REL2 overexpression in the Anopheles gambiae midgut causes major transcriptional changes but fails to induce an immune response

  1. Astrid Hoermann
  2. Paolo Capriotti
  3. Giuseppe Del Corsano
  4. Maria Grazia Inghilterra
  5. Tibebu Habtewold
  6. Julia A. Cai
  7. Gauri Sachiko Saini
  8. Huong Nguyen
  9. Nikolai Windbichler
  10. George K. Christophides

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

The NF-κB-like transcription factor, REL2, is a key player in the mosquito Immunodeficiency (Imd) pathway and holds promise for controlling malaria parasite infections in genetically modified Anopheles gambiae mosquitoes. We engineered transgenic mosquitoes overexpressing REL2 from within the bloodmeal-inducible zinc carboxypeptidase A1 (CP) host gene in the adult posterior midgut. Our results confirmed elevated REL2 expression in the posterior midgut following a bloodmeal, with the corresponding protein localized within epithelial cell nuclei. While this induced overexpression triggered substantial transcriptional changes, accompanied by notable fitness costs, the resultant reduction in Plasmodium falciparum infection was modest. An in-depth analysis of regulatory regions of differentially regulated genes allowed us to identify direct REL2 target genes and revealed signatures indicative of potential transcriptional repressors. To account for potential impacts of host gene modification, we also created a CP knockout line that caused marginal effects on mosquito fitness. These findings shed light on the observed absence of transcriptional activation and, in some cases, induced repression of antimicrobial peptides (AMPs) presumed to be under Imd pathway control. In conclusion, our study suggests that elevated REL2 expression in the posterior midgut may induce the upregulation of negative immune regulators, facilitating control over an otherwise unrestrained immune response, and that concurrent transcriptional derepression may be needed to effectively induce the mosquito immune response. This work contributes valuable insights into the intricate regulation of midgut immunity in malaria vector mosquitoes.

Article activity feed

  1. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/12768797.

    Summmary

    In this paper from Hoermann et al., the authors engineer transgenic Anopheles gambiae mosquitoes with bloodmeal-induced overexpression of REL2. REL2 is a member of the mosquito immunodeficiency (Imd) pathway, and previous work has demonstrated that A. gambiae REL2 overexpression in another mosquito species, A. stephensi, significantly reduced Plasmodium falciparum infection. To assess the antiplasmodial activity of REL2 overexpression in A. gambiae itself, the authors used CRISPR/Cas9 to generate a markerless REL2-CP line wherein a second copy of REL2 is integrated into the carboxypeptidase (CP) locus which is upregulated upon blood feeding. Via immunofluorescence assays, the authors show that REL2 is strongly upregulated following a blood meal and shows nuclear localization as expected for its activity as a transcription factor. However, across three biological replicates there was no significant impact on the number of P. falciparum oocysts per mosquito midgut relative to WT. Unexpectedly, there was also a significant decrease in the number of REL2-CP eggs and mosquito survival over time, indicating that there were strong fitness costs associated with this transgenic line.

    To assess whether these fitness costs were due to possible disruption of endogenous CP activity in the REL2-CP line, the authors generated a CP knockout mosquito line. Surprisingly, this locus was amenable to knockout and other than a slight reduction in the hatch rate of CPKO eggs, there were no apparent fitness costs, suggesting that this was not responsible for the effects seen in the REL2-CP line. The authors also found minor effects on P. falciparum infection in the CPKO line, including a slight reduction in the number of oocysts per midgut and a slight increase in oocyst size. To understand why REL2 overexpression had a limited impact on P. falciparum infection, the authors performed RNA-sequencing of WT and REL2-CP midguts before blood feeding and at 6 and 20 hours post-blood feeding. The authors found that some antimicrobial peptides with likely REL2 transcription factor binding sites were downregulated in the REL2-CP line, suggesting that REL2 overexpression may actually suppress its targets and provide a potential explanation for the lack of antiplasmodial activity in this line. However, mechanistic work remains to more conclusively support this hypothesis. The data in this paper largely supports the authors' claims, but the manuscript could be improved by more consistent data analysis methods, additional validation of the RNAseq, and further interpretation of this work in the context of future genetically engineered vector control strategies.

    Major points:

    • One of the major findings of this paper is that REL2 overexpression does not impact P. falciparum infection. However, the authors are inconsistent in their evaluation and statistical interpretation of infection data between the REL2-CP and CPKO lines. When assessing oocysts/midgut in the REL2-CP line, the authors evaluate each replicate separately whereas in the CPKO line the authors pool all three replicates. In the latter, they ultimately detect a significant decrease in oocyst numbers relative to WT, but this may also be the case for REL2-CP if the replicates were pooled and would change the interpretation of the data. The authors also do not look at oocyst size in the REL2-CP line, although they do quantify oocyst diameter for the CPKO line. The authors should still have midgut images from their infections as raw data, so I would suggest measuring the oocyst diameters from the three REL2-CP infection experiments to assess whether there is any impact on parasite size as seen in the CPKO line.

