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Parasitology Section Molecular Basis of Miltefosine Resistance in Leishmaniasis Zusammenfassung Wir streben die Identifizierung der genetischen Grundlagen <strong>für</strong> die Resistenz von Leishmania-Parasiten gegen den neuen Antileishmania-Wirkstoff Miltefosine an. Zu diesem Zweck haben wir ein screening durch funktionelle Komplementation suszeptibler Parasiten durchgeführt. Bis jetzt konnten wenigstens zwei unabhängige Genloci identifiziert werden, deren Amplifikation zur Miltefosin-Reisistenz in vitro führt. Ziel ist die Entwicklung proaktiver Strategien gegen die Entwicklung von klinischer Therapieresistenz. Summary We aim at the identification of genetic traits that lead to resistance to the new antileishmanial drug miltefosine. To this end, we have implemented a screen using functional complementation genetics. So far, at least two independent gene loci were identified that can, upon overexpression, confer miltefosine resistance in vitro. The goal is the development of a proactive strategy against the development of clinical resistance to this newly approved drug. 38 Introduction Visceral leishmaniasis (VL) or Kala Azar (KA) is caused by Leishmania donovani and L. infantum and is reported from about 47 countries around the world. It has an annual incidence of 500,000, and 90% of the cases occur in just four countries: India, Nepal, Bangladesh and Sudan. In India, the neighbour states of Bihar, Uttar Pradesh and West Bengal are highly endemic foci of KA where periodic epidemics are common due to anthroponotic transmission cycles. Over 60% of the cases in Northern Bihar are resistant to traditional antimony therapies and respond only to the expensive Amphotericin B treatment. In India and in other endemic regions, the number of cases of Leishmania-HIV co-infections is increasing due to the simultaneous spread and geographic overlap of both diseases. This new clinical entity is refractory to chemotherapy. This and the increased transmission of Leishmania spp. through contaminated syringes may further contribute to the spread of therapy resistance. A new, orally administered drug, miltefosine, has recently been approved for treatment of KA in India. Miltefosine had been developed as an anti-breast cancer drug, but it shows good efficacy against Leishmania donovani both in vitro and in vivo. However, first reports have emerged of miltefosine resistant breast cancer cell lines and a resistant Leishmania strain. Miltefosine appears to be stable in the human organism at subtherapeutic doses for prolonged periods after treatment, raising concerns about the possible development of resistance in anthroponotic VL. Figure 1: Growth of L. infantum promastigotes in vitro. 1 x 10 6 ml -1 promastigotes were seeded into medium with different concentrations of the drug (µM). Cell density was determined 48 hours later. L. infantum [pcosTL] is a control strain transfected with the vector cosmid, L. infantum [pcosM1], L. infantum [pcosM2], and L. infantum [pcosM12], are strains transfected with cosmids that were selected under miltefosine challenge.
39 Parasitology Section Figure 2: Scanning electron microscopy of L. infantum promastigotes. L. infantum [pcosTL] cells were cultivated in vitro with either 0 µM (A) or 25 µM (B) of miltefosine. Note the morphological alterations and aggregation of cells under miltefosine. By contrast, L. infantum [pcosM2] show very similar morphology to the untreated control and no aggregation either at 0 µM or at 25 µM miltefosine. Project Description and Results With this in mind, we have initiated a comprehensive screening for genes that may confer clinical drug resistance to Leishmania donovani and L. infantum. Cosmid libraries of genomic DNA from both species were prepared in a shuttle vector, pcosTL. DNA from these libraries were electrotransfected into L. donovani and L. infantum, respectively, and the recombinant parasite populations were subjected to miltefosine challenge. Cosmid DNA from the surviving populations was prepared and used to transform competent E. coli bacteria for clonal analysis. Cosmid DNA isolates from individual bacterial clones were then subjected to analytical cleavage with restriction endonucleases to test whether specific cosmids were selected. Indeed, a small number of cosmid prototypes, 4 from each screen, were characterised in L. donovani and L. infantum. This is indicative of the selective pressure applied and implies that only specific DNA sequences can mediate miltefosine resistance. L. infantum promatigotes transfected with individual cosmids indeed showed an increase of the IC50 for miltefosine in vitro by a factor of 2-3 (Figure 1A). Two cosmids derived from the L. infantum screen, pcosM1 and pcosM2, actually possess overlapping genomic DNA segments. This allowed us to narrow down the number of candidate genes on both cosmids to just one, an open reading frame that encodes a 299 kDa protein and that is found on both cosmids. This hypothetical protein does not show conservation of any known sequence motifs, nor did we find homologues genes in any species outside the leishmaniae, so far. We are in the process of expressing this protein in L. infantum and E. coli to verify its function in miltefosine resistance and to produce antibodies that will help to analyse its expression kinetics and subcellular location. Scanning electron microscopy reveals morphological changes, indicative of cellular damage, induced by miltefosine in L. infantum control strains (Figure 2, A-B). By contrast, the presence of the cosmid pcosM2, derived from L. infantum, abrogates these negative effects (Fig- ure 2, C-D). This is further proof of the protective potential of a gene located on this cosmid. In perspective, we aim at the identification of Leishmania genes that are involved in miltefosine resistance. With the emergence of European VL patients that are refractory to miltefosine treatment it should be possible to analyse parasites isolated from such cases for the amplification or mutation of the identified genes. This should help, in the future, to identify drug resistant parasites by genetic markers prior to treatment and thus reduce treatment failures and the spread of drug resistance. Investigators • Joachim Clos • Kohelia Choudhury, • Andrea Macdonald • Manfred Krömer
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Diary 2004/2005 12.-14. November 20
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