Wolbachia uses host microRNAs to manipulate host gene expression and facilitate colonization of the dengue vector Aedes aegypti.
ABSTRACT: The obligate endosymbiont Wolbachia pipientis is found in a wide range of invertebrates where they are best known for manipulating host reproduction. Recent studies have shown that Wolbachia also can modulate the lifespan of host insects and interfere with the development of human pathogens in mosquito vectors. Despite considerable study, very little is known about the molecular interactions between Wolbachia and its hosts that might mediate these effects. Using microarrays, we show that the microRNA (miRNA) profile of the mosquito, Aedes aegypti, is significantly altered by the wMelPop-CLA strain of W. pipientis. We found that a host miRNA (aae-miR-2940) is induced after Wolbachia infection in both mosquitoes and cell lines. One target of aae-miR-2940 is the Ae. aegypti metalloprotease gene. Interestingly, expression of the target gene was induced after Wolbachia infection, ectopic expression of the miRNA independent of Wolbachia, or transfection of an artificial mimic of the miRNA into mosquito cells. We also confirmed the interaction of aae-miR-2940 with the target sequences using GFP as a reporter gene. Silencing of the metalloprotease gene in both Wolbachia-infected cells and adult mosquitoes led to a significant reduction in Wolbachia density, as did inhibition of the miRNA in cells. These results indicate that manipulation of the mosquito metalloprotease gene via aae-miR-2940 is crucial for efficient maintenance of the endosymbiont. This report shows how Wolbachia alters the host miRNA profile and provides insight into the mechanisms of host manipulation used by this widespread endosymbiont.
Project description:West Nile virus (WNV) is an enveloped virus with a single-stranded positive-sense RNA genome from the Flaviviridae family. WNV is spread by mosquitoes and able to infect humans, causing encephalitis and meningitis that can be fatal; it therefore presents a significant risk for human health. In insects, innate response to RNA virus infection mostly relies on RNA interference and JAK/SAT pathways; however, some evidence indicates that it can also involve microRNAs (miRNAs). miRNAs are small noncoding RNAs that regulate gene expression at posttranscriptional level and play an important role in a number of processes, including immunity and antiviral response. In this study, we focus on the miRNA-mediated response to WNV in mosquito cells. We demonstrate that in response to WNV infection the expression of a mosquito-specific miRNA, aae-miR-2940, is selectively downregulated in Aedes albopictus cells. This miRNA is known to upregulate the metalloprotease m41 FtsH gene, which we have also shown to be required for efficient WNV replication. Correspondingly, downregulation of aae-miR-2940 reduced the metalloprotease level and restricted WNV replication. Thus, we have identified a novel miRNA-dependent mechanism of antiviral response to WNV in mosquitoes.A detailed understanding of vector-pathogen interactions is essential to address the problems posed by vector-borne diseases. Host and viral miRNAs play an important role in regulating expression of viral and host genes involved in endogenous processes, including antiviral response. There has been no evidence to date for the role of mosquito miRNAs in response to flaviviruses. In this study, we show that downregulation of aae-miR-2940 in mosquito cells acts as a potential antiviral mechanism in the mosquito host to inhibit WNV replication by repressing the expression of the metalloprotease m41 FtsH gene, which is required for efficient WNV replication. This is the first identification of an miRNA-dependent antiviral mechanism in mosquitoes, which inhibits replication of WNV. Our findings should facilitate identification of targets in the mosquito genome that can be utilized to suppress vector population and/or limit WNV replication.
Project description:The endosymbiont Wolbachia is known to block replication of several important arboviruses, including dengue virus (DENV), in the mosquito vector Aedes aegypti. So far, the exact mechanism of this viral inhibition is not fully understood. A recent study in Drosophila melanogaster has demonstrated an interaction between the pelo gene and Drosophila C virus. In this study, we explored the possible involvement of the pelo protein, that is involved in protein translation, in Wolbachia-mediated antiviral response and mosquito-DENV interaction. We found that pelo is upregulated during DENV replication and its silencing leads to reduced DENV virion production suggesting that it facilities DENV replication. However, in the presence of Wolbachia, specifically in female mosquitoes, the pelo protein is downregulated and its subcellular localization is altered, which could contribute to reduction in DENV replication in Ae. aegypti. In addition, we show that the microRNA aae-miR-2940-5p, whose abundance is highly enriched in Wolbachia-infected mosquitoes, might mediate regulation of pelo. Our data reveals identification of pelo as a host factor that is positively involved in DENV replication, and its suppression in the presence of Wolbachia may contribute to virus blocking exhibited by the endosymbiont.
