MOLECULAR DIAGNOSIS OF RESISTANCE TO ISOMETAMIDIUM IN TRYPANOSOMA CONGOLENSE

DIAGNOSTIC MOLECULAIRE DE LA RESISTANCE A L’ISOMETAMIDIUM CHEZ TRYPANOSOMA CONGOLENSE

V. Delespaux, D. Geysen & S. Geerts

Institute of Tropical Medicine, Animal Health Department,
Nationalestraat 155, B-2000 , Antwerpen, Belgium 

Résumé

            Le diagnostic de la résistance aux trypanocides a été simplifié et normalisé, mais le recours à l'usage de souris ou de bovins, ou encore l'adaptation des souches aux systèmes de culture in vitro restent nécessaires. Comme les tests in vivo et in vitro pour la détection de la résistance aux trypanocides sont laborieux et de longue durée, il faut de toute urgence recourir à des techniques de diagnostic moléculaire. Un test PCR/ RFLP (Polymerase chain réaction/Restriction fragment length polymorphism) a été mis au point ; il permet de faire la distinction entre les isolats de T. congolense résistants et sensibles à l’isométamidium. Bien que la corrélation entre le test sur souris (pour la détection de la résistance à l’isométamidium) et la PCR/RFLP avoisinait les 85 % pour les 35 isolats testés, certains isolats résistants ne pouvaient pas être identifiés par cette méthode, ce qui montre l’existence d’autres mécanismes de résistance à l’isométamidium chez T. congolense.  Les techniques basées sur la PCR/RFLP constituent des outils intéressants tant dans le cadre de l’évaluation de la chimiorésistance à grande échelle que dans le cadre de la caractérisation individuelle de souches,  mais elles doivent encore être validées et affinées pour permettre la détection de mécanismes alternatifs de résistance à l’isométamidium. L’état actuel et l’avenir du diagnostic moléculaire de la résistance à l’isométamidium seront discutés.

Summary

            The diagnosis of trypanocidal resistance has been simplified and standardised, but tests have to be done  either in mice or in cattle or in vitro. Since both in vivo and in vitro tests for the detection of trypanocidal drug resistance are laborious and time consuming, molecular diagnostic methods for the detection of drug resistance are urgently needed. A polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) test was developed which allows distinguishing isometamidium resistant T. congolense isolates from susceptible isolates. Although the correlation between the mouse test (for isometamidium resistance) and the PCR-RFLP was consistent in about 85.1 % of the 47 isolates tested, some resistant isolates could not be identified, which suggests the existence of more than one mechanism of resistance to isometamidium in T. congolense.  PCR-RFLP based tests are rapid and convenient tools, suitable for large-scale surveys of livestock as well as for individual strain characterization. Further research is necessary, however, to validate the PCR-RFLP test and to develop other molecular tests to detect alternative mechanisms of resistance to isometamidium.

Introduction

            Trypanocidal drug resistance remains a serious problem in trypanosomes of livestock (Geerts et al., 2001). Although several techniques are available for the detection of drug resistance (Geerts & Holmes, 1998), only a few of them are routinely used: the test in mouse or in cattle (Eisler et al., 2001) and the field test (Eisler et al., 2000). These tests are very laborious and time consuming. It takes 2 to 3 months before an answer can be given whether or not a trypanosome isolate is susceptible or resistant to trypanocidal drugs.

            Therefore, a polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) test was developed which allows distinguishing isometamidium resistant T. congolense isolates from susceptible isolates.

Molecular diagnosis of isometamidium resistance

            Recent fingerprint studies (based on Amplified Fragment Length Polymorphism, AFLP) of two isogenic clones of Trypanosoma congolense, the parent clone being sensitive to isometamidium (CD50 equal to 0,018mg/kg in mice) and the derived resistant one presenting a CD50 94-fold higher, showed that at least 58 polymorphic fragments were only present in the derived resistant clone (Delespaux et al., 2005). This indicates that this is likely to be the minimum number of loci at which those two clones differ. Even though those fragments are not all related to isometamidium resistance, response to this trypanocide is most probably a multigenic trait under influence of genes most likely located at different loci as it is the case for chloroquine resistance of the malaria parasite (Dye and Williams, 1997, Babiker et al., 2001).

