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Microbiota activates IMD pathway and limits Sindbis infection in Aedes aegypti.

ABSTRACT: BACKGROUNDAedes aegypti is the main vector of important arboviruses such as dengue, Zika and chikungunya. During infections mosquitoes can activate the immune pathways Toll, IMD and JAK/STAT to limit pathogen replication.RESULTSHere, we evaluate the immune response profile of Ae. aegypti against Sindbis virus (SINV). We analyzed gene expression of components of Toll, IMD and JAK/STAT pathways and showed that a blood meal and virus infection upregulated aaREL2 in a microbiota-dependent fashion, since this induction was prevented by antibiotic. The presence of the microbiota activates IMD and impaired the replication of SINV in the midgut. Constitutive activation of the IMD pathway, by Caspar depletion, leads to a decrease in microbiota levels and an increase in SINV loads.CONCLUSIONTogether, these results suggest that a blood meal is able to activate innate immune pathways, through a nutrient induced growth of microbiota, leading to upregulation of aaREL2 and IMD activation. Microbiota levels seemed to have a reciprocal interaction, where the proliferation of the microbiota activates IMD pathway that in turn controls bacterial levels, allowing SINV replication in Ae. aegypti mosquitoes. The activation of the IMD pathway seems to have an indirect effect in SINV levels that is induced by the microbiota.

SUBMITTER: Barletta AB

PROVIDER: S-EPMC5324288 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Microbiota activates IMD pathway and limits Sindbis infection in Aedes aegypti.

Barletta Ana Beatriz Ferreira AB Nascimento-Silva Maria Clara L MC Talyuli Octávio A C OA Oliveira José Henrique M JH Pereira Luiza Oliveira Ramos LO Oliveira Pedro L PL Sorgine Marcos Henrique F MH

Parasites & vectors 20170223 1

<h4>Background</h4>Aedes aegypti is the main vector of important arboviruses such as dengue, Zika and chikungunya. During infections mosquitoes can activate the immune pathways Toll, IMD and JAK/STAT to limit pathogen replication.<h4>Results</h4>Here, we evaluate the immune response profile of Ae. aegypti against Sindbis virus (SINV). We analyzed gene expression of components of Toll, IMD and JAK/STAT pathways and showed that a blood meal and virus infection upregulated aaREL2 in a microbiota-de ...[more]

PMID: 28231846

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Project description:Arboviruses are transmitted by specific vectors, and the reasons for this specificity are not fully understood. One contributing factor is the existence of tissue barriers within the vector such as the midgut escape barrier. We used microRNA (miRNA) targeting of Sindbis virus (SINV) to study how replication in midgut cells contributes to overcoming this barrier in the mosquito Aedes aegypti. SINV constructs were designed to be attenuated specifically in midgut cells by inserting binding sites for midgut-specific miRNAs into either the 3' untranslated region (MRE3'miRT) or the structural open reading frame (MRE-ORFmiRT) of the SINV genome. Both miRNA-targeted viruses replicated less efficiently than control viruses in the presence of these miRNAs. When mosquitoes were given infectious blood meals containing miRNA-targeted viruses, only around 20% (MRE3'miRT) or 40% (MRE-ORFmiRT) of mosquitoes developed disseminated infection. In contrast, dissemination occurred in almost all mosquitoes fed control viruses. Deep sequencing of virus populations from individual mosquitoes ruled out selection for mutations in the inserted target sequences as the cause for dissemination in these mosquitoes. In mosquitoes that became infected with miRNA-targeted viruses, titers were equivalent to those of mosquitoes infected with control virus in both the midgut and the carcass, and there was no evidence of a threshold titer necessary for dissemination. Instead, it appeared that if infection was successfully established in the midgut, replication and dissemination were largely normal. Our results support the hypothesis that replication is an important factor in allowing SINV to overcome the midgut escape barrier but hint that other factors are also likely involved. IMPORTANCE When a mosquito ingests an arbovirus during a blood meal, the arbovirus must escape from the midgut of the vector and infect the salivary glands in order to be transmitted to a new host. We used tissue-specific miRNA targeting to examine the requirement for Sindbis virus (SINV) to replicate in midgut epithelium in order to cause disseminated infection in the mosquito Aedes aegypti. Our results indicate that specifically reducing the ability of SINV to replicate in the mosquito midgut reduces its overall ability to establish infection in the mosquito, but if infection is established, replication and dissemination occur normally. These results are consistent with an importance for replication in the midgut epithelium in aiding arboviruses in crossing the midgut barrier.

