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. 2021 Feb 16:12:633667.
doi: 10.3389/fmicb.2021.633667. eCollection 2021.

Novel Asaia bogorensis Signal Sequences for Plasmodium Inhibition in Anopheles stephensi

Christina Grogan  1 , Marissa Bennett  1 , Shannon Moore  1 , David Lampe  1
Affiliations

Novel Asaia bogorensis Signal Sequences for Plasmodium Inhibition in Anopheles stephensi

Christina Grogan et al. Front Microbiol. .

Abstract

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.

Keywords: Anopheles; Asaia; Plasmodium; malaria; paratransgenesis; secretion.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Genetic constructs for the creation of transgenic Asaia strains. (A) pNB92 vector backbone for secretion signal insertion (Bongio and Lampe, 2015). The first 150 nucleotides of predicted signal sequence were inserted between the NdeI and PacI restriction sites for fusion with the reporter E. coli PhoA protein lacking its native signal sequence (phoA). pBBR, origin of replication; KanR, kanamycin resistance; oriT, origin of transfer; PnptII, promoter. (B) Construct for antiplasmodial effector strains. The ORF encoding the antimicrobial scorpine effector as well as a (GGGGS)3 flexible linker were inserted between the PacI and SbfI restrictions sites for fusion between the signal sequences and ‘phoA coding sequence.
FIGURE 2
FIGURE 2
Alkaline phosphatase localization in transgenic Asaia strains using ELISA. (A) A representative ELISA that utilized an anti-PhoA-HRP antibody to detect the presence of the alkaline phosphatase protein in the supernatant, cell surface/periplasm, and the cell lysate fractions of PhoA-only Asaia strains. SF2.1, wild-type Asaia; Sider, previously identified siderophore receptor signal (Bongio and Lampe, 2015). (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-PhoA antibody in the supernatant fraction 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; Sider, previously identified siderophore receptor signal (Bongio and Lampe, 2015).
FIGURE 3
FIGURE 3
Abundance of alkaline phosphatase protein in the supernatants from Asaia using predicted N-terminal signal peptides. (A) Representative western blot of supernatants from transgenic Asaia strains that showed localization of the PhoA reporter protein to the supernatant in the ELISA. The PhoA protein was detected using a monoclonal anti-PhoA antibody. (B) PhoA protein abundance in the supernatant. Western blots for all strains were repeated three times, and the signal intensities for the PhoA band were quantified without using a correction for lane to lane variation in total protein as an internal control. 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. No protein was detected in the SF2.1 or the PhoA (with no signal sequence) lanes. For the other strains, only significant comparisons are shown. SF2.1, wild-type Asaia; PhoA, alkaline phosphatase with no signal sequence; Sider, previously identified siderophore receptor signal. The same analysis correcting for variations in total protein between lanes as an internal control is shown in Supplementary Figure 2.
FIGURE 4
FIGURE 4
Fitness assessments of alkaline phosphatase Asaia strains. (A) Growth curves and maximum growth rates (μmax) were calculated from 10 individual isolates of each strain grown over log phase of the bacteria using the package growthrates59 (Petzoldt, 2017) in RStudio. Only Hyp4 showed a significantly lower μmax than the Sider strain. (B) Relative colonization of mosquito midguts by Asaia strains. Asaia strains were fed to mosquitoes and 15 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. The Sider strain had the highest rate of colonization, followed by the Hyp2 strain. 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 = 10, B. n = 3). Only significant comparisons are shown.
FIGURE 5
FIGURE 5
Antiplasmodial effector abundance in the supernatants from Asaia. (A) Representative western blot of supernatants from antiplasmodial Asaia strains. The PhoA segment of the fusion protein was detected using a monoclonal anti-PhoA antibody. Three bands were detected with the antibody. The larger band (57 kDa) corresponds to the size of the scorpine-PhoA protein, while we interpret the other two bands (51 and 47 kDa) as most likely corresponding to cleavage products. (B) Fusion protein abundance in the supernatant. Western blots for all strains were repeated four times, and the signal intensities for the bands were quantified without using a correction for lane to lane variation in total protein as an internal control and reported as a single value. 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. Only significant comparisons are shown. SF2.1, wild-type Asaia; Siders, scorpine antiplasmodial strain using the previously identified siderophore receptor signal. Similar analyses to this figure using a correction for variations in total protein in each lane and quantifying only the largest reactive band in each lane are shown in Supplementary Figures 4–6.
FIGURE 6
FIGURE 6
Fitness assessments of antiplasmodial Asaia strains. (A) Maximum growth rates (μmax) were calculated from 10 individual isolates of each strain grown over log phase of the bacteria using RStudio. Only Hyp1s showed a significantly lower μmax than the Siders strain. (B) Relative colonization of mosquito midguts by antiplasmodial Asaia strains. Antiplasmodial Asaia strains were fed to mosquitoes and 15 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. Siders showed the highest 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 ≥ 10, B. n = 3). Only significant comparisons are shown.
FIGURE 7
FIGURE 7
Suppression of P. berghei development by paratransgenic Asaia strains. 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 Siders is the scorpine antiplasmodial strain using the previously identified siderophore receptor signal (positive control). All antiplasmodial strains significantly reduced the median number of oocysts (horizontal bars) compared to the wild-type strain (quantile regression, P < 0.001). The Hyp1s and the Hyp4s strains showed a significant reduction in the median number of oocysts (the bottom set of horizontal comparisons in the figure) compared to the Siders strain (quantile regression, P ≤ 0.00187). The prevalence of infection (the top set of horizontal comparisons in the figure) was also significantly different between the wild-type and all antiplasmodial strains (χ2, 1 df). There was no significant difference in prevalence of infection between the Siders strain and any of the new antiplasmodial strains (χ2, 1 df). P-values: *P < 0.05, **P < 0.01, ***P < 0.001.
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References

    1. Adelman Z. N., Tu Z. (2016). Control of mosquito-borne infectious diseases: sex and gene drive. Trends Parasitol. 32 219–229. 10.1016/j.pt.2015.12.003 - DOI - PMC - PubMed
    1. Alauzet C., Teyssier C., Jumas-Bilak E., Gouby A., Chiron R., Rabaud C., et al. (2010). Gluconobacter as well as Asaia species, newly emerging opportunistic human pathogens among acetic acid bacteria. J. Clin. Microbiol. 48 3935–3942. 10.1128/JCM.00767-10 - DOI - PMC - PubMed
    1. Alphey L. (2014). Genetic control of mosquitoes. Ann. Rev. Entomol. 59 205–224. 10.1146/annurev-ento-011613-162002 - DOI - PubMed
    1. An Y., Wang J., Li C., Revote J., Zhang Y., Naderer T., et al. (2017). SecretEPDB: a comprehensive web-based resource for secreted effector proteins of the bacterial types III, IV and VI secretion systems. Sci. Rep. 7:41031. - PMC - PubMed
    1. Bagos P. G., Nikolaou E. P., Liakopoulos T. D., Tsirigos K. D. (2010). Combined prediction of Tat and Sec signal peptides with hidden Markov models. Bioinformatics 26 2811–2817. 10.1093/bioinformatics/btq530 - DOI - PubMed