            Nevertheless, this AFLP study has identified a putative protein that could be involved in the transport of isometamidium. This protein presents 8 transmembrane domains, a signal sequence, a putative ATP binding site, a LSGG motif, a Walker B motif, some homology with the family of the ABC transporters and a conserved GAA codon insertion in the resistant phenotype (Delespaux et al., 2005) that can be revealed by PCR-RFLP as shown in Figure 1.

            The correlation between the single dose mouse test  (for isometamidium resistance) carried out according to the protocol described by Eisler et al (2001) and  the PCR-RFLP test was consistent in  85.1 % of the 47 isolates tested. The origin of the isolates is shown in Table 1. (above).Table 2 shows that in 7 cases isolates, which were identified as resistant in the mouse test, could not be identified as such by the PCR-RFLP test, which suggests the existence of more than one mechanism of resistance to isometamidium in T. congolense

            Interestingly, all sensitive strains described in this study appeared to be heterozygous for the insertion unlike the resistant strains that were all homozygous for the specified insertion. To the best of our knowledge, an increased affinity of a drug efflux transporter was never described as a resistance mechanism in Trypanosoma spp. On the contrary, a lack or a decreased affinity of an influx transporter was extensively described in the literature as it is the case for the purine transporters in T. brucei for example. Thus, in the case of a transporter with decreased affinity, only the homozygous organisms will present an altered transport of the drug whatever the status of expression of the gene. More isolates should be screened to confirm the absence of a sensitive phenotype homozygous for the insertion which would compromise these speculations. A deeper insight in the exact physiological role of this transporter, its expression in presence or absence of isometamedium would certainly bring interestinginformation about this particular resistance mechanism.

Figure 1. Examples of PCR-RFLP profiles of sensitive field strains in L1 to L3, isogenic sensitive strain IL1180 in L6, isogenic resistant strain IL3343 in L7, cloned resistant reference strain TRT57c1 in L8, resistant field strains in L9 to 11. DNA size markers are in lanes L 4, 5 and 12. Arrows show the 384 bp band specific for sensitive strains (Delespaux et al., 2005).

Table 1. Origins of the Trypanosoma congolense isolates used in the study. All isolates are T. congolense savannah type except the Dind isolate which is riverine/forest type.

(a) Institute of Tropical Medicine of Antwerp, (b) Centre International de Recherche - Développement sur l'Elevage en zone Subhumide, (c) International Laboratory for Research on Animal Diseases, (d) International Livestock Research Institute.

(e) x for any number, (f) Democratic Republic of the Congo.

(**)for any letter or number

Table 2. Correlation between sensitivity or resistance to isometamidium chloride based on the single dose mouse test (1 mg/kg) and the presence or absence of the GAA codon (PCR-RFLP test) in 47 T. congolense isolates

Shown in brackets is the proportion of mice which relapsed after treatment with isometamidium chloride at 1 mg/kg

(a) (b) (c)   Not tested in mouse, resistant to 1 mg/kg in cattle: a: Afewerk et al., 2000; b: Clausen et al., 1992; c: Tanenbe, 2005.

*: No GAA insertion means sensitive to isometamidium according to PCR-RFLP. According to the mouse or cattle test, however,  these 7 isolates were identified as resistant

Alternative mechanism of isometamidium resistance

            The main mode of action of isometamidium chloride is the cleavage of kDNA-topo-isomerase complexes causing the desegregation of the minicircle network within the kinetoplast. It was recently shown that two amino acid substitutions within the topo-isomerase I gene conferred resistance to camptothecin, an antitumor compound, in Leishmania donovani (Marquis et al., 2005). This led us to investigate a possible alteration of a T. congolense gene highly similar (Score 5447, smallest sum probability 0) to the gene of T. brucei located on chromosome 11 and coding for a topo-isomerase II. The topo-isomerase II genes of three strains, one sensitive and two resistant to isometamidium were sequenced from the start to the termination codon. DNA sequences were translated to their corresponding amino acid sequences and then compared through clustal alignment (Fig 2).

Figure 2. Translation of the T. congolense gene coding for a Topoisomerase II enzyme corresponding to the T. brucei gene situated on chromosome 11 (Score 5547, Smallest Sum Probability 0), temporary ID Tb11.01.3390.

