P-glycoprotein-9-mediated multidrug tolerance in Caenorhabditis elegans

Abstract

Background: The active drug efflux pumps P-glycoproteins (PGPs) are the cornerstones of multidrug resistance in many organisms. In parasitic helminths, resistance to macrocyclic lactones (MLs) has been associated with pgp regulation and structural defects in amphids. In Caenorhabditis elegans, the nuclear hormone receptor (NHR)-8 also influences xenobiotic tolerance by regulating pgp genes. However, the specific contribution of individual transporters and their regulation remain poorly defined. We recently demonstrated that PGP-9 specifically contributes to ivermectin (IVM) tolerance in an IVM-resistant C. elegans strain. This study aimed to explore the role of PGP-9 in drug efflux in C. elegans.

Methods: We used the IVM-resistant and dye-filling defective (Dyf) C. elegans strain IVR10 and a pgp-9 IVR10 mutant to assess larval development under MLs (eprinomectin (EPR) and moxidectin (MOX)) and tunicamycin (TM). We evaluated whether the Dyf phenotype was affected by pgp-9 deletion. We investigated the role of NHR-8 in regulating pgp-9 via reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and by assessing ML sensitivity in an IVR10 nhr-8 mutant. Additional candidate regulators of pgp-9 were also tested.

Results: IVR10 displayed resistance to MLs and to TM, while pgp-9 deletion restored full drug sensitivity despite the persistence of the Dyf phenotype. Although nhr-8 deletion in IVR10 increased IVM sensitivity, pgp-9 expression was not significantly altered in that strain or IVR10. Interfering RNA (RNAi) targeting pgp-9 in the nhr-8 mutant further increased IVM sensitivity, uncoupling PGP-9 from NHR-8 regulation. Candidate NHRs did not affect IVM tolerance in N2B.

Conclusions: These results provide the first direct evidence that PGP-9 is necessary for multidrug tolerance in C. elegans, independently of amphid structural defects and NHR-8 regulation. These findings uncover a novel mechanism supporting drug resistance and highlight PGP-9 as a potential therapeutic target to improve ML treatments.

Keywords: Caenorhabditis elegans; Amphids; Anthelmintic; Macrocyclic lactones; NHR-8; Nuclear hormone receptor; P-glycoproteins; PGP-9; Resistance.

Fig. 1

Effects of loss of pgp-9 on cross-tolerance of IVR10. Sensitivities of IVR10(Δpgp-9) to moxidectin (MOX) (A), eprinomectin (EPR) (B), and tunicamycin (TM) (C) on larval development assay (LDA). Dose–response curves to each compound (MOX, EPR, and TM) are compared with N2B and IVR10 as controls. Values represent the percentage of L1 reaching the young adult stage in the presence of increasing doses of the drug. Data are mean ± SD from 3–7 independent experiments. IC₅₀ values for each strain are presented in Table 2

Fig. 2

Dye-filling defect phenotype. Young adults N2B (A), IVR10 (B) and IVR10(Δpgp-9) (C) were examined by fluorescence stereomicroscopy to visualize the dye filling of the amphids following staining with DiIC₁₂(3) (DiI). The arrow indicates a set of amphids dyed red because of Dil absorption. D Percentage of dyed amphids, evaluated as the number of stained amphids normalized to the total number of worms treated. Data are expressed as mean ± SD from at least three independent experiments. ns P > 0.05; **** P < 0.0001 (one-way ANOVA followed by Tukey’s post hoc test)

Fig. 3

NHR-8 role in ivermectin tolerance and pgp-9 regulation. A Effects of loss of nhr-8 on tolerance of IVR10 to ivermectin (IVM) on larval development assay (LDA). Dose–response curves are compared with N2B and IVR10 as controls. For N2B and IVR10, values have already been published within a larger dataset [23]. B Quantification of pgp-9 transcripts in N2B, IVR10, and IVR10(Δnhr-8) by RT-qPCR. Data are expressed as fold change to the expression level of pgp-9 in N2B and are normalized against the housekeeping gene tba-1. mRNA transcript levels are presented as mean ± SD from four independent experiments. mRNA levels were compared using a one-way ANOVA followed by Tukey’s post hoc multiple-comparisons test. C Additional effect of pgp-9 silencing on sensitivity to IVM of IVR10(Δnhr-8) on LDA. Worms were fed on HT115 bacteria transformed with either the L440 vector producing double-stranded RNA against pgp-9 or an empty plasmid as a control. Dose–response curve of pgp-9 silencing on IVR10(Δnhr-8) is compared with IVR10(Δnhr-8) fed on HT115-control. Values represent the percentage of L1 reaching the young adult stage in the presence of increasing doses of IVM. Data are mean ± SD from 3 or 4 independent experiments. IC₅₀ values for each strain are presented in Table 3 (A) and Table 4 (B)

Fig. 4

Contributions of NHR-210, NHR-226, and NHR-115 to ivermectin sensitivity. The effects of loss of nhr-210, nhr-226, and nhr-115 in N2B on tolerance to ivermectin (IVM) were assessed on liquid larval development assay (LDA). Values represent the percentage of L1 reaching the young adult stage in the presence of increasing concentrations of IVM. Data are mean ± SD from 3 or 4 independent experiments. IC₅₀ values for each strain are presented in Table 5