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Role of protein disulfide isomerase (PDI) on Toxoplasma host interplay immunolocalization assays using anti human PDI monoclonal antibodies (MAbs) on in vitro culture systems

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Protistology
Roger A.J.1
1
- Dept. of Biochemistry and Molecular Biology,
Dalhousie University, Halifax, Canada
2
- BIOCEV Group, Department of Parasitology,
Faculty of Science, Charles University in Prague
3
- Laboratory of Genomics and Bioinformatics,
Institute of Molecular Genetics, Academy of Sciences
of the Czech Republic
4
- Robert Cedergren Centre for Bioinformatics and
Genomics, Département de Biochimie, Université de
Montréal, Montréal, Canada
5
- Department of Biology and Ecology, Faculty of
Science, University of Ostrava, Czech Republic
m.leger@dal.ca
Bacterial division initiates at the site of a contractile
Z-ring composed of polymerized FtsZ. The location
of the Z-ring in the cell is controlled by a system of
three mutually antagonistic proteins, MinC, MinD,
and MinE. Plastid division is also known to be
dependent on homologs of these proteins, derived
from the ancestral cyanobacterial endosymbiont that
gave rise to plastids. In contrast, the mitochondria
of model systems such as Saccharomyces cerevisiae,
mammals, and Arabidopsis thaliana seem to have
replaced the ancestral alphaproteobacterial Minbased division machinery with host-derived dynamin-related proteins that form outer contractile
rings. By mining transcriptome and genome data,
we show that the mitochondrial division system of
these model organisms is the exception, rather than
the rule, for eukaryotes. We describe endosymbiontderived, bacterial-like division systems comprising
FtsZ and Min proteins in diverse less-studied
eukaryote protistan lineages, including jakobid
and heterolobosean excavates, a malawimonad,
stramenopiles, amoebozoans, a breviate, and an
apusomonad. For two of these taxa, the amoebozoan Dictyostelium purpureum and the jakobid
Stygiella incarcerata, we confirm a mitochondrial
localization of these proteins by their heterologous
expression in Saccharomyces cerevisiae. The discovery of a proteobacterial-like division system in
mitochondria of diverse eukaryotic lineages suggests
that it was the ancestral feature of all eukaryotic
mitochondria and has been supplanted by a hostderived system multiple times in distinct eukaryote
lineages.
DIVERSITY OF PHOTOSYNTHETIC PAULINELLA SPECIES INCLUDING A COMPARATIVE PLASTID GENOME ANALYSIS
Lhee D.H.1, Yang E.C.2, Kim J.I.3, Kim S.4, Park
M.G.4, Andersen R.A.5, Yoon H.S.1
1
- Department of Biological Science, Sungkyunkwan
University, Suwon 16419, Republic of Korea
· 41
2
- Marine Ecosystem Research Division, Korea
Institute of Ocean Sciences & Technology, Ansan
15627, Republic of Korea
3
- Department of Biology, Chungnam National
University, Daejeon 34134, Republic of Korea
4
- Department of Oceanography, Chonnam National
University, Gwangju 61186, Republic of Korea
5
- Friday Harbor Laboratories, University of Washington, WA, 98250, USA
duckhyunlhee@gmail.com
The thecate filose amoeba Paulinella chromatophora
is a good model organism for understanding plastid
organellogenesis because its plastid was derived
from a Synechococcus-Cyanobium type of alphacyanobacterium. Recent studies have shown species-level of divergence after the acquisition of
the organelle; however, a full investigation has not
been conducted for the photosynthetic species. We
surveyed the biodiversity of this interesting alga using
samples collected from around the world. Using four
gene markers (18S rRNA, 16S rRNA, dnaK1, psaL),
two distinct lineages with high genetic variation were
identified, including one new species candidate (i.e.,
P. microporus). In addition, the chromatophore
genome was fully sequenced from P. microporus
strain KR01 and the recently reported marine P.
longichromatophora. Comparative genomic analysis
showed 0.17% of sequence divergence between the
Korean strain KR01 and the Japanese strain FK01.
Among 1,626 variable sites, the divergence was
converged on noncoding regions at a rate seven times
higher than for coding regions. The chromatophore
genome of P. longichromatophora, when compared
to other photosynthetic Paulinella species, showed
a higher mutation rate. These results suggest that
the diversification of the photosynthetic Paulinella
species has occurred at a rapid rate and that the
diversification is still ongoing.
