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. 2017 Jan 12;11(1):e0005187.
doi: 10.1371/journal.pntd.0005187. eCollection 2017 Jan.

Engineered Aedes aegypti JAK/STAT Pathway-Mediated Immunity to Dengue Virus

Natapong Jupatanakul  1 , Shuzhen Sim  1 , Yesseinia I Angleró-Rodríguez  1 , Jayme Souza-Neto  1 , Suchismita Das  1 , Kristin E Poti  1 , Shannan L Rossi  2 , Nicholas Bergren  2 , Nikos Vasilakis  2 , George Dimopoulos  1
Affiliations

Engineered Aedes aegypti JAK/STAT Pathway-Mediated Immunity to Dengue Virus

Natapong Jupatanakul et al. PLoS Negl Trop Dis. .

Abstract

We have developed genetically modified Ae. aegypti mosquitoes that activate the conserved antiviral JAK/STAT pathway in the fat body tissue, by overexpressing either the receptor Dome or the Janus kinase Hop by the blood feeding-induced vitellogenin (Vg) promoter. Transgene expression inhibits infection with several dengue virus (DENV) serotypes in the midgut as well as systemically and in the salivary glands. The impact of the transgenes Dome and Hop on mosquito longevity was minimal, but it resulted in a compromised fecundity when compared to wild-type mosquitoes. Overexpression of Dome and Hop resulted in profound transcriptome regulation in the fat body tissue as well as the midgut tissue, pinpointing several expression signatures that reflect mechanisms of DENV restriction. Our transcriptome studies and reverse genetic analyses suggested that enrichment of DENV restriction factor and depletion of DENV host factor transcripts likely accounts for the DENV inhibition, and they allowed us to identify novel factors that modulate infection. Interestingly, the fat body-specific activation of the JAK/STAT pathway did not result in any enhanced resistance to Zika virus (ZIKV) or chikungunya virus (CHIKV) infection, thereby indicating a possible specialization of the pathway's antiviral role.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Generation of transgenic Ae. aegypti over-expressing Dome and Hop under the control of the Vg promoter.
(A) Schematic of the piggyBac (pBac) transformation plasmids used to generate the VgDome and VgHop lines. pBacL, pBacR: pBac left and right arms, 3xP3: eye-specific promoter with either EGFP or DsRed as markers, Vg promoter: vitellogenin promoter, Dome: Dome coding sequence, Hop: Hop coding sequence, TrypT: trypsin terminator sequence. (B) Transcript abundance of transgenes and effector genes in the fat body of VgDome and VgHop lines from before blood feeding (0 hr) up to 48 hpbm. Each bar represents the relative fold change of Dome, Hop or DVRF1 (DENV restriction factor 1), compared between transgenic lines and WT Ae. aegypti. The S7 ribosomal gene was used to normalize cDNA templates. Error bars indicate standard error of the mean.
Fig 2
Fig 2. Effect of JAK/STAT pathway activation on DENV infection in transgenic Ae. aegypti.
The JAK/STAT pathway was induced in the transgenic lines by providing them a naïve bloodmeal; 2 days later, JAK/STAT-activated mosquitoes were orally infected with DENV2 or DENV4. DENV2 titers of the VgDome and VgHop lines were determined for (A) midgut infection at 7 dpibm, (B) disseminated infection at 14 dpibm, and (C) salivary gland infection at 21 dpibm. (D) Midgut DENV2 infection without prior activation of the JAK/STAT pathway through a naïve blood meal at 7 dpibm. (E) Disseminated DENV2 infection of the JAK/STAT pathway-activated hybrid VgDomexVgHop line at 14 dpibm. (F) Disseminated DENV4 infection of the JAK/STAT-activated VgDome and VgHop lines at 14 dpibm. WT mosquitoes were used as a control in parallel in all experiments. Horizontal red lines indicate medians. (G) Prevalence of DENV infection represents data from graphs A-F. Data are pools of results from at least three replicates. Statistical analyses comparing median virus titers were performed using either the Mann-Whitney test or Kruskal-Wallis test with Dunn’s post-test, using Prism software. Statistical analyses comparing virus prevalence were determined by chi-square test. *: p<0.05, **: p<0.01, ***: p<0.001 compared to WT. Descriptive statistics is presented in supplementary S7 Table.
Fig 3
Fig 3. Impact of transgenesis on mosquito fitness.