Project description:BackgroundThe mosquito Aedes aegypti is a major vector for the arthropod-borne viruses (arboviruses) chikungunya, dengue, yellow fever and Zika viruses. Vector immune responses pose a major barrier to arboviral transmission, and transgenic insects with altered immunity have been proposed as tools for reducing the global public health impact of arboviral diseases. However, a better understanding of virus-immune interactions is needed to progress the development of such transgenic insects. Although the NF-?B-regulated Toll and 'immunodeficiency' (Imd) pathways are increasingly thought to be antiviral, relevant pattern recognition receptors (PRRs) and pathogen-associated molecular patterns (PAMPs) remain poorly characterised in A. aegypti.Methodology/principle findingsWe developed novel RT-qPCR and luciferase reporter assays to measure induction of the Toll and Imd pathways in the commonly used A. aegypti-derived Aag2 cell line. We thus determined that the Toll pathway is not inducible by exogenous stimulation with bacterial, viral or fungal stimuli in Aag2 cells under our experimental conditions. We used our Imd pathway-specific assays to demonstrate that the viral dsRNA mimic poly(I:C) is sensed by the Imd pathway, likely through intracellular and extracellular PRRs. The Imd pathway was also induced during infection with the model insect-specific virus cricket paralysis virus (CrPV).Conclusions/significanceOur demonstration that a general PAMP shared by many arboviruses is sensed by the Imd pathway paves the way for future studies to determine how viral RNA is sensed by mosquito PRRs at a molecular level. Our data also suggest that studies measuring inducible immune pathway activation through antimicrobial peptide (AMP) expression in Aag2 cells should be interpreted cautiously given that the Toll pathway is not responsive under all experimental conditions. With no antiviral therapies and few effective vaccines available to treat arboviral diseases, our findings provide new insights relevant to the development of transgenic mosquitoes as a means of reducing arbovirus transmission.

Project description:BackgroundEmerging and re-emerging vector-borne diseases (VBDs) pose a recurring threat to tropical countries, mainly due to the abundance and distribution of the Aedes aegypti mosquito, which is a vector of the Zika, dengue, chikungunya, and yellow fever arboviruses.MethodsFemale 3-5 day-old Ae. aegypti were distributed into two experimental groups: group I-survey of cultivable bacteria; sucrose group: fed only on sucrose, i.e., non-blood-fed (UF); blood-fed group: (i) fed with non-infected blood (BF); (ii) fed with blood infected with the Zika virus (BZIKV); (iii) pretreated with penicillin/streptomycin (pen/strep), and fed with non-infected blood (TBF); (iv) pretreated with pen/strep and fed blood infected with ZIKV, i.e., gravid with developed ovaries, (TGZIKV); group II-experimental co-infections: bacteria genera isolated from the group fed on sucrose, i.e., non-blood-fed (UF).ResultsUsing the cultivable method and the same mosquito colony and ZIKV strain described by in a previous work, our results reveled 11 isolates (Acinetobacter, Aeromonas, Cedecea, Cellulosimicrobium, Elizabethkingia, Enterobacter, Lysinibacillus, Pantoea, Pseudomonas, Serratia, and Staphylococcus). Enterobacter was present in all evaluated groups (i.e., UF, BF, BZIKV, TBF, and TGZIKV), whereas Elizabethkingia was present in the UF, BZIKV, and TBF groups. Pseudomonas was present in the BZIKV and TBF groups, whereas Staphylococcus was present in the TBF and TGZIKV groups. The only genera of bacteria that were found to be present in only one group were Aeromonas, Lysinibacillus, and Serratia (UF); Cedacea, Pantoea and Acinetobacter (BF); and Cellulosimicrobium (BZIKV). The mosquitoes co-infected with ZIKV plus the isolates group fed on sucrose (UF) showed interference in the outcome of infection.ConclusionsWe demonstrate that the distinct feeding aspects assessed herein influence the composition of bacterial diversity. In the co-infection, among ZIKV, Ae. aegypti and the bacterial isolates, the ZIKV/Lysinibacillus-Ae. aegypti had the lowest number of viral copies in the head-SG, which means that it negatively affects vector competence. However, when the saliva was analyzed after forced feeding, no virus was detected in the mosquito groups ZIKV/Lysinibacillus-Lu. longipalpis and Ae. aegypti; the combination of ZIKV/Serratia may interfere in salivation. This indicates that the combinations do not produce viable viruses and may have great potential as a method of biological control.

Project description:Background Mosquito-borne dengue virus (DENV) causes major disease worldwide, impacting 50–100 million people every year, and is spread by the major mosquito vector Aedes aegypti. Understanding mosquito physiology, including antiviral mechanisms, and developing new control strategies have become an important step towards the elimination of DENV disease. In the study reported here, we focused on autophagy, a pathway suggested as having a positive influence on virus replication in humans, as a potential antiviral target in the mosquito. Methods To understand the role played by autophagy in Ae. aegypti, we examined the activation of this pathway in Aag-2 cells, an Ae. aegypti-derived cell line, infected with DENV. Rapamycin and 3-methyladenine, two small molecules that have been shown to affect the function of the autophagy pathway, were used to activate or suppress, respectively, the autophagy pathway. Results At 1-day post-DENV infection in Aag-2 cells, transcript levels of both the microtubule-associated protein light chain 3-phosphatidylethanolamine conjugate (LC3-II) and autophagy-related protein 1 (ATG1) increased. Rapamycin treatment activated the autophagy pathway as early as 1-h post-treatment, and the virus titer had decreased in the Aag-2 cells at 2 days post-infection; in contrast, the 3-methyladenine treatment did not significantly affect the DENV titer. Treatment with these small molecules also impacted the ATG12 transcript levels in DENV-infected cells. Conclusions Our studies revealed that activation of the autophagy pathway through rapamycin treatment altered DENV infection in the mosquito cells, suggesting that this pathway could be a possible antiviral mechanism in the mosquito system. Here we provide fundamental information needed to proceed with future experiments and to improve our understanding of the mosquito’s immune response against DENV. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13071-021-05066-w.

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Microbiota activates IMD pathway and limits Sindbis infection in Aedes aegypti.

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Microbiota activates IMD pathway and limits Sindbis infection in Aedes aegypti.

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