Comparative study of transcription profiles of the P-glycoprotein transporters of two Haemonchus contortus isolates: Susceptible and resistant to ivermectin

https://doi.org/10.1016/j.molbiopara.2020.111281Get rights and content

Highlights

  • H. contortus resistant and sensible to IVM was characterized by in vitro mortality.

  • Gene expression of 10 P-gp´s to H. contortus stages resistant to IVM was different.

  • P-gp 1, 11 and 12 are important in H. contortus stages to IVM resistance.

  • P-gp16 was the most frequent upregulated gen, suggesting its role in IVM resistance.

  • P-gp16 might be use as molecular marker to IVM resistance in Mexican isolates.

Abstract

The objective of this study was to analyze the mRNA transcription levels of ten functional genes of P-glycoproteins (P-gp) in free life stages, eggs and infective larvae (L3) and in endoparasitic stages, fourth larval stage (L4) and adult males of two native isolates of Haemonchus contortus: resistant and susceptible to IVM. The IVM resistant isolate was obtained from sheep naturally infected with H. contortus, and the susceptible isolate (with no pressure to IVM) conserved since 1990. The lethal effect of IVM was evaluated under in vitro conditions, which showed significant differences between susceptible and resistant H. contortus L3 isolates (P < 0.01). The IVM susceptible isolate revealed a lethal effect of 79.22% at 11.42 mM, whereas that resistant isolate showed no lethal effect at any of the four assessed concentrations (1.43, 2.85, 5.71 and 11.42 mM) of IVM. The expression levels of ten Hco-pgp genes (1, 2, 3, 4, 9, 10, 11, 12, 14, and 16) were evaluated in the resistant isolate of H. contortus and compared to the susceptible isolate (as control), using two constitutive genes (GAPDH and β-tubulin). Up-regulation at two statistical significant values (P ≤ 0.05, 0.1) was the criterion to associate IVM resistance with the free life and endoparasitic stages of H. contortus. The expression levels in H. contortus adult nematodes showed 5.64 to 127.56-fold increase for Hco-pgp genes 1, 9, 12, 14, and 16, followed by an increase for Hco-pgp-2 (49.75-fold) and Hco-pgp-10 (106.40-fold) in L4, and for Hco-pgp-16 (2.90-fold) in eggs (P ≤ 0.05). In addition, high expression levels with P < 0.1 were detected in H. contortus L3, L4, and adults for Hco-pgp genes 1, 4, 11, 12, and 16, with changes ranging from 2.17 to 29.72-fold. In conclusion, the highest expression was observed in the adult stage of H. contortus, and the most frequent gene with a significant P-value was Hco-pgp-16, revealing it plays an important role in IVM resistance.

Introduction

The parasitic diseases caused by gastrointestinal nematodes (GIN) are a serious animal health problem in tropical regions, which is aggravated by the increased anthelmintic resistance (AR) observed in the sheep industry [[1], [2], [3]]. The nematode Haemonchus contortus is the primary GIN that affects small ruminants and has a major impact on health and production [4,5]. Since this parasite has high prevalence and resistance to several drugs, it is considered a model for studying nematodiasis control with different types of drugs [6]. The macrocyclic lactones (ML) family comprises two class of compounds: avermectins and milbemycins, with broad-spectrum against endoparasites and ectoparasites, known as “endectocides” [7,8]. Ivermectin (IVM) is the most common xenobiotic drug and it is a compound derived from type B1 avermectins, consisting of 2223-dihydro-avermectin B1a and dihydro-avermectin B1b in a ratio of 80% and 20%, respectively [7,8]. Moreover, cross-anthelmintic resistance has been generated for drugs of the same family with similar action mechanisms, like IVM and moxidectin (MOX), which belong to the ML family. It is known that the ML resistance among GIN populations is the result of different molecular mechanisms, such as: 1) changes in the binding sites on the chloride ion channels (GluCls), 2) enzymatic detoxification processes, and 3) xenobiotic molecule detoxification by P-glycoproteins (P-gp) [9,10,8].

