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Ecdysone signaling mediates the trade-off between immunity and reproduction via suppression of amyloids in the mosquito Aedes aegypti [1]

['Mao Wang', 'State Key Laboratory Of Integrated Management Of Pest Insects', 'Rodents', 'Institute Of Zoology', 'Chinese Academy Of Sciences', 'Beijing', 'People S Republic Of China', 'Cas Center For Excellence In Biotic Interactions', 'University Of Chinese Academy Of Sciences', 'Yanhong Wang']

Date: 2022-11 

The balance between immunity and reproduction is essential for many key physiological functions. We report that to maintain an optimal fertility, 20-hydroxyecdysone (20E) and the ecdysone receptor (EcR) downregulate the immune deficiency (IMD) pathway during the post blood meal phase (PBM) of the Aedes aegypti reproductive cycle. RNA interference-mediated depletion of EcR elicited an increased expression of the IMD pathway components, and these mosquitoes were more resistant to infection by Gram-negative bacteria. Moreover, 20E and EcR recruit Pirk-like, the mosquito ortholog of Drosophila melanogaster Pirk. CRISPR-Cas9 knockout of Pirk-like has shown that it represses the IMD pathway by interfering with IMD-mediated formation of amyloid aggregates. 20E and EcR disruption of the amyloid formation is pivotal for maintaining normal yolk protein production and fertility. Additionally, 20E and its receptor EcR directly induce Pirk-like to interfere with cRHIM-mediated formation of amyloid. Our study highlights the vital role of 20E in governing the trade-off between immunity and reproduction. Pirk-like might be a potential target for new methods to control mosquito reproduction and pathogen transmission.

To reproduce, hematophagous female mosquitoes require repeated blood feeding that consequentially makes them crucial vectors of many devastating diseases. A steroid hormone 20-hydroxyecdysone (20E) plays a critical role in mosquito reproduction. It is also essential for maintaining a balance between reproduction and immunity. Following a blood meal, the ecdysone receptor (EcR) directly induces an inhibitor Pirk-like, affecting the immune deficiency (IMD) pathway. Pirk-like downregulates the IMD pathway by disrupting the amyloid formation and preventing the nuclear translocation of the NF-κB transcriptional factor Rel2. The EcR downregulation of the immune response and the amyloid formation is vital for maintaining the mosquito fertility. This study identified the Pirk-like–amyloid immune axis that could be a target for new mosquito control methods.

Funding: This work was supported by the National Key Plan for Scientific Research and Development of China No. 2021YFC2600100 (ZZ), National Science Foundation of China Grant No. 32090011 (YW), Strategic Priority Research Program of Chinese Academy of Sciences (Grant No. XDPB16) (ZZ), and NIH grant RO1 AI036959 (ASR). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2022 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The pleiotropic effects of JH and 20E make them prime candidates for controlling the allocation of resources among different physiological processes [ 26 , 27 ]. In D. melanogaster, mated females are more likely to die from septic injury and have less ability to induce an immune response. These defects are rescued when JH signaling is suppressed [ 28 ]. JH downregulates the expression of immunity-related genes (IMRGs) in Ae. aegypti during the PE stage [ 29 ]. However, the role of 20E in the trade-off between reproduction and immunity remains elusive in Ae. aegypti. Here, we have identified that 20E and EcR in Ae. aegypti downregulate the IMD pathway to protect reproductive output. Moreover, we provide evidence that, for this action, the 20E-EcR-USP complex directly activates the expression of Pirk-like, a negative regulator of the IMD pathway [ 30 ]. Our study highlights that the regulatory role of 20E in the IMD pathway is crucial for mosquito fertility, which may further affect the coordination of innate immune and reproductive responses.

Immunity and reproduction are highly demanding processes and, thus, a balance must be maintained between the two. Reduced reproductive output and capacity have been observed after a bacterial challenge or the immune signaling activation in D. melanogaster [ 16 , 17 ]. In septically injured Anopheles mosquitoes, apoptosis of follicular cells occurs, resulting in a reduced oviposition [ 18 – 20 ]. Conversely, the increased reproductive output can downregulate the constitutive and inductive immunity of female insects. For example, mating leads to a reduced survival rate of D. melanogaster females in response to infection with various pathogens, with an observed higher pathogen load and decreased AMP expression level [ 21 , 22 ]. Mating reduces cellular encapsulation and melanization in crickets Acheta domesticus and Allonemobius socius [ 23 – 25 ]. These important observations inspired us to decipher the molecular interactions between reproduction and immunity in Aedes aegypti, a mosquito vector of numerous human viral diseases.

