Fig 1.
Aminophospholipid composition is altered throughout DENV infection in mosquitoes.
(A) Schematic of the multidimensional strategy deployed to profile the mosquito metabolome after DENV infection. Aag2 mosquito cells were infected with DENV at an MOI of 5 and collected at 6, 12, 24 and 48 h post infection (hpi). Mosquitoes were orally infected with 107 pfu/ml of DENV. Midguts and whole mosquitoes were collected at 1, 7 and 14 days post infection (dpi). Metabolic extracts were analyzed by LC-HRMS as in S2 Fig. (B) Venn diagrams show regulated metabolites at the different time points and bars indicate class distribution within tissue levels. Triangles indicate direction of regulation and are color-coded with regards to the time point. Uninfected condition was used as control. (C) Fold changes of annotated and significantly regulated metabolites (|log2 fold change| > 1 and p-value < 0.05) by DENV in cells, midguts and mosquitoes as compared to mock infection. Only metabolites from the general metabolism (i.e., lipid, carbohydrate, amino acid and peptide, nucleotide and nucleoside, sialic acid) are shown. *, indicates metabolite annotations determined by spectral similarity. Carbo., carbohydrate; SA, sialic acid; PL, phospholipid; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PS, phosphatidylserine; PA, phosphatidic acid; PI, phosphatidylinositol; PG, phosphatidylglycerol; LysoPS, lysophosphatidylserine; LysoPC, lysophosphatidylcholine; LysoPE, lysophosphatidylethanolamine; LysoPG, lysophosphatidylglycerol; LysoPI, lysophosphatidylinositol; SM, Sphingomyelin; DAG, Diacylglycerol; MAG, Monoacylglycerol; FAHFA, Fatty Acid ester of Hydroxyl Fatty Acid; NAE, N-acylethanolamine; HEA, Heneicosanoic acid; pep., peptides.
Table 1.
Summary of metabolites detected across mosquito tissues.
¶MS Finder (HMDB, ChEBI, LipidMAPS, LipidBlast) score annotation ≥ 5; * p-value < 0.05 as indicated by unpaired t-test and |log2 Fold Change| ≥ 1.
Fig 2.
DENV infection decreases AGPAT1 but not AGPAT2 expression.
(A) An overview of phospholipid biogenesis. Sequential additions of two acyl-coA to one G3P produce a LysoPA and a PA. Several AGPATs mediate the second addition. PA forms either DAG or CDP-DAG, each generating a different set of phospholipids. DAG produces aminophospholipids by addition of a Cho or Eth group. Aminophospholipids are also produced by acylation of lysoPL and base group modifications. CDP-DAG produces PI and PG, the latter being transformed in CL by combination with a second PG. Phospholipases cleave off an acyl chain from PL to produce lysoPL. G3P, glycerol-3-phosphate; LysoPA, lysophosphatidate; PA, phosphatidate; AGPAT, acyl-sn-glycerol-3-phosphate acyltransferases; DAG, diacylglycerol; CDP-DAG, cytidine diphosphate diacylglycerol; PC, phosphatidylcholine; LPC, lysophosphatidylcholine; Cho, choline; PE, phosphatidylethanolamine; LPE, lysophosphatidylethanolamine; Eth, ethanolamine; PS, phosphatidylserine; LPS, lysophosphatidylserine; PI, phosphatidylinositol; LPI, lysophosphatidylinositol; PG, phosphatidylglycerol; LPG, lysophosphatidylglycerol; CL, cardiolipin; LCL, lysocardiolipin. (B) Maximum likelihood tree between AGPATs from Ae. aegypti and humans. (C) AGPAT1 and AGPAT2 expressions in DENV-infected cells at 24 and 48 hpi with an MOI of 5. Expressions of AGPAT1 and AGPAT2 (D) in midguts at 1, 7 dpi and (E) in whole mosquitoes at 1, 7 and 14 dpi with DENV at 107 pfu/ml. (C-E) Actin expression was used for normalization. Bars show means ± s.e.m from 4 independent wells or 3 pools of 5 midguts or 5 mosquitoes. *, p-value < 0.05; **, p-value<0.01; as indicated by unpaired t-test.