Project description:The endosymbiont Wolbachia is common among insects and known for the reproductive manipulations it exerts on hosts as well as inhibition of virus replication in their hosts. Recently, we showed that Wolbachia uses host microRNAs to manipulate host gene expression for its efficient maintenance in the dengue mosquito vector, Aedes aegypti. Cytosine methylation is mediated by a group of proteins called DNA (cytosine-5) methyltransferases, which are structurally and functionally conserved from prokaryotes to eukaryotes. The biological functions of cytosine methylation include host defense, genome stability, gene regulation, developmental promotion of organs, and lifespan regulation. Ae. aegypti has only one DNA methyltransferase gene (AaDnmt2) belonging to the cytosine methyltransferase family 2, which is the most deeply conserved and widely distributed gene among metazoans. Here, we show that in mosquitoes the introduced endosymbiont, Wolbachia, significantly suppresses expression of AaDnmt2, but dengue virus induces expression of AaDnmt2. Interestingly, we found that aae-miR-2940 microRNA, which is exclusively expressed in Wolbachia-infected mosquitoes, down-regulates the expression of AaDnmt2. Reversely, overexpression of AaDnmt2 in mosquito cells led to inhibition of Wolbachia replication, but significantly promoted replication of dengue virus, suggesting a causal link between this Wolbachia manipulation and the blocking of dengue replication in Wolbachia-infected mosquitoes. In addition, our findings provide an explanation for hypomethylation of the genome in Wolbachia-infected Ae. aegypti.
Project description:BACKGROUND:The bacterial endosymbiont Wolbachia pipientis has been shown to increase host resistance to viral infection in native Drosophila hosts and in the normally Wolbachia-free heterologous host Aedes aegypti when infected by Wolbachia from Drosophila melanogaster or Aedes albopictus. Wolbachia infection has not yet been demonstrated to increase viral resistance in a native Wolbachia-mosquito host system. METHODOLOGY/PRINCIPAL FINDINGS:In this study, we investigated Wolbachia-induced resistance to West Nile virus (WNV; Flaviviridae) by measuring infection susceptibility in Wolbachia-infected and Wolbachia-free D. melanogaster and Culex quinquefasciatus, a natural mosquito vector of WNV. Wolbachia infection of D. melanogaster induces strong resistance to WNV infection. Wolbachia-infected flies had a 500-fold higher ID50 for WNV and produced 100,000-fold lower virus titers compared to flies lacking Wolbachia. The resistance phenotype was transmitted as a maternal, cytoplasmic factor and was fully reverted in flies cured of Wolbachia. Wolbachia infection had much less effect on the susceptibility of D. melanogaster to Chikungunya (Togaviridae) and La Crosse (Bunyaviridae) viruses. Wolbachia also induces resistance to WNV infection in Cx. quinquefasciatus. While Wolbachia had no effect on the overall rate of peroral infection by WNV, Wolbachia-infected mosquitoes produced lower virus titers and had 2 to 3-fold lower rates of virus transmission compared to mosquitoes lacking Wolbachia. CONCLUSIONS/SIGNIFICANCE:This is the first demonstration that Wolbachia can increase resistance to arbovirus infection resulting in decreased virus transmission in a native Wolbachia-mosquito system. The results suggest that Wolbachia reduces vector competence in Cx. quinquefasciatus, and potentially in other Wolbachia-infected mosquito vectors.
Project description:The gram-negative, pleomorphic endosymbiont Wolbachia is known to infect a large number of insects and other arthropods naturally. This bacterium modifies the host biology, mainly causing reproductive alterations including feminization, death of male, parthenogenesis, and importantly cytoplasmic incompatibility. Wolbachia-induced cytoplasmic incompatibility results in nonviable offspring and vector population suppression. In addition, this bacterium rapidly spreads and propagates within the host population. This study is the first report on Wolbachia detection and characterization from Culex quinquefasciatus collected from Lahore, Pakistan. For this purpose, mosquito adults were collected from different localities of Lahore and identified at the species level. A total of 145 pairs of ovaries were individually subjected to DNA isolation, and polymerase chain reaction amplification of three (wsp, 16S rRNA, and ftsZ) genes were investigated. In all, 128 females were found positive, representing 82.3% infection rate. The phylogenetic analysis indicated that the detected endosymbiont had 100% homology with Wolbachia pipientis wPip strain and supergroup B. The detection of the local strain of Wolbachia (wPip) will be useful in investigating its potential for the control of dengue vector (Aedes aegypti) and reducing dengue transmission in Pakistan.
