Dataset Information

Functional Validation of Endogenous Redox Partner Cytochrome P450 Reductase Reveals the Key P450s CYP6P9a/-b as Broad Substrate Metabolizers Conferring Cross-Resistance to Different Insecticide Classes in Anopheles funestus.

ABSTRACT: The versatility of cytochrome P450 reductase (CPR) in transferring electrons to P450s from other closely related species has been extensively exploited, e.g., by using An. gambiae CPR (AgCPR), as a homologous surrogate, to validate the role of An. funestus P450s in insecticide resistance. However, genomic variation between the AgCPR and An. funestus CPR (AfCPR) suggests that the full metabolism spectrum of An. funestus P450s might be missed when using AgCPR. To test this hypothesis, we expressed AgCPR and AfCPR side-by-side with CYP6P9a and CYP6P9b and functionally validated their role in the detoxification of insecticides from five different classes. Major variations were observed within the FAD- and NADP-binding domains of AgCPR and AfCPR, e.g., the coordinates of the second FAD stacking residue AfCPR-Y⁴⁵⁶ differ from that of AgCPR-His⁴⁵⁶. While no significant differences were observed in the cytochrome c reductase activities, when co-expressed with their endogenous AfCPR, the P450s significantly metabolized higher amounts of permethrin and deltamethrin, with CYP6P9b-AfCPR membrane metabolizing α-cypermethrin as well. Only the CYP6P9a-AfCPR membrane significantly metabolized DDT (producing dicofol), bendiocarb, clothianidin, and chlorfenapyr (bioactivation into tralopyril). This demonstrates the broad substrate specificity of An. funestus CYP6P9a/-b, capturing their role in conferring cross-resistance towards unrelated insecticide classes, which can complicate resistance management.

SUBMITTER: Ibrahim SS

PROVIDER: S-EPMC11311542 | biostudies-literature | 2024 Jul

REPOSITORIES: biostudies-literature

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Functional Validation of Endogenous Redox Partner Cytochrome P450 Reductase Reveals the Key P450s CYP6P9a/-b as Broad Substrate Metabolizers Conferring Cross-Resistance to Different Insecticide Classes in Anopheles funestus.

Ibrahim Sulaiman S SS Kouamo Mersimine F M MFM Muhammad Abdullahi A Irving Helen H Riveron Jacob M JM Tchouakui Magellan M Wondji Charles S CS

International journal of molecular sciences 20240725 15

The versatility of cytochrome P450 reductase (CPR) in transferring electrons to P450s from other closely related species has been extensively exploited, e.g., by using An. gambiae CPR (AgCPR), as a homologous surrogate, to validate the role of An. funestus P450s in insecticide resistance. However, genomic variation between the AgCPR and An. funestus CPR (AfCPR) suggests that the full metabolism spectrum of An. funestus P450s might be missed ...[more]

PMID: 39125661

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Project description:Cross-resistance to insecticides in multiple resistant malaria vectors is hampering resistance management. Understanding its underlying molecular basis is critical to implementation of suitable insecticide-based interventions. Here, we established that the tandemly duplicated cytochrome P450s, CYP6P9a/b are driving carbamate and pyrethroid cross-resistance in Southern African populations of the major malaria vector Anopheles funestus. Transcriptome sequencing revealed that cytochrome P450s are the most over-expressed genes in bendiocarb and permethrin-resistant An. funestus. The CYP6P9a and CYP6P9b genes are overexpressed in resistant An. funestus from Southern Africa (Malawi) versus susceptible An. funestus (Fold change (FC) is 53.4 and 17 respectively), while the CYP6P4a and CYP6P4b genes are overexpressed in resistant An. funestus in Ghana, West Africa, (FC is 41.1 and 17.2 respectively). Other up-regulated genes in resistant An. funestus include several additional cytochrome P450s (e.g. CYP9J5, CYP6P2, CYP6P5), glutathione-S transferases, ATP-binding cassette transporters, digestive enzymes, microRNA and transcription factors (FC<7). Targeted enrichment sequencing strongly linked a known major pyrethroid resistance locus (rp1) to carbamate resistance centering around CYP6P9a/b. In bendiocarb resistant An. funestus, this locus exhibits a reduced nucleotide diversity, significant p-values when comparing allele frequencies, and the most non-synonymous substitutions. Recombinant enzyme metabolism assays showed that both CYP6P9a/b metabolize carbamates. Transgenic expression of CYP6P9a/b in Drosophila melanogaster revealed that flies expressing both genes were significantly more resistant to carbamates than controls. Furthermore, a strong correlation was observed between carbamate resistance and CYP6P9a genotypes with homozygote resistant An. funestus (CYP6P9a and the 6.5kb enhancer structural variant) exhibiting a greater ability to withstand bendiocarb/propoxur exposure than homozygote CYP6P9a_susceptible (e.g Odds ratio = 20.8, P<0.0001 for bendiocarb) and heterozygotes (OR = 9.7, P<0.0001). Double homozygote resistant genotype (RR/RR) were even more able to survive than any other genotype combination showing an additive effect. This study highlights the risk that pyrethroid resistance escalation poses to the efficacy of other classes of insecticides. Available metabolic resistance DNA-based diagnostic assays should be used by control programs to monitor cross-resistance between insecticides before implementing new interventions.

