Schlüsselwort: Plasmids

Identification and characterisation of p-glycoproteins in small strongyles Cyathostomins or small strongyles are considered to belong to the most important infectious agents of horses due to their high prevalence, widely distributed anthelmintic resistance and the pathogenicy of their third and fourth stage larvae. Resistance has been first recorded for benzimidazoles (BZ). Due to widespread resistance BZ are nowadays only used with limitations to treat cyathostomin infections. Currently macrocylic lactones (ML) are used most frequently for the treatment of worm infections in horses. In spite of their intensive use the efficacy of ML is apparently almost unaltered in Europe. However, first cases of resistance have been reported in South America. Expression of p-glycoproteins (Pgps), which are able to export various xenobiotics out of cells, has been shown to be associated with increased tolerance towards anthelmintics in nematodes such as Haemonchus contortus and Teladorsagia circumcincta. The study presented herein describes the identification and functional analysis of Pgps in small strongyles. Using RT-PCR, full-length cDNA sequences of Pgp-3 were identified in Cylicocyclus elongatus, Cylicocyclus insigne and Cylicostephanus goldi. Additionally, amplification and sequencing of a full length cDNA sequence of Pgp-9 has been accomplished for C. elongatus. All deduced proteins show the characteristics of the ABC transporter super family with two transmembrane and nucleotide-binding domains. Their molecular mass was aprox. 140 kDa. For the first time, nematode Pgps, i.e. CegPgp-3 and CegPgp-9, were expressed in two Saccaromyces cerevisae strains (AD1-7, JRY8012) for functional characterization and identification of Pgp substrates. In both strains different numbers of endogenous ABCtransporters are deleted. The transcription of both Pgps was confirmed by RT-PCR. The detection of Pgp expression by Western Blot failed. Alternatively fluorescence activated cell scanning (FACS) was performed to demonstrate Pgp-expression. The percentage of positive cells was highest for AD1-7 CegPgp-9 and reached max. 20%. For CegPgp-3 only 2.3 – 2.7% positive cells could be detected. JRY8012 showed no expression of CegPgp-3. CegPgp-9 was only expressed in 0.58 – 1.99% of the analyzed cells. Using an automated yeast growth inhibition assay in a 96 well plate, susceptibility of transformed yeast cells to the BZ thiabendazole and the Pgp-inhibitor and –substrate ketoconazol (KCON) was investigated. The assay consists of repeated measurements of OD600 of yeast cultures every 10 min over 48 h. All resulting growth curves were statistically analyzed. A four parametric logistic regression was used to compare relative growth of yeast strains with and without Pgp-expression and to calculate EC50 values. No significant differences were detected for any of the JRY8012 transformants compared to the control strain with LacZ-expression, possibly due to low Pgp-expression in this yeast strain. EC50-values of AD1-7 expressing CegPgp-9 or LacZ differed significantly. Yeasts with expression of CegPgp-9 tolerated a KCON concentration of 0.72 μM without any growth reduction in contrast to the control strain, which showed normal growth in presence of max. 0.18 μM KCON. Furthermore putative interaction of different ML with the heterogously expressed Pgp was analyzed. The results showed a concentration dependent, growth-reducing effect of EPM and IVM on AD1-7 CegPgp-9. MOX did not induce any growth change. For the control strain none of the tested ML concentrations was able to affect growth rates. The combination of KCON (0.18 or 0.73 μM) and ML resulted in a significantly increased and concentration dependent susceptibility to KCON for all tested strains. Putative synergies between KCON and ML could not be analyzed via the applied method. Among other mechanisms a competitive replacement of KCON at one substrate binding site or a ML-induced inhibition of Pgp-activity were conceivable explications. The established, automated, competitive growth assay constitutes a simple and cost-efficient method for functional analysis of heterologous Pgps on protein level. Identification of Pgp substrates and drug-interactions could be of advantage to achieve sustained drug efficacies and the development of new drugs, that are e.g. unaffected by Pgp expression.