Insect humoral immunity is composed of two evolutionarily conserved NF-κB pathways, Toll and immune deficiency (IMD), which lead to the production of antimicrobial peptides (AMPs) in response to peptidoglycan (PGN) infection from bacteria [ 8 – 10 ]. Diaminopimelic acid-containing PGN specifically triggers the IMD pathway by its most proximal components—peptidoglycan recognition proteins (PGRP-LC or PGRP-LE) [ 11 ]. There is a conserved sequence motif in the N-terminal domains of PGRP-LC and PGRP-LE [ 12 ] that has weak homology to the mammalian receptor interacting protein (RIP) homotypic interaction motifs (RHIM) and is referred to as a cryptic RHIM (cRHIM) in Drosophila melanogaster. The cRHIMs in PGRP-LC and PGRP-LE form amyloid fibrils and trigger the IMD pathway [ 13 ]. As a result, the downstream NF-κB transcriptional factor Relish (Rel2) is translocated to the nucleus and initiates the transcription of multiple immune effectors targeting invading pathogens [ 14 , 15 ].

Hematophagous female mosquitoes utilize vertebrate blood as their source of nutrition and energy for egg production. Blood feeding on humans allows them to transmit virus, Plasmodium, and nematode pathogens. In blood-feeding mosquitoes, gonadotrophic cycle consists of a post-eclosion phase (PE) in the first cycle and a post blood meal phase (PBM) that are regulated by two major insect hormones, juvenile hormone (JH) and 20-hydroxyecdysone (20E), respectively [ 1 ]. JH controls preparatory previtellogenic events, while 20E is the principal hormone regulating vitellogenesis, egg maturation and ovulation [ 2 ]. 20E binds to the heterodimeric receptor complex of the ecdysone receptor (EcR) and Ultraspiracle (USP) [ 3 , 4 ], and initiates the transcriptional cascade responsible for the expression of vitellogenin (Vg) genes [ 5 , 6 ]. Vg proteins are secreted from the fat body and subsequently taken up by oocytes for egg maturation [ 1 ]. The fat body tissue is the central site for both vitellogenesis and immune defense [ 7 ].

Results

Pirk-like responds to 20E at the PBM reproductive phase To identify potential factors of the IMD pathway responding to 20E signaling, we first analyzed the overall expression of genes involved in the IMD pathway using custom-made Agilent microarray chips at nine time points (3–72 h) during the PBM phase [34,35]. Hierarchical clustering analysis revealed that PGRP-LC, IMD, Ben, IAP2 and Rel2 with upregulated expressions were positively correlated with 20E titer (Fig 3A). However, we found that the protein level of PGRP-LC was consistent during the PBM phase, and Rel2 was barely detected in the nucleus from 24 h to 60 h PBM (Fig 3B). Therefore, the inhibitors of the IMD pathway might respond to 20E signaling. Through sequence alignment, we found 29 reported IMD inhibitors in Ae. aegypti. Among them, an ortholog of D. melanogaster Pirk (29% similarity) was identified. It has 105 amino acid residues and does not contain any functional domain or cRHIM motif; thus, it was designated as Pirk-like (S3A and S3B Fig). PPT PowerPoint slide

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TIFF original image Download: Fig 3. Pirk-like expression is regulated by 20E signaling. (A) Heat map of transcriptional patterns of the IMD pathway component genes during PBM phases. Gene expression at each time point was normalized to PE 72 h. (B) Protein levels of PGRP-LC and Rel2 determined using western blots. For each time point, both cytoplasmic and nuclear protein extracts were prepared from ten female mosquito fat bodies. GAPDH and Histone H3 were used as controls. (C) Putative EcRE of the IMD pathway inhibitors aligned with reported Aedes EcRE sequences. The three core amino acids are highlighted with yellow. (D) Heatmap showing the expression pattern of 25 IMD inhibitor genes in iEcR mosquitoes compared with iEGFP mosquitoes upon E. cloacae infection. (E) mRNA abundance of Pirk-like in iEcR mosquitoes infected by E. cloacae. Bar plots are shown as mean ± SEM. ****p < 0.0001 (one-way ANOVA followed by Bartlett’s test). Data are from three biological replicates. (F) mRNA abundance of Pirk-like in Aag2 cells co-transfected EcR and USP. (G) Expression of Pirk-like and EcR in the PE and PBM phases. Dot plots represent mean ± SEM. Data are from three biological replicates. https://doi.org/10.1371/journal.ppat.1010837.g003 Jaspar program analysis revealed that the promoter regions of Pirk-like, DNR1, STAT92E and Caspar contain highly conserved EcRE (Fig 3C), whereas only Pirk-like was significantly decreased in non-infected iEcR mosquitoes (Fig 3D and 3E). In addition, Pirk-like increased in iEcR mosquitoes after septic injury, but was lower than in infected iEGFP mosquitoes, suggesting that Pirk-like might be co-regulated by 20E and immune stimulation in the PBM phase. Moreover, Pirk-like was induced by 20E in EcR- and USP-overexpressed Ae. aegypti (Aag2) cells (Fig 3F). The time course of Pirk-like expression showed that Pirk-like was very low throughout the PE phase, but significantly increased during the PBM phase, with an expression peak coincident with that of EcR (Fig 3G). Pirk-like appears to be regulated by 20E to inhibit the IMD pathway during the PBM phase.