MAQRTVEEIYQKKTQHEHILARPDMYIGTIEPVTEDMWVYDEADCVMKLQRC
TWTPGLYKIFDEILVNAADNKVRDPHGQTAIKVWIDTERGVIRVYNNGEGIPV
QRHREHDLWVPEMIFGHLLTSSNYDDTEAKVTGGRNGFGAKLTNVFSTCFEL
ETVHSRSRKKFFMRWRNNMLESEEPVITSCDGPDYTMVTFYPDFAKFKLQG
LSEDMALIMKRRVYDIAGCTEKSLSCYLNGEKIPCRSFAEYVDLYPTMGEERR
PGSYSRVNDRWEVCVRVSNIGFQQVSFVNSIATTRGGTHVKYVVEQIIAKVTE
QAMRKSKTEVKPHMIRPHMFVFVNCLIENPSFDSQTKETLNTPKTRFGSVCDL
PASLIDCVLKSSIVERAVEMANSKLSREMAMKLRNSNRKQVLGIPKLDDANEA
GGKNSYRCTLILTEGDSAKALCTAGLAVENRDYFGVFPLRGKPLNVRDASIKK
VMSCAEFQAVSKIMGLDVTQKYTSVEGLRYGHLMIMSDQDHDGSHIKGLIINM
IHNYWPDLLKVPGFLQQFITPIVKARKKSRGNGDEGAISFFSMPDYFEWKNAV
GDNIKNYELRYYKGLGTSGAKEGREYFENIDRHRLNFVYEDKKDDDDIIMAFA
KDKVDERKRWITDFKANTNINESMNYNVRNVTYSEFVHKELILFSVADCERSI
PSVVDE(1)LKPGQRKIMFSAFKRNLVRSIKVAQLAGYVSEHAAYHHGEQSLVQ
TIVGLAQDYVGANNIPLLHRDGQFGTRLQGGKDHAAGRYIFTRLTNIARRIYHP
SDDFVVDYKDDDGLSVEPFYYVPVIPMVLVNGTAGIGTGFATNIPNYSPLDVID
NLRRLLSGDDLRPMKPWYFGFTGTIEEREKGKFVSSGRYTVRPDGVVCITELPI
GTWTSQYKKFLEDLREREVVVQYREHNTDVTVDFEVFIHPEVLQQWTNQGCL
EDKLQLREYIHATNIIAFDREGRITKYLDAESVLKEFYLVRLEYYARRREFLLEQ
LQRSVLKLENMVRFVNEVVNGTFVVTRRPMKEVLADLQQRGYTPFPPQQKK
KVSSTTINDGEEEEAERRHAAANSADAEEAIVLQPDELMGSSEGEGEAPALKR
SARDYDYLLGLRLWNLTAEMSARLLAQLEAARAKYETIAKCSPKDLWREDL
DLLQPEMEKLFDERTKEIAIIQRKKREKKRPFDCSRLRVPLLSDKAREVLRREV
VKEEKKSGRGDASVKNEDGSATGAGRGARGNSGTARRKRKKRSSDDEGDED
FEDFFGESDDDHNFDFGSGMADTAPVPKTSRAKAPAQPRAPRKV(2)EGKTRA
PQVKELKSEQDGKGAIDVEDFDLDCFGIEALTGTVSEHKNTTPRPPLSGTQSK
EKKTGPVGTSAPKAPPRSQAAKAPARSTGKKAPKKRRRAGSEDDSFVVDDS
EDEDEDDDDDNDDDSFNFSD

With square 1 E (glutamate) for the sensitive strain and G (glycine) for the resistant strains JM158 and SA95, square 2 V (valine) for the sensitive IL1180 and G (glycine) for the resistant strains JM158 and SA95.

            The same mutations found in the two resistant strains were sought by PCR-RFLP in the genomes of 25 strains previously characterised for isometamidium sensitivity in the single dose mouse test (Eisler et al., 2001) and by the recently developed PCR-RFLP technique (Delespaux et al., 2005). It appeared that the topo-isomerase II gene corresponding to the gene located on chromosome 11 of T. brucei is highly conserved among the different isolates examined in this study albeit originating from different countries of Africa and presenting a wide range of sensitivity to isometamidium chloride. Results clearly indicate that the sequence differences observed between the sensitive and resistant reference strains cannot be extrapolated to other strains with a known sensitivity profile to isometamidium. As opposed to what happens in Leishmania donovani and in certain cancer cells (Lin et al., 2001), changes in the topo-isomerase II appears thus not to be a strategy adopted by T. congolense to develop resistance to phenanthridines.