    • The authors' primary explanation for the lack of antiplasmodial activity of the REL2-CP line is that REL2 overexpression may in fact suppress the expression of key antimicrobial peptides, including cecropins 1 and 3 and defensin 1. However, no experiments were performed to validate this hypothesis. The authors could perform knockdowns of key AMPs to define their role in P. falciparum infection. If further experiments to mechanistically support this hypothesis are outside of the scope of this paper, the authors could also perform additional analyses of their existing RNAseq data. For example, the authors could examine whether there is a conserved correlation between the number of REL2 transcription factor binding sites and gene expression. In addition to the select genes the authors have analyzed in the text, these more global analyses would provide further quantitative evidence to support their suppression hypothesis.

    • The title of this paper does not accurately reflect its immune response findings. The authors' RNAseq data shows differential gene expression of a number of immune related genes in the Rel2-CP line, the majority of which are upregulated (Fig. 5D). Although the desired P. falciparum reduction was not observed, it is inaccurate to claim that no immune response is induced. The authors should update the title to better match their data.

    Minor points:

    • The temporal flow of the paper is at times confusing. For example, the authors note that CP gene expression is reduced in the REL2-CP line in the text of the results section for Figure 4, but there is no data presented to demonstrate this until figure 5. The authors should more clearly reference figures when the text refers to their data, and could consider reordering their figures to ensure data is presented in an order that matches their text.

    • In figure 2, images should include a scale bar and the authors should note the timepoint of images taken in figure 2B.

    • Across all figures, authors should label the median/mean values for ease of interpretation by the reader.

    • Oocyst counts are often not normally distributed, in which case the median should be plotted rather than the mean. Authors should consider conducting a test for normality and plotting means or medians as appropriate for these data, or just plotting medians for consistency.

    • In figure 3C, the survival curve for the REL2-CP males briefly goes up around 28 days, which should not be possible. The authors should check their raw data and correct the figure as needed.

    • The authors observe a significant mortality phenotype in their REL2-CP line. It is possible that the immune response varies between those mosquitoes that die quickly versus those that have a more standard lifespan, and thus only assessing parasite burden at 8 or 9 days may be obscuring an effect on parasite burden in the mosquitoes that die early. To evaluate this, the authors could quantify parasite burden at an earlier timepoint, such as ookinetes or early oocysts, when most of their mosquitoes are still alive.

    • The incorrect p-value significance key is provided in the legend for figure 3A. It should be ****, p <0.0001

    • Infection prevalence does not need to be its own separate graph in figure 4H. It can just be shown as a part of 4G, as was done by the authors in figure 3D. The p-value significance key (e.g. ****, p <0.0001) should also be included for relevant panels in figure 4.

    • There is a typo in the "Knockout of the CP host gene reveals no significant fitness cost" text. In the second sentence, "any" should be deleted in the phrase "…which exhibited no any obvious fitness issues…"

    • In the "Bloodmeal induced overexpression of REL2 causes broad transcriptional changes" section, the authors should provide a citation for the assertion "The transcriptional activity of the CP locus is known to peak at about 3h PBF…"

    • Most of the RNAseq data is described in the text and supplemental tables, but this analysis is critical to the claims of the paper. The authors could consider moving some of this analysis to a main text figure, such as the GO term enrichment or key DEG analyses (either as additional panels in Figure 5 or a new figure altogether). Figure 5D is not referenced or discussed in the text, so this analysis could be moved to a supplemental figure.

    • The authors should provide additional clarification on how the list of 288 genes involved in immunity was curated.

    • CEC1 should be defined when it is first introduced. Currently, it is described in the text in a later paragraph.

    • In the results text, authors state that DEF2-5 are either downregulated or not among DEGs ("Similarly, DEF2-5 that showed no recognizable REL2 binding sites were either downregulated or not among DEGs…"), while in the discussion the authors state that DEF2-5 were all not differentially expressed ("CEC2 and DEF2-5, all lacking REL2 binding sites in their regulatory regions, as well as GAM1, with only a single REL2 binding site, did not show any response."). The authors should confirm which statement is accurate and update the text accordingly.

    • The authors could consider adding a brief discussion of what their findings mean for future gene drive control strategies.

    Competing interests

    The authors declare that they have no competing interests.

  2. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/12775134.