The 20E-EcR-USP complex binds to the regulatory region of the pirk-like gene The induction of Pirk-like in response to the 20E pulse in the PBM phase prompted us to identify whether Pirk-like is a direct target of 20E and its receptor EcR. We identified two conserved EcRE motifs located at -1590 bp and -818 bp in the Pirk-like gene regulatory region (Figs 3C and 4A). To assess the binding interaction of the Pirk-like gene promoter with EcR, we conducted chromatin immunoprecipitation (ChIP) coupled with quantitative polymerase chain reaction (qPCR). ChIP signals were enriched at the EcRE2 of Pirk-like in EcR- and USP-expressed cells (Fig 4B). The enrichment of EcRE1 was barely detected. PPT PowerPoint slide

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TIFF original image Download: Fig 4. 20E-EcR-USP directly binds to the EcRE motif of Pirk-like. (A) Schematic showing Pirk-like promoter and putative 20E-EcR-USP binding motifs. EcRE1 and EcRE2 represent regional target sites tested in ChIP-qPCR analysis. (B) ChIP-qPCR analysis with EcR and USP co-expressed Aag2 cells using V5 antibody. pAc5.1b lysates incubated with mouse IgG antibody (N-pAc5.1b) or anti-V5 (IP-pAc5.1b) and EcR-USP lysates incubated with mouse IgG antibody (N-EcR-USP) were included as controls. n = 3 biological replicates. (C) EMSA results showing the binding of EcR to a biotin-labeled EcRE2 probe. Nuclear protein extracts from EcR- and USP-overexpressed Aag2 cells. Competitor (unlabeled 26-bp probe), mutant competitor and anti-EcR antibody were added as indicated. (D) Dual-luciferase reporter assay revealed the transcriptional activation of Pirk-like promoter in EcR- and USP-overexpressed S2 cells with 20E treatment. S2 cells were co-transfected with the reporter vector pGL4.10-Pirk (100 ng), the overexpression vectors (pAc5.1b-EcR/V5 and pAc5.1b-USP/HA, 100 ng), and the pGL4.73 plasmid (15 ng). The relative luciferase activity was detected at 48 h after transfection. Western blots were used to analyze the protein level of EcR and USP using V5 and HA antibodies. GAPDH was used as the control. https://doi.org/10.1371/journal.ppat.1010837.g004 Next, we performed an electrophoretic gel mobility assay (EMSA) to analyze the interaction between EcR-USP and Pirk-like. The addition of Aag2 lysates containing co-expressed V5-EcR and V5-USP fusion proteins produced clear shifts of two biotin-labeled oligonucleotide fragments, derived from the EcRE1 and EcRE2 of Pirk-like (Figs 4C and S4). Control (pAc5.1b) lysates showed a slight binding band, which could come from endogenous EcR and USP in Aag2 cells. The specific interaction of protein and DNA was confirmed by a competitor with the unlabeled specific probe. More importantly, the shift after pre-incubating the nuclear extract with anti-EcR antibody was higher than others (Figs 4C and S4). This indicated that the binding of the anti-EcR antibodies and the antigen (V5-EcR) interfered with the migration of the probes. The EcRE2 mutant competitor considerably reduced the intensity of the specific band biotin-labeled Pirk-like probe bound to the V5-EcR-USP complex (Fig 4C). This verified the functionality of the EcRE2 of Pirk-like, confirming direct binding between EcR-USP and the Pirk-like promoter in vitro. In addition, we performed a dual-luciferase reporter assay to study the interaction between EcR-USP and the Pirk-like promoter in vivo. Co-expressed V5-EcR and HA-USP resulted in a 5.6-fold increased induction of the Pirk-like-luciferase reporter after 20E treatment (Fig 4D), and showed the ability of EcR and USP to induce the Pirk-like expression in a dose-dependent manner. These data demonstrate that the 20E-EcR-USP heterodimer directly binds to the EcRE2 of Pirk-like, mediating its expression in the presence of 20E.