Project description:Wolbachia pipientis, a maternally transmitted bacterium that colonizes arthropods, may affect the general aspects of insect physiology, particularly reproduction. Wolbachia is a natural endosymbiont of Aedes fluviatilis, whose effects in embryogenesis and reproduction have not been addressed so far. In this context, we investigated the correlation between glucose metabolism and morphological alterations during A. fluviatilis embryo development in Wolbachia-positive (W+) and Wolbachia-negative (W-) mosquito strains. While both strains do not display significant morphological and larval hatching differences, larger differences were observed in hexokinase activity and glycogen contents during early and mid-stages of embryogenesis, respectively. To investigate if glycogen would be required for parasite-host interaction, we reduced Glycogen Synthase Kinase-3 (GSK-3) levels in adult females and their eggs by RNAi. GSK-3 knock-down leads to embryonic lethality, lower levels of glycogen and total protein and Wolbachia reduction. Therefore, our results suggest that the relationship between A. fluviatilis and Wolbachia may be modulated by glycogen metabolism.
Project description:Wolbachia pipientis is an insect endosymbiont known to limit the replication of viruses including dengue and Zika in their primary mosquito vector, Aedes aegypti. Wolbachia is being released into mosquito populations globally in a bid to control the diseases caused by these viruses. It is theorized that Wolbachia's priming of the insect immune system may confer protection against subsequent viral infection. Other hypotheses posit a role for competition between Wolbachia and viruses for host cellular resources. Using an A. aegypti cell line infected with Wolbachia, we tested the effects of targeting siRNAs against the major innate immune pathways on dengue virus loads. We show that while Wolbachia infection induces genes in the Toll, JAK/STAT and RNAi pathways, only reduced expression of RNAi leads to a rebound of dengue virus loads in Wolbachia-infected cells. The magnitude of the effect explained less than 10% of the total DENV load, demonstrating that blocking must be dependent on other factors in addition to the expression of RNAi. The findings bode well for the long-term stability of blocking given that immunity gene expression would likely be highly plastic and susceptible to rapid evolution.
Project description:The Flavivirus genus contains some of the most prevalent vector-borne viruses such as dengue, Zika and yellow fever viruses that cause devastating diseases in humans. However, the insect-specific clade of flaviviruses is restricted to mosquito hosts; albeit they have retained the general features of the genus such as genome structure and replication. The interaction between insect-specific flaviviruses (ISFs) and their mosquito hosts are largely unknown. Pathogenic flaviviruses are known to modulate host-derived microRNAs (miRNAs), a class of non-coding RNAs that are important in controlling gene expression. Alteration in miRNAs may represent changes in host gene expression and provide understanding of virus-host interactions. The role of miRNAs in ISF-mosquito interactions is largely unknown. A recently discovered Australian ISF, Palm Creek virus (PCV), has the ability to suppress medically relevant flaviviruses. Here, we investigated the potential involvement of miRNAs in PCV infection using the model mosquito Aedes aegypti. By combining small RNA sequencing and bioinformatics analysis, differentially expressed miRNAs were determined. Our results indicated that PCV infection hardly affects host miRNAs. Out of 101 reported miRNAs of Ae. aegypti, only aae-miR-2940-5p had significant altered expression over the course of infection. However, further analysis of aae-miR-2940-5p revealed that this miRNA does not have any direct impact on PCV replication in vitro. Thus, the results overall suggest that PCV infection has a limited effect on the mosquito miRNA profile and therefore, they may not play a significant role the PCV- Ae. aegypti interaction. Overall design: To obtain RNA samples from PCV infections, 4 day old Ae. aegypti mosquitoes (Townsville, Australia strain) were injected with 102.3 virus/ml (106 TCID50/ml) in 200 nl of stock PCV diluted in Opti-MEM medium (Gibco, Invitrogen Corporation, Grand Island, NY) with 3% FBS without antibiotics or antimycotics. Control group mosquitoes were injected with the same growth medium without PCV. Mosquitoes were collected and stored at -80°C on days 2, 6 and 12 post-inoculation. For each day and treatment, 15 mosquitoes were collected and divided into three groups of five mosquitoes, which were pooled to create three biological replicates.