Project description:BACKGROUND:It is anticipated that malaria elimination efforts in Africa will be hampered by increasing resistance to the limited arsenal of insecticides approved for use in public health. However, insecticide susceptibility status of vector populations evaluated under standard insectary test conditions can give a false picture of the threat, as the thermal environment in which the insect and insecticide interact plays a significant role in insecticide toxicity. METHODS:The effect of temperature on the expression of the standard WHO insecticide resistance phenotype was examined using Anopheles arabiensis and Anopheles funestus strains: a susceptible strain and the derived resistant strain, selected in the laboratory for resistance to DDT or pyrethroids. The susceptibility of mosquitoes to the pyrethroid deltamethrin or the carbamate bendiocarb was assessed at 18, 25 or 30 °C. The ability of the pyrethroid synergist piperonyl-butoxide (PBO) to restore pyrethroid susceptibility was also assessed at these temperatures. RESULTS:Temperature impacted the toxicity of deltamethrin and bendiocarb. Although the resistant An. funestus strain was uniformly resistant to deltamethrin across temperatures, increasing temperature increased the resistance of the susceptible An. arabiensis strain. Against susceptible An. funestus and resistant An. arabiensis females, deltamethrin exposure at temperatures both lower and higher than standard insectary conditions increased mortality. PBO exposure completely restored deltamethrin susceptibility at all temperatures. Bendiocarb displayed a consistently positive temperature coefficient against both susceptible and resistant An. funestus strains, with survival increasing as temperature increased. CONCLUSIONS:Environmental temperature has a marked effect on the efficacy of insecticides used in public health against important African malaria vectors. Caution must be exercised when drawing conclusions about a chemical's efficacy from laboratory assays performed at only one temperature, as phenotypic resistance can vary significantly even over a temperature range that could be experienced by mosquitoes in the field during a single day. Similarly, it might be inappropriate to assume equal efficacy of a control tool over a geographic area where local conditions vary drastically. Additional studies into the effects of temperature on the efficacy of insecticide-based interventions under field conditions are warranted.

Project description:BackgroundThe Democratic Republic of the Congo (DRC) is characterized as a holoendemic malaria area with the main vectors being Anopheles funestus and members of the Anopheles gambiae complex. Due to political instability and socio-economic challenges in the region, knowledge of insecticide resistance status and resistance mechanisms in these vectors is limited. Mosquitoes were collected from a mining site in the north-eastern part of the country and, following identification, were subjected to extensive testing for the target-site and biochemical basis of resistance. Quantitative real-time PCR was used to assess a suite of 10 genes frequently involved in pyrethroid and dichlorodiphenyltrichloroethane (DDT) resistance in An. gambiae females and males. In An. funestus, gene expression microarray analysis was carried out on female mosquitoes.ResultsIn both species, deltamethrin resistance was recorded along with high resistance and suspected resistance to DDT in An. gambiae and An. funestus, respectively. A total of 85% of An. gambiae carried the kdr mutations as either homozygous resistant (RR) (L1014S, L1014F or both) or heterozygous (RS), however only 3% carried the rdl mutant allele (RS) and no ace-1 mutations were recorded. Synergist assays indicated a strong role for P450s in deltamethrin resistance in both species. In An. gambiae, analysis of transcription levels showed that the glutathione-S-transferase, GSTS1-2, produced the highest fold change in expression (7.6-fold in females and 31-fold in males) followed by GSTE2, thioredoxin peroxidase (TPX2), and cytochrome oxidases (CYP6M2 and CYP6P1). All other genes tested produced fold change values below 2. Microarray analysis revealed significant over-transcription of cuticular proteins as well as CYP6M7, CYP6P9a and CYP6P9b in insecticide resistant An. funestus.ConclusionsThese data show that high levels of deltamethrin resistance in the main malaria vector species, conferred by enzymatic detoxification, are present in the DRC.

Dataset Information

Functional Validation of Endogenous Redox Partner Cytochrome P450 Reductase Reveals the Key P450s CYP6P9a/-b as Broad Substrate Metabolizers Conferring Cross-Resistance to Different Insecticide Classes in Anopheles funestus.

Publications

Functional Validation of Endogenous Redox Partner <i>Cytochrome P450 Reductase</i> Reveals the Key P450s <i>CYP6P9a</i>/-<i>b</i> as Broad Substrate Metabolizers Conferring Cross-Resistance to Different Insecticide Classes in <i>Anopheles funestus</i>.

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