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BIOGRAPHY:
Dr. Briones is Associate Professor of the Department of Microbiology, Immunology and Parasitology at the Federal University of Sao Paulo in Brazil. His Ph.D. in 1994 in molecular biology is also from the Federal University of Sao Paulo. He did postdoctoral research in the Pathology Department at the New York University Medical Center until 1996. In 1999 he was appointed Sequencing Center Coordinator in the Human Cancer Genome Project FAPESP-LICR because of his participation in the Xylella fastidiosa bacteria genome project. His HHMI-funded project concentrates on the evolution and epidemiology of Trypanosoma cruzi and Candida spp. in the Americas from the perspective of molecular phylogenetics.
RESEARCH ABSTRACT SUMMARY:
Molecular Systematics Tools Provide New Insights on Candida
spp. and Trypanosoma cruzi Gene Expression and Epidemiology
1) Application of the penalized likelihood method to a Bayesian
phylogeny of fungi provides a new perspective on phylogenetic
relationships and divergence dates of Ascomycota groups.
The depiction of evolutionary relationships within the Ascomycota
phylum is still controversial because of unresolved polytomies in the
radiation of major taxa. We generated a dataset of 166 small subunit
(18S) rDNA sequences, representative of all groups of fungi, and used
as input in a Bayesian phylogenetic analysis. This phylogeny suggests
that Discomycetes are a basal group of filamentous
Ascomycetes and probably include ancestral characters once their
representatives are intermingled among other filamentous fungi. Also,
we show that the evolutionary rate heterogeneity within Ascomycota
precludes the assumption of a global molecular clock. Accordingly, we
used the penalized likelihood method and included a 400
million-year-old Pyrenomycete fossil and the plant-animal-fungi
split of 1576 Myr for calibration. Our estimates are generally older
than dates proposed in previous studies based on small subunit rDNA
sequences but corroborate estimates from multi-protein analysis,
suggesting that the radiation of the major Ascomycota groups occurred
into the Proterozoic era.
2)Insertional inactivation of CaYLL34, a novel Candida
albicans AAA ATPase–encoding gene, impairs morphology, biofilm, and
secreted aspartyl proteinases activity.
Proper morphology is essential for the ability of Candida
albicans to switch between yeast and hyphae and therefore sustain
its virulence. We identified, by differential screening, a novel C.
albicans AAA ATPase–encoding gene, CaYLL34, with enhanced
expression in hyphae. Phylogenetic analysis suggests that
CaYLL34 belongs to a nuclear VCP-like subgroup of AAA
ATPases and is essential for yeast viability. Inactivation of one copy
of CaYLL34, by the URA3-HisG cassette method, generated
the heterozygous mutant strain M61, which has severe phenotypic
alterations, such as a highly enlarged vacuole, abnormal cell shape,
and reduced growth under different conditions. Also, major
pathogenicity factors are affected in M61, as for example, a
significant decrease in hyphae formation (>90%), surface biofilm
adhesion (86%), and secreted aspartyl proteinases activity (76.5%). The
results show that the partial impairment of CaYLL34p cellular levels is
sufficient to affect appropriate cellular morphology and pathogenicity
factors, suggesting that the product of CaYLL34 gene could be
tested as a novel target for antifungal drugs.
3) Satellite DNA provides direct evidence of Trypanosoma
cruzi genome hybridization.
The evolutionary significance of clonal structure versus genetic
exchange or hybrid strains in natural populations of Trypanosoma
cruzi has long been debated. It has been shown experimentally that
T. cruzi can exchange DNA segments, although the influence and
significance of this observation in Chagas disease epidemiology is
still controversial. Based on indirect evidence from two nuclear gene
haplotypes, it has been suggested that CL Brener, the strain used in
the T. cruzi genome project, is a hybrid. Here we present direct
evidence, based on satellite DNA genealogy, that the genome of T.
cruzi CL Brener strain is composed of two sets of chromosomes, one
set inherited from the major phylogenetic lineage T. cruzi I and
the other set inherited from T. cruzi II, which diverged from
T. cruzi I between 9 and 88 million years ago. Our data also
indicate that T. cruzi satellite sequences are not
chromosome-specific, contrary to what is observed in primates. For the
first time, these results provide direct evidence, using satellite DNA
markers, of genetic introgression in natural populations of T.
cruzi.
4) Phylogenetic evidence of introgression in the T. cruzi
and T. cruzi II complex using direct sequence comparison of five
genetic markers of the hybrid subgroups rDNA 1/2 and Z3.
Trypanosoma cruzi exhibits sufficient genetic diversity to be
considered a complex of at least two different species, T. cruzi
I and T. cruzi II, which were defined by phylogeny and not
merely by typing studies. Additional subgroups of strains have been
described, such as 1/2 (rDNA 1/2) and Z3 (zymodeme 3), whose
positioning within the T. cruzi I and II complex is still
controversial. We cloned and sequenced five genes that encode EF-1alpha
(Elongation Factor-1 alpha); actin; DHFR-TS (dihydrofolate
reductase-thymidylate synthase); TR (trypanothione reductase); and 18S
rRNA from 25 T. cruzi strains. Sequences were used to infer
phylogenies and polymorphisms. Our phylogenetic study shows that T.
cruzi I and T. cruzi II groups are monophyletic, different
from the 1/2 and Z3 subgroups, which cluster with either T.
cruzi I or T. cruzi II strains, as also observed for CL
Brener. The results suggest that strains 1/2 and zymodeme III are
hybrids of T. cruzi I and T. cruzi II parental lineages.
The location of these hybrid subgroup haplotypes in the phylogenies and
their identity suggest that the 1/2 and zymodeme III subgroups have
resulted from introgression of T. cruzi II into T. cruzi
I. Our study shows, furthermore, a higher polymorphism in subgroups
1/2, Z3, and CL Brener. The hybrid nature, high polymorphism, and wide
geographic distribution of subgroups 1/2 and Z3 suggest that the
hybrids, generated by sporadic introgression, have a selective
advantage in the T. cruzi species complex.
Photo: Kent Kallberg, Kallberg Studios
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