase of the bacteria. 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; c Antiplasmodial Asaia strains were fed to mosquitoes and 10 midguts from each sample were pooled and plated on selective media. Transgenic CFUs were counted for each isolate and taken as a ratio of the total across all strains. For each panel, box bars are medians. The top and bottom of the boxes represent the first and third quartile of the data spread. The lower and upper bounds of the whiskers are the lowest datum still within 1.5X interquartile range (IQR) of the lower quartile, and the highest datum still within 1.5X IQR of the upper quartile, respectively. Data lying beyond the upper or lower 1.5X IQR ranges are shown as single points. 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 (n = 10, a; n ≥ 10, b; n = 3, c). Only significant comparisons are shown

Fig. 6

BMI strains significantly reduce the prevalence of Plasmodium infection in mosquito midguts compared to the constitutively expressing strain. In three 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) and ACG18 is the constitutively-expressing positive control. All strains secreting scorpine significantly reduced the median number of oocysts (horizontal bars) compared to the wild type strain (quantile regression, P«0.001). There was no significant difference in median oocyst number between the constitutive paratransgenic strain (ACG18) and any of the BMI strains (quantile regression, P > 0.05). The prevalence of infection (horizontal comparisons in the figure) was not significantly different between the wild-type and the positive constitutive control, while all of the BMI strains were significantly different (χ², 1 df). All of the BMI strains except ACG18.HF showed a significant decrease in prevalence compared to the constitutive positive control ACG18 (χ², 1 df). P -values: *P < 0.05, **P < 0.01, ***P < 0.001