Predictive chemoproteomics and functional validation reveal Coeae6g-mediated insecticide cross-resistance in the malaria vector Anopheles gambiae

The study addresses the urgent challenge of rising insecticide resistance in African malaria vectors, particularly Anopheles gambiae, which threatens frontline interventions such as pyrethroid-treated nets. Pirimiphos-methyl (PM), an organophosphate used in indoor residual spraying, has been increasingly deployed, yet resistance is emerging in West Africa. The research question is whether a predictive, functionally driven chemoproteomics approach can identify enzyme-mediated resistance mechanisms to PM and related chemistries before they are established in field populations. The authors propose activity-based protein profiling (ABPP) to directly map active serine hydrolases (SHs) and their interactions with PM and its bioactive metabolite PMO in a susceptible An. gambiae strain, aiming to anticipate resistance liabilities and guide resistance-informed vector control strategies.

The paper situates PM as a pro-insecticide requiring P450-mediated bioactivation to PMO, which inhibits acetylcholinesterase (AChE; Ace1). Resistance mechanisms include Ace1 mutations/duplications and overexpression of detoxification enzymes (P450s, carboxylesterases), with CCEs either hydrolyzing ester bonds or sequestering OPs via phosphorylation. In An. gambiae, a large CCE family (>50 genes) is implicated in resistance to organophosphates, carbamates, and sometimes pyrethroids. Conventional genomic/transcriptomic approaches are retrospective, revealing mechanisms after resistance becomes established. Prior work has linked CNVs in CCE clusters (e.g., Coeae2g–Coeae6g) and Ace1 variants to PM resistance. ABPP has been used in pharmacology/toxicology and to profile pyrethroid-metabolizing enzymes and P450 activities, suggesting its potential utility in vector biology to functionally map resistance-associated enzymes. The authors highlight gaps in predicting liabilities to new chemistries and the need for proactive functional mapping in susceptible populations.

• Dual-probe ABPP in An. gambiae (Kisumu strain): TAMRA-tagged FP (T-FP) for in-gel fluorescence and desthiobiotin-FP for affinity enrichment followed by LC-MS/MS. Homogenates from 3–5-day-old females were prepared, normalized, and incubated with PM, PMO, FP probes, or DMSO, with heat-denatured controls to confirm activity-dependent labeling.
• Competitive ABPP: Pre-incubation with PMO (near-saturating conditions; 100 µM) followed by FP labeling to detect proteins with reduced probe binding (indicative of PMO target engagement). Streptavidin enrichment, on-bead reduction/alkylation, trypsin digestion, and LC-MS/MS on Orbitrap Exploris 480. Data processed with MaxQuant (MaxLFQ), statistics via LFQ-Analyst (log2 transform, MNAR imputation, BH-adjusted p ≤ 0.05, |log2FC| ≥ 1).
• Subcellular localization: Crude fractionation from thoraxes to separate cytosolic and mitochondrial fractions; Western blots with anti-Coeae6g, anti-ATP5A (mitochondrial), and anti-alpha-tubulin (cytosolic).
• Recombinant expression: An. coluzzii Coeae6g cloned into baculovirus vector; expression in Sf9 cells; verification by Western blot. Esterase activity measured against α-NA, β-NA, and p-NPA; kinetic parameters (Km, Vmax) determined for α-NA and β-NA.
• Inhibition kinetics: α-NA hydrolysis by recombinant Coeae6g challenged with PM, PMO, malathion, malaoxon, bendiocarb, propoxur, permethrin, deltamethrin; IC50 values calculated.
• Transgenic validation: Construction of UAS-Coeae6g responder lines (An. gambiae and An. coluzzii coding sequences) via ΦC31-mediated cassette exchange into Ubi-GAL4 or A11 docking lines; crosses with Ubi-GAL4 driver for ubiquitous expression.
• Expression verification: qPCR for transcription (fold-change vs controls), Western blot for protein abundance, total esterase activity assays (p-NPA, β-NA) in overexpressing vs parental lines.
• WHO tube bioassays: Diagnostic doses to assess PM resistance; probit analyses to estimate LD50 (dose for 50% mortality) or LT50 (time for 50% mortality); calculation of resistance ratios (RR50) vs control lines. Cross-resistance tested for bendiocarb, propoxur, malathion, permethrin, deltamethrin with time-response assays where needed.

