Vacuolar
H+-Pyrophosphatase
Trypanosomatids and apicomplexans
contain subcellular organelles, rich in calcium and
pyrophosphate, which are called acidocalcisomes (Scott et
al, 1998; Docampo and Moreno, 2001). These organelles share
properties with the vacuoles of plants and they all are
characterized by the presence in their membranes of two
proton pumps, a vacuolar H+-ATPase, and a
vacuolar H+-pyrophosphatase (V-PPase). The
V-PPase is an enzyme previously described only in plants and
bacteria but a recent phylogenetic analysis carried out with
the homologous sequences revealed that three available
trypanosomatid sequences were monophyletic with the
sequences of the apicomplexans P. falciparum and
Toxoplasma gondii. Moreover, all the above protists
formed a single clade of type-I V-PPases together with the
green algae Chlamydomonas reinhardtii and Chara
corallina and with plants (see also Drozdowicz
and Rea, 2001) . Further details of the analysis
The alignment
in Phylip format was used for
likelihood
mapping as implemented in
PUZZLE version 4.0.1. The result indicated that the dataset
contained a strong phylogenetic signal with 95.5% of the
quartets fully resolved and only 2.5% of all quartets that
were unresolved. A bootstrapped neigbor-joining tree
(1000 bootstrap samplings), a maximum likelihood (10 000
puzzling steps) and a bootstrapped maximal parsimony tree
(100 bootstrap samplings) were created using the alignment
in Phylip format. All
three trees were robust and
clustered the trypanosomatid sequence together with that of
Toxoplasma, and Plasmodium. In the NJ
tree T. cruzi was paraphyletic with the other two
protists, while in the ML and MP trees all three protists
behaved as a monophyletic group. Clustering together with
the plants and the two chlorophyte alga received 100 %
support in all three phylogenetic inference methods.
Conclusion
A Trypanosoma cruzi vacuolar
H+-dependent pyrophosphatase (VHPPASE)(
Q9NDF0
) was compared with all the sequences of the
Trembl/SwissProt
database indexed at European
Bioinformatics Institute (EBI, Hinxton UK), using the NCBI
BLASTP program and the BLOSUM 62 matrix (http://www2.ebi.ac.uk/blastall/).
Click
here to inspect the BLASTP
output file. The best scoring sequences after that of
T.
cruzi were those of the
plant VHPPASEs and those of Chlamydomonas
reinhardtii (CAC44451) and
Toxoplasma
gondii (Q9BK07), an
incomplete sequence of Leishmania
major (Q9GVF8)and
Plasmodium
falciparum (O97154)
(scores ranging from 779-622, or E=0 - e-177). The best
scoring sequences (E values of 0 to 1e-12 ) were selected
for the creation of an alignment
by Clustal. The alignment was
freed of duplicate and incomplete sequences and gaps and the
resulting alignment
of 31 sequences with 546 sites
was used for further analyses. The program Clustal was used
to calculate a table
with uncorrected pairwise distances.
Pairwise percentages of identity of the T. cruzi
sequence with that of the plants, including the
chlorophyte alga Chlamydomonas reinhardtii was
58-56%, while the sequences of Toxoplasma and
Plasmodium shared between 50% identical residues. All
other sequences shared 44-35% identical residues.
The trypanosome VHPPASE is robustly monophyletic with the
plant and the Apicomplexan VHPPASEs. Support for this comes
from three phylogenetic inference methods and distance
data.