The P-gp are membrane transporters present in many organisms including nematodes, these proteins play an important role in the elimination of toxins and hydrophobic molecules such as IVM and other drugs through the cell efflux pump mechanism. This mechanism depends on ATP hydrolysis [11,12]. Due to the efflux of several molecules, the P-gp are also associated with multidrug resistance family (MDR) and ATP-binding cassette (ABC) [7,13]. In H. contortus 11 functional P-gp genes were identified as P-gp 1, 2, 3, 4, 9, 10, 11, 12, 13, 14 y 16 and they are homologues in the free-living nematode Caenorhabditis elegans except P-gp 16 [[14], [15], [16]]. Among these, P-gp genes 1, 2, 9, and 16 have been identified to have a high association with IVM resistance than others due to the frequent and increased expression levels, likewise they have been functionally characterized with ML transport [13,[17], [18], [19], [20], [21], [22]].

Regarding the H. contortus specie, several genetic expression changes occur in different development stages [23,4], and their study is significant as reference biological material for AR diagnoses and control in GIN [13,14,18,24]. The objective of this study was to analyze the mRNA transcription levels of 10 functional genes of P-gp (1, 2, 3, 4, 9, 10, 11, 12, 14, and 16) in two native isolates of H. contortus, one resistant and a susceptible to IVM in free life stages (eggs and L3) and endoparasitic stages (L4 and adult males) in order to identify the participation of the P-gp genes in the AR.

Section snippets

Haemonchus contortus

Two H. contortus isolates were examined in this study. Each isolate was obtained and its IVM resistance and susceptibility was characterized as follows: The IVM susceptible isolate of H. contortus was kept in cryopreservation as per Campos-Ruelas et al. [25]. It was previously characterized as benzimidazole (BZ) resistant under field conditions in naturally infected sheep with H. contortus in Hueytamalco, Puebla, Mexico (a tropical region). The IVM resistant isolate was obtained from the GIN

Larval mortality in vitro assays

Table 2 shows the result of the IVM concentrations for the in vitro assays, indicating significant differences between isolates of H. contortus L3 (P < 0.01). In control treatments the highest larval mortalities were 7.33% for triton X-100 and 2.66% for water (P > 0.05).

The IVM susceptible isolate revealed a lethal effect of 79.22% in H. contortus L3 at 11.42 mM, with significant differences (P < 0.01) in comparison to other concentrations (5.71, 2.85, 1.43 mM). The IVM resistant isolate showed

Discussion and conclusion

In this study, the mortality ratio of IVM at different concentrations was calculated in vitro for two H. contortus isolates (L3), IVM resistant and IVM susceptible, as germplasm used for reference diagnose in Mexico. Due to the lipid solubility limitations posed by IVM [33], the highest concentration with lethal effect was 11.42 mM, using EtOH-Triton as diluent, reaching a mortality rate of 79.22 and 5% for the susceptible and resistant isolates of H. contortus L3, respectively. The lethal

Declaration of competing interest

None.

Funding

M. Sc. Reyes-Guerrero DE received a scholarship number 384195/631929 from the Mexican grant institution CONACYT. This project received financial support from the Mexican grants institutions CONACYT-SEP and CENID-SAI, INIFAP under grant numbers 287598 and 20454534898, respectively.

CRediT authorship contribution statement

David E. Reyes-Guerrero: Conceptualization, Formal analysis, Writing - original draft. Rogelio Alonso-Morales: Conceptualization, Formal analysis. Miguel Alonso-Díaz: Conceptualization, Formal analysis. Agustín Olmedo-Juárez: Formal analysis. Pedro Mendoza-de-Gives: Formal analysis. Ma. Eugenia López-Arellano: Conceptualization, Formal analysis, Writing - original draft.

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