Project description:Mosquito-borne arboviruses are a major source of human disease. One strategy to reduce arbovirus disease is to reduce the mosquito's ability to transmit virus. Mosquito infection with the bacterial endosymbiont Wolbachia pipientis wMel is a novel strategy to reduce Aedes mosquito competency for flavivirus infection. However, experiments investigating cyclic environmental temperatures have shown a reduction in maternal transmission of wMel, potentially weakening the integration of this strain into a mosquito population relative to that of other Wolbachia strains. Consequently, it is important to investigate additional Wolbachia strains. All Zika virus (ZIKV) suppression studies are limited to the wMel Wolbachia strain. Here we show ZIKV inhibition by two different Wolbachia strains: wAlbB (isolated from Aedes albopictus mosquitoes) and wStri (isolated from the planthopper Laodelphax striatellus) in mosquito cells. Wolbachia strain wStri inhibited ZIKV most effectively. Single-cycle infection experiments showed that ZIKV RNA replication and nonstructural protein 5 translation were reduced below the limits of detection in wStri-containing cells, demonstrating early inhibition of virus replication. ZIKV replication was rescued when Wolbachia was inhibited with a bacteriostatic antibiotic. We observed a partial rescue of ZIKV growth when Wolbachia-infected cells were supplemented with cholesterol-lipid concentrate, suggesting competition for nutrients as one of the possible mechanisms of Wolbachia inhibition of ZIKV. Our data show that wAlbB and wStri infection causes inhibition of ZIKV, making them attractive candidates for further in vitro mechanistic and in vivo studies and future vector-centered approaches to limit ZIKV infection and spread.IMPORTANCE Zika virus (ZIKV) has swiftly spread throughout most of the Western Hemisphere. This is due in large part to its replication in and spread by a mosquito vector host. There is an urgent need for approaches that limit ZIKV replication in mosquitoes. One exciting approach for this is to use a bacterial endosymbiont called Wolbachia that can populate mosquito cells and inhibit ZIKV replication. Here we show that two different strains of Wolbachia, wAlbB and wStri, are effective at repressing ZIKV in mosquito cell lines. Repression of virus growth is through the inhibition of an early stage of infection and requires actively replicating Wolbachia Our findings further the understanding of Wolbachia viral inhibition and provide novel tools that can be used in an effort to limit ZIKV replication in the mosquito vector, thereby interrupting the transmission and spread of the virus.
Project description:The ability of the endosymbiont Wolbachia pipientis to restrict RNA viruses is presently being leveraged to curb global transmission of arbovirus-induced diseases. Past studies have shown that virus replication is limited early in arthropod cells colonized by the bacterium, although it is unclear if this phenomenon is replicated in mosquito cells that first encounter viruses obtained through a vertebrate blood meal. Furthermore, these cellular events neither explain how Wolbachia limits dissemination of viruses between mosquito tissues, nor how it prevents transmission of infectious viruses from mosquitoes to vertebrate host. In this study, we try to address these issues using an array of mosquito cell culture models, with an additional goal being to identify a common viral target for pathogen blocking. Our results establish the viral RNA as a cellular target for Wolbachia-mediated inhibition, with the incoming viral RNA experiencing rapid turnover following internalization in cells. This early block in replication in mosquito cells initially infected by the virus thus consequently reduces the production of progeny viruses from these same cells. However, this is not the only contributor to pathogen blocking. We show that the presence of Wolbachia reduces the per-particle infectivity of progeny viruses on naïve mosquito and vertebrate cells, consequently limiting virus dissemination and transmission, respectively. Importantly, we demonstrate that this aspect of pathogen blocking is independent of any particular Wolbachia-host association and affects viruses belonging to Togaviridae and Flaviviridae families of RNA viruses. Finally, consistent with the idea of the viral RNA as a target, we find that the encapsidated virion RNA is less infectious for viruses produced from Wolbachia-colonized cells. Collectively, our findings present a common mechanism of pathogen blocking in mosquitoes that establish a link between virus inhibition in the cell to virus dissemination and transmission.