• ABPP mapping: 380 proteins identified; 298 significantly enriched by FP vs heat-denatured controls (adjusted p ≤ 0.05, log2FC ≥ 1). Of these, 73 were serine hydrolases (SHs), representing 24.5% of the FP-labeled proteome. Enriched domains included Alpha/Beta hydrolase folds; known OP targets (AChE1/2), CCEs, lysophospholipases, and fatty acid synthases were detected.
• Competitive ABPP with PMO: 23 proteins showed significantly reduced FP labeling; 18 were putative SHs. AChE1/2 were strongly inhibited. PMO targeted eight CCEs; Coeae5g, Coebe2o, Coe09916, and Coeae6g exhibited the most profound inhibition (near background levels).
• Coeae6g localization: Western blot fractionation indicated cytosolic localization, contradicting some computational predictions of mitochondrial targeting.
• Recombinant Coeae6g kinetics (α-NA, β-NA): Kmα-NA = 68.49 µM (95% CI: 57.19–81.73); Vmaxα-NA = 307.7 nmol min⁻¹ mg⁻¹ (95% CI: 297.3–318.3). Kmβ-NA = 369.4 µM (95% CI: 306.0–446.5); Vmaxβ-NA = 253.0 nmol min⁻¹ mg⁻¹ (95% CI: 238.2–269.3). Coeae6g expression in Sf9 increased esterase activity 8–12-fold vs controls.
• Inhibition of Coeae6g by insecticides (IC50, µM): PMO 0.03 (0.024–0.031); malaoxon 0.24 (0.23–0.25); bendiocarb 7.27 (6.43–8.19); propoxur 3.52 (3.06–4.05); permethrin 129.7 (127.5–131.9). No inhibition detected up to solubility limits for PM (≤450 µM), malathion (≤45 µM), deltamethrin (≤450 µM).
• Transgenic overexpression: qPCR showed 71-fold (An. gambiae) and 81-fold (An. coluzzii) Coeae6g overexpression vs controls (p < 0.01). Total carboxylesterase activity increased 2.9-fold against p-NPA and 2.3-fold against β-NA (p < 0.0001).
• PM resistance in vivo: WHO bioassays showed reduced mortality in Coeae6g-overexpressing lines (Ang: 67% mortality; Anc: 79% mortality). Probit analyses: Ang LD50 = 2.4% (95% CI: 1.5–7.4) vs control 0.28% (0.25–0.32), RR = 8.6 (6.0–23.1). Anc LT50 = 39.6 min (34.2–45.3) vs control 5.2 min (4.0–6.4), RR = 7.6 (6.4–8.8).
• Cross-resistance (Anc line): Time-response RRs—bendiocarb RR = 2.2 (1.9–2.5), malathion RR = 4.6 (3.8–5.5), permethrin RR = 3.0 (2.2–4.1). No defined resistance to deltamethrin; short exposures (10–15 min) yielded 100% mortality in both strains.

By directly profiling the active SH proteome in a susceptible An. gambiae strain using FP-based ABPP, the study provides forward-looking evidence of enzyme-mediated liabilities to PM and other insecticides before resistance is phenotypically apparent. The identification of 18 PMO-inhibited SHs, including canonical targets (AChE1/2) and multiple CCEs, demonstrates ABPP’s specificity and functional relevance. Coeae6g emerged as a central candidate showing strong PMO sensitivity, cytosolic localization consistent with xenobiotic metabolism, and biochemical inhibition by oxon forms (PMO, malaoxon) but not thio-phosphate precursors (PM, malathion), supporting a sequestration/inactivation mechanism. In vivo, Coeae6g overexpression confers PM resistance above WHO thresholds and mediates cross-resistance to permethrin and carbamates, aligning with genomic signals (CNVs in the Coeae2g–Coeae6g cluster) observed in field populations. While laboratory effects are low-to-moderate, field resistance may be amplified via synergy with target-site mutations (e.g., Ace1), underscoring the need to integrate ABPP insights with genomic surveillance. The approach complements sequencing-based methods by capturing functional protein activity and enzyme target engagement, bridging genotype–phenotype gaps and informing insecticide rotation and dual-chemistry strategies.

The study establishes a predictive chemoproteomic framework using FP-based ABPP to functionally profile serine hydrolase activity and insecticide–protein interactions in An. gambiae. It identifies both established and previously underappreciated enzymes associated with PM toxicity and resistance, validates Coeae6g as a mediator of PM resistance and cross-resistance to malathion (via malaoxon), carbamates, and permethrin, and demonstrates in vivo resistance in transgenic mosquitoes. ABPP provides actionable, species-agnostic insights that complement genomic surveillance, offering a scalable tool to anticipate resistance liabilities and guide resistance-informed vector control. Future work should expand ABPP to diverse field populations, developmental stages, tissues, and additional enzyme classes (e.g., P450s, GSTs) using tailored probes, enabling comprehensive resistance mechanism mapping and improved operational decisions.

The demonstration is limited to serine hydrolase activity in a laboratory-susceptible An. gambiae strain, which may not capture the full complexity and variability of resistance mechanisms in genetically diverse field populations. ABPP here focuses on SHs; contributions from other enzyme families (e.g., P450s, GSTs) were not profiled. The resistance conferred by Coeae6g overexpression was low-to-moderate under laboratory conditions and may depend on synergy with other mechanisms (e.g., Ace1 mutations) in the field. Computational predictions of Coeae6g localization were contradicted by experimental data, highlighting potential annotation uncertainties. Validation across operational settings and longitudinal studies is needed to assess robustness and predictive utility.