Increased transcription of Glutathione S-transferases in acaricide exposed scabies mites

Abstract

Background: Recent evidence suggests that Sarcoptes scabiei var. hominis mites collected from scabies endemic communities in northern Australia show increasing tolerance to 5% permethrin and oral ivermectin. Previous findings have implicated detoxification pathways in developing resistance to these acaricides. We investigated the contribution of Glutathione S-transferase (GST) enzymes to permethrin and ivermectin tolerance in scabies mites using biochemical and molecular approaches.

Results: Increased in vitro survival following permethrin exposure was observed in S. scabiei var. hominis compared to acaricide naïve mites (p < 0.0001). The addition of the GST inhibitor diethyl maleate restored in vitro permethrin susceptibility, confirming GST involvement in permethrin detoxification. Assay of GST enzymatic activity in mites demonstrated that S. scabiei var. hominis mites showed a two-fold increase in activity compared to naïve mites (p < 0.0001). Increased transcription of three different GST molecules was observed in permethrin resistant S. scabiei var. canis- mu 1 (p < 0.0001), delta 1 (p < 0.001), and delta 3 (p < 0.0001). mRNA levels of GST mu 1, delta 3 and P-glycoprotein also significantly increased in S. scabiei var. hominis mites collected from a recurrent crusted scabies patient over the course of ivermectin treatment.

Conclusions: These findings provide further support for the hypothesis that increased drug metabolism and efflux mediate permethrin and ivermectin resistance in scabies mites and highlight the threat of emerging acaricide resistance to the treatment of scabies worldwide. This is one of the first attempts to define specific genes involved in GST mediated acaricide resistance at the transcriptional level, and the first application of such studies to S. scabiei, a historically challenging ectoparasite.

Figure 1

In vitro bioassays of S. scabiei var. hominis permethrin sensitivity. a) Differences in in-vitro permethrin susceptibility in S. scabiei with different acaricide exposure histories: Permethrin naïve S. scabiei var. suis (n = 100), exposed S. scabiei var. hominis (n = 40) and permethrin resistant S. scabiei var. canis (n = 100). Survival curves are significantly different (p < 0.0001). b) Synergistic activity of DEM. S. scabiei var. hominis mites exposed to permethrin combined with DEM (n = 40) show increased susceptibility compared to mites exposed to permethrin alone (n = 40) (p < 0.0001). Control mites exposed to mineral oil (n = 20) or DEM alone (n = 20) show little mortality over eight hours.

Figure 2

Comparison of glutathione S-transferase enzymatic activity between mite populations. GST activity was determined by a fluorometric assay measuring the conjugation of monochlorobimane to reduced glutathione. Permethrin resistant mites and human mites both display significantly (***, p < 0.0001) elevated GST activity compared to permethrin naïve mites. Bars represent median +/- SEM, n = 3 (protein extracts from female mites).

Figure 3

Up-regulation of GST transcription in permethrin resistant Sarcoptes scabiei. Total RNA was extracted from pools of female mites (n = 4 pools per population) and reverse transcribed; GST transcripts were amplified using GST gene-specific primers in quantitative PCR, and normalised using levels of β-actin transcription. Bars represent mean +/- SE. ***: p < 0.001, **: p < 0.01 when compared to permethrin sensitive controls (b): Crude (non-normalised) fold-changes in expression in resistant compared to sensitive mites, showing little change in β-actin transcription between groups.

Figure 4

Comparison of life-stage specific expression between permethrin sensitive and resistant S. scabiei. Levels of scabies mite GST transcripts from permethrin resistant and sensitive populations were compared across all life stages. GST mu 1 (a), delta 1 (b) and delta 3 (c) are expressed constitutively throughout the mite life-cycle. Resistant mites show increased mRNA at all life stages, with this trend reaching significance in female mites. *:<0.05, n = 3 pools per population.

Figure 5

Up-regulation of gene transcription in S. scabiei following clinical ivermectin exposure. Changes in transcription of GST mu 1, GST delta 3 (a) and P-glycoprotein (b) in adult female mites exposed to ivermectin. Pools of female mites were collected from a crusted scabies patient prior to treatment (n = 4), after one dose of ivermectin (IVM, n = 4), and after two doses of IVM (n = 2). **:p < 0.01, ***:p < 0.001.