    In Hoermann et al., they examine the role of over-expressing the IMD regulatory protein, REL2, in Anopheles gambiae mosquitoes to aid in elimination of malaria parasites. The over-expression of REL2 has been previously shown to be effective in blocking Plasmodium transmission and altering reproductive mosquito behavior in favor of gene drive dynamics in Anopheles Stephensi. Thus, this work presented here addresses an important gap in our knowledge in A. gambiae mosquitoes towards generating additional control tools for an important malaria vector. Using previously established Integral Gene Drive (IGD) lines, they integrate REL2 into the midgut-specific bloodmeal-inducible zinc carboxypeptidase (CP) A1 locus and examine the impact on Plasmodium transmission and vector fitness. As a control, they knock out the CP locus and subsequently measure the same fitness parameters. Finally, they perform RNA-seq analysis to examine transcriptional changes resulting from REL2 overexpression and examined transcriptional changes that contained a REL2 DNA-binding site in the promoter region. 

    The introduction provides adequate background on the Immune Deficient Pathway and the role of Relish as transcriptional regulator for immunity in the previously published work in Anopheles stephensi. The authors then provide the conceptional framework for their experiments using their previously published Integral Gene Drive (IGD) system with REL2 in A. gambiae. The final statements of the introduction section regarding the major findings should be moved to the discussion and further expanded on in that section. The experimental methods (transgenic mosquitoes, parasite infections and RNA-seq analysis) used are appropriate in the study and a few concerns regarding the statistical analysis for certain experiments are detailed in the points below. Both the results section and figure legends could use improvements to improve clarity on the described experiment. Much of the discussion reads as a re-statement of the results section and could be replaced with a short summary of the overall findings and then expanding upon why the authors think there may be differences between the previously published findings using a different mosquito species and potential future directions to overcome this challenge.

    Major Points:   A major gap in the study is the lack of confirmation for REL2 function in the IMD pathway for the over-expression line. One of the key findings from the previous work done in Anopheles Stephensi is the impact of REL2 on the microbiome of the vector, which is subsequently missing from this study. As REL2 is part of the anti-microbial pathway, this should be thoroughly examined through either 16S quantitative PCR or plating of midguts for CFU counts after a blood meal. This is essential as the over-expression of REL2 cannot be traced back to the specific upregulation of antimicrobial peptides (and as the RNA-seq data seems to indicate leads to the down-regulation of multiple AMPs) as it does in the Drosophila system.   A large focus of the manuscript (almost half) is based on RNA-seq data generated from the CP-REL2 line and then subsequently examining potential gene candidates regulated by REL2 by computation analysis. However, for this data to justify such a significant portion of the manuscript, additional experiments (luciferase assay, EMSA DNA binding) should be done to support REL2 binding to target promoter regions for gene activity. Minor Points: The methodology behind the generation of the REL2-S over-expression line is confusing for readers who are not familiar with the laboratories' previous publication on generating the IGD lines. A more detailed figure, including a map of the HDR donor plasmid pD-REL2-CP, should be included in Figure 1. It would be beneficial to measure REL2 gene expression levels between the WT line and REL2-CP lines to confirm the construct results in enhancing REL2 expression. For the phenotypic characteristics of the CP-REL2 line in Figure 3, it would better to exclude the zero's (egg numbers and oocysts infection intensity) and plot them as prevalence %. The zero's can easily skew the mean/median of the data set and thus should be analyzed separately. For example in Figure 3A, the mean of eggs / female by REL2-CP group ~50 eggs; however, that is because ~20% of mosquitoes fail to produce any eggs, lowering the overall group average. Thus, if a female end up producing eggs, it will be ~70 eggs / female, which closer to the WT group than is represented in the current graph.

    For the survival curves in Figure 3C, A log-rank (Mantel–Cox) test is the appropriate test to be used to compare survival distribution. The infection plots (Fig. 3D) should display the median (oocysts intensity is a non-normal distribution) and a generalized linear model should be used to pool the data and consider replicate effects between the groups for statistical analysis. It seems like all three replicates have a small reduction in oocyst numbers in the CP-REL2 line, and if analyzed this way it would possibly reach significance.  Several studies have correlated a reduction in egg numbers with an increase in parasite size/development. Since there was a reduction in egg numbers in the CP-REL2 line, was there any change to parasite size as noted in Figure 4 with the KO-CP line? Is it possible that the female mosquitoes that died before examination are the ones that display fewer oocysts? If mosquitoes were examined at day 4-5 rather than day 7 would there be a difference in oocysts intensity?

    Would an alternative to over-expressing REL2 be to over-express the individual AMPs genes of interest under the CP Promoter? This could be discussed as a future potential project in the discussion.

    Competing interests

    The author declares that they have no competing interests.

  3. Version published to 10.1101/2024.02.05.578852v1 on bioRxiv