Conclusion

            Since more than one mechanism of isometamidium resistance seems to exist, new tools have to be developed which allow to detect T. congolense isolates that show an alternative mechanism of resistance. Furthermore, in analogy to the PCR-RFLP test for the detection of isometamidium resistance in T. congolense, similar tests should be developed to detect resistance in T. brucei and T. vivax.

Acknowledements

            This study was financed by the Fund for Scientific Research, Flanders, Belgium. Our thanks go to ILRI, CIRDES and the Free University of Berlin for providing some of the T. congolense isolates

References

Afewerk, Y., Clausen, P. H., Abebe, G. Tilahun, G. and  Mehlitz, D. ( 2000). Multiple drug resistant Trypanosoma congolense populations in village cattle of Metekel district, north-west Ethiopia. Acta Tropica 76:. 231-238.

Babiker, H. A., Pringle, S. J., Abdel-Muhsin A., Mackinnon M., Hunt P., and Walliker D. (2001). High-level chloroquine resistance in sudanese isolates of Plasmodium falciparum is associated with mutations in the chloroquine resistance transporter gene pfcrt and the multidrug resistance  gene pfmdr1. Journal of Infectious Diseases  183  1535-1538.

Clausen, P.H., Sidibe, I., Kabore, I. and Bauer, B. (1992). Development of Multiple Drug Resistance of Trypanosoma Congolense in Zebu Cattle Under High Natural Tsetse-Fly Challenge in the Pastoral Zone of Samorogouan, Burkina-Faso. Acta Tropica 51,  229-236.

Delespaux, V., Geysen, D. Majiwa, P. A. O. and Geerts, S. (2005). Identification of a genetic marker for isometamidium chloride resistance in Trypanosoma congolense. International Journal for Parasitology 35  235-243.

Dye, C. and Williams, B. G. (1997). Multigenic drug resistance among inbred malaria parasites. Proc R Soc Lond B Biol Sci.  264, 61-67.

Eisler, M.C., McDermott, J.J., Mdachi, R., Murilla, G.A., Sinyangwe, L., Mubanga, J., Machila, N., Mbwambo, H., Coleman, P.G., Clausen, P.H., Bauer, B., Sidibé, I., Geerts, S., Holmes, P.H. and Peregrine, A.S. (2000). Rapid method for the assessment of trypanocidal drug resistance of the field. In: The proceedings of the 9th Symposium of the International Society for Veterinary Epidemiology and Economics (ISVEE 9), Breckenridge, Colorado, 6-11 August.

Eisler, M C, Brandt, J., Bauer, B., Clausen, P. H., Delespaux, V., Holmes, P. H., Ilemobade, A., Machila, N., Mbwambo, H., McDermott, J., Mehlitz, D., Murilla, G., Ndung'u, J. M., Peregrine, A. S., Sidibe, I., Sinyangwe, L. and Geerts, S. (2001). Standardised tests in mice and cattle for the detection of drug resistance in tsetse-transmitted trypanosomes of African domestic cattle. Vet Parasitol.  97,  171-182.

Geerts, S., Holmes P. H. (1998). Drug management and parasite resistance in bovine trypanosomiasis in Africa. PAAT Technical Scientific Series,1, FAO, Rome

Geerts, S. and Holmes, P.H., Diall, O., Eisler, M.C. (2001). African bovine trypanosomiasis: the problem of drug resistance. Trends Parasitol 17, 25-28.

Lin, D. M., Chen, T. Y., Wang, L. F., Hui, C. F., and Hwang, J. (2001).  Characterization of drug resistance to VM-26 in A2780 ovarian carcinoma cells. Zoological Studies (40): 71-78.

Marquis, J. F., Hardy, I. and Olivier, M. (2005). Topoisomerase I amino acid substitutions, Gly185Arg and Asp325Glu, confer camptothecin resistance in Leishmania donovani. Antimicrobial Agents and Chemotherapy. 49, 1441-1446.

Tanenbe, C. (2005). Detection de la résistance à l’isometamidium chez Trypanosoma congolense au Cameroun en utilisant un test de terrain et PCR-RFLP Thèse de Master, No. 32, IMT, Anvers, 26 pp.