Pirk-like interacts with and disrupts the formation of amyloid aggregates The core motif IQIG or VQVG of mammalian RHIMs fold into cross-β sheet conformations and shape functional amyloid fibrils, which is related to necrosis signaling [36–38]. In D. melanogaster, the cRHIM of PGRP-LC, PGRP-LE, and IMD can form amyloid fibrils [13,39]. We found that Ae. aegypti PGRP-LE and IMD have the VHIG motif, a cRHIM in D. melanogaster [13], and that the cRHIMs of PGRP-LC are highly conserved with mammalian RHIMs, having the third position (P3) V substituted to N (S5A Fig). This sequence similarity suggested that amyloidal aggregates could be formed in Ae. aegypti. Thus, we used thioflavin T (ThT) fluorescence to measure amyloids in Aag2 cells. The cells with these overexpressed factors of V5-tagged PGRP-LC, PGRP-LE or IMD showed strong ThT fluorescence (Fig 5A). These results demonstrate that Ae. aegypti cRHIM can form amyloid fibrils, despite the sequence variability. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Pirk-like interferes with amyloidal aggregates formed by PGRP-LC, PGRP-LE, and IMD. (A) Amyloidal aggregate staining of Aag2 cells transiently transfected with V5-tagged PGRP-LC, PGRP-LE or IMD. Scale bar: 10 μm. (B) GST pull-down assay between PGRP-LC, PGRP-LE, IMD, and Pirk-like. The interactions of His-Pirk-like with GST-PGRP-LC, GST-PGRP-LE or GST-IMD were observed in the pull-down assay and confirmed using western blots with anti-GST and anti-His antibodies. (C-E) ThT fluorescence in Aag2 cells co-expressed with Ha-tagged PGRP-LC (C), PGRP-LE (D) or IMD (E), and either an empty vector (mock) or a V5-Pirk-like. Scale bar: 10 μm. (F) Expression of Rel2, Dpt, DefC and CecB in iEGFP and iEcR mosquitoes infected with E. cloacae in the presence of ThT. Acetic acid was used as control. Data are shown as mean ± SEM. ****p < 0.0001; ns, not significant (one-way ANOVA followed by Bartlett’s test). https://doi.org/10.1371/journal.ppat.1010837.g005 In D. melanogaster, Pirk directly interacts with PGRP-LC, PGRP-LE, and IMD via cRHIM [13]. Since cRHIM was not present in Ae. aegypti Pirk-like (S3A Fig), we wanted to investigate whether Pirk-like interacts with these proteins. The yeast two-hybrid (Y2H) assay showed that Pirk-like could bind to PGRP-LE (S5B–S5D Fig). PGRP-LC and IMD were self-activated when co-transformed with pGBKT7 (S5D Fig). Furthermore, a pull-down assay identified the presence of His-tagged-Pirk-like (His-Pirk-like) in the PGRP-LC (GST-PGRP-LC), PGRP-LE (GST-PGRP-LE), and IMD (GST-IMD) immunoprecipitation, confirming their interaction in vitro (Fig 5B). We then determined whether Pirk-like could affect the IMD signaling by blocking amyloid formation. To test this, V5-tagged Pirk-like was co-transfected along with HA-tagged PGRP-LC, PGRP-LE, or IMD into Aag2 cells. Formaldehyde-fixed cells were stained with ThT, and the presence of the amyloidal aggregates was determined using confocal microscopy. The expression of PGRP-LC, PGRP-LE, and IMD, as detected by HA antibody staining, was similar in both mock and Pirk-like co-transfected cells. However, ThT signaling was extremely weak in Pirk-like co-expressed cells (Fig 5C–5E), indicating that Pirk-like terminates the formation of amyloids in Aag2 cells. Collectively, Pirk-like interacts with and suppresses the amyloid formation from PGRP-LC, PGRP-LE, and IMD. In the next step, we examined the effect of EcR silencing on cRHIMs-mediated amyloid formation in fat bodies at PBM 24 h. EcR was translocated into the nuclei of iEGFP_PBS and iEGFP_Ec fat bodies with little detectable ThT signals. However, the dsRNA silencing of EcR significantly promoted amyloid formation, with or without E. cloacae infection (S5E Fig). ThT not only binds to amyloid fibrils but also inhibits further aggregation of amyloid [40]. We therefore postulated that ThT could rescue the expression of AMPs in iEcR mosquitoes. To examine this, iEcR mosquitoes were injected with ThT at 12 h PBM and then infected with E. cloacae. ThT caused a noticeable decrease in mRNA abundance of Rel2, Dpt, and DefC in infected iEcR mosquitoes, except for CecB (Fig 5F). ThT also reduced the expression of Dpt and DefC in infected iEGFP mosquitoes. In contrast, the mRNA level of CecB was strongly increased after ThT injection. These data re-affirm that 20E inhibits the IMD pathway by modulating the formation of amyloid and indicate that ThT specifically inhibits the IMD pathway.

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010837

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