(A) Lifespans of male and female mosquitoes maintained on 10% sucrose solution, or of female mosquitoes that were provided a bloodmeal to induce transgene expression. Statistical analyses of survival curves was performed using the log rank test with Prism software. ***: p<0.001. (B) Fecundity of WT and transgenic Ae. aegypti, as represented by the number of eggs produced by each female mosquito. Statistical analyses were performed using the Mann-Whitney test with Prism software **: p<0.01 as compared to WT. (C) Expression of vitellogenin at 24 hpbm in the transgenic lines as compared to WT. mRNA levels were measured by real-time PCR, with ribosomal gene S7 as the normalization control. Error bars indicate standard error of the mean.
Fig 4
Fig 4. Fat body transcriptomic profiles of transgenic mosquitoes compared to WT at 24 hpbm.
(A) Number of differentially expressed transcripts between the fat body of transgenic and WT mosquitoes., classified according to functional groups as previously described [8,15]. Abbreviations: CS, cytoskeletal and structural; CSR, chemosensory reception; DIV, diverse functions; DIG, blood and sugar food digestive; IMM, immunity; MET, metabolism; PROT, proteolysis; RSM, redox, stress, and mitochondrion; RTT, replication, transcription, and translation; TRP, transport; UKN, unknown functions. (B) Percentage of genes enriched or depleted, in the fatbody of transgenic lines compared to the WT, for each functional group (C) Venn diagram showing genes significantly regulated in VgDome and VgHop mosquitoes. Green arrows and circle represent the VgDome strain, and red arrows and circle represent the VgHop strain. Upward arrows represent genes significantly enriched, downward arrows represent genes significantly depleted in each strain when compared to WT mosquitoes.
Fig 5
Fig 5. Effect of putative host and restriction factor silencing on DENV susceptibility.
DENV2 titers at 14 dpibm in the disseminated infection (whole mosquito body except midgut) of WT Ae. aegypti after silencing of putative HFs or RFs, as compared to the GFP dsRNA-injected control. Data are pools of three biological replicates, and statistical analyses were performed using the Mann-Whitney test, **: p<0.01, ***: p<0.001 vs. WT mosquitoes. Descriptive statistics is presented in supplementary S8 Table.
Fig 6
Fig 6. Midgut transcriptomic profiles of transgenic mosquitoes compared to WT at 24 hpbm.
Relative gene expression of (A) Fold change in Dome and Hop gene expression (fat body/midgut) (B) Fold change in DVRF1 gene expression in the midgut of the transgenic lines as compared to WT. (C) Number of differentially expressed transcripts in the midgut of the transgenic lines as compared to WT mosquitoes, classified according to functional groups as previously described [8,15]. Abbreviations: CS, cytoskeletal and structural; CSR, chemosensory reception; DIV, diverse functions; DIG, blood and sugar food digestive; IMM, immunity; MET, metabolism; PROT, proteolysis; RSM, redox, stress, and mitochondrion; RTT, replication, transcription, and translation; TRP, transport; UKN, unknown functions. (D) Percentage of genes enriched or depleted in each functional group in the midguts of the VgDome or VgHop mosquitoes as compared to WT.
Fig 7
Fig 7. Effect of JAK/STAT pathway activation on CHIKV and ZIKV infection in the VgHop line.
The JAK/STAT pathway was induced in the VgHop mosquitoes by providing them a naïve bloodmeal; 2 days later, JAK/STAT-activated mosquitoes were orally infected with CHIKV or ZIKV. CHIKV titers of the VgHop line were determined for (A) midgut infection at 7 dpibm, (B) disseminated infection at 7 dpibm, (C) midgut infection at 14 dpibm, and (D) disseminated infection at 14 dpibm. (E) Prevalence of CHIKV infection represents data from Fig 7A–D. ZIKV titers of the VgHop line were determined for (F) midgut infection at 7 dpibm, (G) disseminated infection at 7 dpibm, (H) midgut infection at 14 dpibm, and (I) disseminated infection at 14 dpibm. (J) Prevalence of ZIKV infection represents data from Fig 7F–I. Data are pools of results from 2 replicates. Statistical analyses comparing median virus titers were performed using the Mann-Whitney test with Prism software. Statistical analyses comparing virus prevalence were determined by chi square test. *: p<0.05 compared to WT mosquitoes. Descriptive statistics for CHIKV and ZIKV infection assays are presented in S5 Table.
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