The role of drug efflux systems in anthelmintic resistance in parasitic nematodes

Abstract

Trichostrongyloid nematodes have adapted various mechanisms to combat the toxic effects of anthelmintic drugs. These mechanisms can be classified as specific (e.g. modified drug receptors) or non-specific (altered drug metabolism or increased drug transport) mechanisms. Our knowledge of non-specific mechanisms is less advanced as most attention to date has been focused on specific drug target-based resistance mechanisms. ATP binding cassette (ABC) transporters are involved in active efflux of anthelmintics, and are considered to be important non-specific mechanisms of resistance. This PhD project aimed to explore the role of ABC transporters in anthelmintic resistance in Haemonchus contortus using a drug-susceptible and two drug-resistant isolates of this nematode. For this purpose, two in vitro parasitological bioassays (larval development and migration assays) were used to explore the effects of multidrug-resistance inhibitors (MDRIs) on the susceptibility of the selected isolates to different anthelmintics. The effects of anthelmintics on expression patterns of ABC transporters were measured by qPCR following pre-exposure of worms to different anthelmintics. The phenotypic consequences of anthelmintic exposure were evaluated using migration assays, and by measuring the effects on efflux of the fluorescent dye Rhodamine-123 (R-123). Finally, a monepantel-resistant isolate (MPL-R) was also phenotypically characterised, and expression patterns of ABC transporters were determined using molecular assays. Significant increases in sensitivity of larvae to anthelmintics were observed in the presence of third generation MDRIs in in vitro assays, particularly with ivermectin (IVM) (synergism ratios up to 6-fold). Several of the inhibitors increased the sensitivity of both a drug-resistant and -susceptible isolate, while others had significant effects on the resistant isolate only. This suggests that some of the inhibitors interact with P-glycoproteins (P-gps) representing intrinsic efflux pathways present across nematode populations with quite different drug sensitivities, while other inhibitors interact with P-gps of significance only to resistant nematodes; hence, most-likely representing an acquired resistance mechanism. Zosuquidar, tariquidar and crizotinib rendered the drug-resistant isolate equally sensitive, or more sensitive, to IVM than the drug-susceptible isolate in migration assays (Chapter 2). Analysis of the data from gene transcription experiments showed that the drug-resistant isolate exhibited over-expression of three P-gp genes (pgp-1, pgp-9.1 and pgp-9.2) as compared to the susceptible isolate. In addition, pre-exposure to IVM and levamisole (LEV) for 3 h significantly increased the expression levels of multiple ABC transporters in the resistant isolate only. In contrast, both isolates showed an increased R-123 efflux following exposure to the drugs, suggesting that the drug exposure stimulated the activity of existing transporter proteins (Chapter 3). In contrast, exposure to monepantel (MPL) significantly increased transcription of multiple ABC transporter genes in both the drug-susceptible and -resistant isolates, not only at 3 h but also at longer drug exposure periods (6 and 24 h). These increases in transcription were consistent for pgp-11, pgp-12 and pgp-14 across all the time points in both isolates. In addition, pgp-11 maintained the elevated levels of upregulation 24 h after the end of 3 h MPL exposure of drug-resistant L3 (Chapter 4). Drug-exposed worms showed an increased ability in a proportion of the larval population to tolerate higher IVM concentrations in subsequent migration assays. This subsequent ability to tolerate higher IVM concentrations following pre-exposure to IVM, LEV and MPL suggests a protective role of some ABC transporters across different chemical entities. The LDA was able to detect resistance to MPL in this isolate and the resistance was shown to exist in two distinct forms, with subpopulations showing resistance factors of 7-fold and 1000-fold compared to the susceptible isolate. This suggests that at least two separate monepantel resistance mechanisms are acting within this isolate, with one or more mechanisms or combinations of mechanisms conferring a much higher level of resistance than the other(s). In the MPL-resistant isolate, the expression level of pgp-11 was significantly decreased as compared to the drug-susceptible isolate, whereas, transcriptions of four ABC transporter genes (pgp-2, pgp-9.2, pgp-11 and mrp-1) were at significantly lower levels in the MPL-resistant isolate as compared to the multi-drug resistant isolate (susceptible to MPL) (Chapter 5). In conclusion, this thesis highlights the capacity of the third generation MDRIs to increase the sensitivity of nematodes to anthelmintics. In addition, the study also demonstrates an interaction between transcription of nematode ABC transporters and anthelmintic drugs. The study is the first published work to indicate an interaction between MPL and ABC transporters in nematodes. The results also show that ABC transport proteins are important in protecting parasitic worms against a variety of structurally unrelated compounds. Further research is needed to: (i) study the in vivo effects of anthelmintic/MDRI combination therapy on the efficacy of anthelmintics, (ii) explore the molecular mechanisms involved in the two MPL-resistant subpopulations, and (iii) discover the physiological functions of ABC transporters to expose their vulnerability as potential drug targets in nematodes.