Research Group Heussler (Malaria I) - Bernhard-Nocht-Institut für ...

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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