Table 2.
Acyltransferase motif comparison between human and Ae. aegypti AGPAT homologues.
Purple indicates amino acid residues highly conserved across all hAGPATs and yellow in hAGPAT1 and 2 only.
Fig 3.
AGPAT1 but not AGPAT2 depletion increases DENV multiplication and aminoPL in cells.
Aag2 cells were transfected with either dsRNA against AGPAT1 or 2 (dsAGPAT1 or 2) or with dsRNA control (dsControl). At 24 h post transfection, cells were infected with DENV at MOI of 1 or mock infected. Supernatant was collected at 48 hpi. Expressions of (A) AGPAT1 and (B) AGPAT2 in mock-infected cells at 72 h post transfection. Actin expression was used for normalization. Bars show mean ± s.e.m. (C) Number of phospholipid-related metabolites significantly regulated. (D) Impact of AGPAT1 or 2 depletion on the lipidome of mock-infected cells at 72 h post transfection. *, p-value <0.05 and |log2 fold change| > 1. (E) Impact of AGPAT1 or 2 depletion on DENV production at 48 hpi as determined by plaque forming unit (pfu) assay. Bars show mean ± s.e.m. and each point represents independent wells. (F) Impact of AGPAT1 or 2 depletion on the lipidome of infected cells at 48 hpi. *, p-value < 0.05 and |log2 fold change| > 1. (A, B, D and F) result from three biological replicates. (A, B) ***; p-value < 0.001; ****, p-value < 0.0001, as indicated by unpaired t-test. (E) **, p-value < 0.01, as indicated by Dunnett’s test. PE, phosphatidylethanolamine; PC, phosphatidylcholine; PS, phosphatidylserine; LPE, lysophosphatidylethanolamine.
Fig 4.
AGPAT1 depletion increases DENV multiplication and consumption of aminoPLs in mosquitoes.
Mosquitoes were injected with either dsRNA against AGPAT1 (dsAGPAT1) or dsRNA control (dsControl). Two days later, mosquitoes were orally fed with non-infectious blood or infectious blood containing DENV at 107 pfu/ml. (A) Validation of AGPAT1 silencing in non-infected mosquitoes at two days post dsRNA injection. Actin expression was used for normalization. Bars show mean ± s.e.m. from 3 pools of 5 mosquitoes each. ****, p-value < 0.0001 as indicated by unpaired t-test. (B) Impact of AGPAT1 depletion on the lipidome of mosquitoes at 2 and 7 days post non-infectious blood feeding (dpb). Impact of AGPAT1 depletion on (C) DENV gRNA copies and (D) viral load measured as pfu/ml at 7 days post oral infection (dpi). Bars indicate mean (C) or geometric means ± 95% CI (D) with each dot representing one mosquito. ***, p-value < 0.001 ****, p-value < 0.0001 as indicated by unpaired t-test (C) or Mann-Whitney test (D). (E) Impact of AGPAT1 depletion on the lipidome of infected mosquitoes at 7 dpi. (F) Impact of infection on the lipidome of AGPAT1-depleted mosquitoes at 7 dpi. (B, E, F) Only regulated metabolites are shown (p-value < 0.05 and |log2 fold change| > 1). PL, phospholipid; FA; fatty acyl; AC, acylcarnitine; DAG, diacylglycerol; PA, phosphatidic acid; PS, phosphatidylserine; LPE, lysophosphatidylethanolamine; LPC, lysophosphatidylcholine; LPS, lysophosphatidylserine; LPG, lysophosphatidylglycerol; LPI, lysophosphatidylinositol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PI, phosphatidylinositol; GPEtn, glycerophosphoethanolamine.