ROLE OF PROTEIN DISULFIDE ISOMERASE
(PDI) ON TOXOPLASMA-HOST INTERPLAY:
IMMUNOLOCALIZATION ASSAYS USING
ANTI-HUMAN PDI MONOCLONAL ANTIBODIES (MABS) ON IN VITRO CULTURE
SYSTEMS
Lobo Maria Luísa1, Novo Carlos2, Matos Olga1
1
- Medical Parasitology Unit, Group of Opportunistic
Protozoa/HIV and Other Protozoa, Global Health
and Tropical Medicine, Instituto de Higiene e Medicina Tropical (IHMT), Universidade Nova de
Lisboa (UNL)
2
- Medical Parasitology Unit, IHMT, UNL, Lisboa,
Portugal.
omatos@ihmt.unl.pt
The main goal of this study is to address the functional
42
· “PROTIST–2016”
roles of PDI on Toxoplasma gondii-host interplay,
in the context of acute/chronic infection, and evaluating their usefulness as drug-targets using antihuman PDI commercial MAbs. The potential crossreactions of PDI anti-human MAbs with T. gondii,
confirming their usefulness for immunolocalization
of PDI not only in human fibroblasts (HFF) target cells, but also in Toxoplasma tachyzoites/cysts
were verified. In order to validate this hypothesis
a bioinformatics analysis was conducted in the
Toxoplasma genome database to search for homologous regions: of the antigens’ complete amino
acid sequence used in the manufacturing processes
of the MAbs utilized in this study; and/or of their
more predicted immunogenic regions in case of
antigens with longer sequences. Confirmation was
obtained by reverse search on human genome for
the predicted T. gondii antigens/peptides sequences.
Results from indirect immunolabeling assays
using immunofluorescence techniques with antihuman PDI MAbs: PDI, PDIA3(ErP57), PDIA6,
Calnexin(CNX), glucose-regulated proteins/immunoglobulin heavy-chain binding protein (GRP78/
BiP) and GRP94 suggest both cross-reaction with
target cell-lines and Toxoplasma, being suitable
for their profile identification and to evaluate
PDI usefulness as a drug-target against T. gondii
infection. Different patterns of immunolabeling
were observed in Toxoplasma infected and noninfected HFF cells according to the MAb used. The
functional confirmation of the characterized PDI
involved in the host-pathogen interaction, in the
presence of PDI inhibitors, is in progress. Further
analyses will be performed by RNA silencing to get
complementary evidence of these specific proteins
in Toxoplasma-host interactions.
Acknowledgments: Supported by FCT ref:VIH/
SAU/0019/2011.
ANALYSIS OF THREE SINGLE-CELL AMPLIFIED GENOMES OF THE CHOANOFLAGELLATE MONOSIGA BREVICOLLIS
REVEALS SINGLE-CELL GENOMICS AS A
LIMITED APPROACH FOR EVOLUTIONARY
PURPOSES
López-Escardó D.1, Ruiz-Trillo I.1,2
1 - Institut de Biologia Evolutiva (CSIC-Universitat
Pompeu Fabra), Passeig Marítim de la Barceloneta
37-49, 08003 Barcelona, Catalonia, Spain
2 - Institució Catalana de Recerca i Estudis Avançats
(ICREA), Barcelona, Catalonia, Spain
david.lopez@upf.edu
Environmental metabarcoding data is providing
a new view of the real protist diversity. This new
diversity often occupies key phylogenetic positions
which can led to important insights in the evolution
of different eukaryotic lineages. This is the case
of the Opisthokonta, in which the identification
of new unicellular lineages close to animals has
the potential to improve our understanding of the
transition towards animal multicellularity. However,
isolating these new organisms is a challenge. Instead,
single-cell genomics (SCG) seems a prominsing
approach to obtain directly the genomes from the
environment. However, SCG technologies, which
have mainly been employed in prokaryotes, are
affected by important biases, specially during the
whole genome amplification step. The potential
of SGC for eukaryotes remains unclear. To test
the potential of this technique for evolutionary
studies in eukaryotes, we here analyze three
environmental single amplified genomes (SAGs)
from the choanoflagellate Monosiga brevicollis,
whose genome is already sequenced. We show the
genome recovery obtained from our SAGs was low
(between 30-5%, around 3,684 out of 9,175 genes).
Interestingly, the percentatge of pylogenomic
markers that our SAGs contains is good (almost
100% for some datasets). We also explored different
techniques to improve the quality and the recovery
of the assemblies obtained. We found that genome
recovery increased up to 48% by pooling the data
from the three different SAGs. Current SCG
technologies seem a limited approach for adressing
some questions but has the potential to provide
enough data for phylogenomic analyses.
DIPLONEMIDS – NEW KIDS ON THE BLOCK
L u ke š Julius 1,2, Flegontova Olga 1,2, Flegontov
Pavel1,2,3, Faktorová Drahomíra1,2, Kaur Binnypreet1,2, Votýpka Jan1,2,4, Tashyreva Daria1,2, Yabuki
Akinori5, Malviya Shruti6, de Vargas Colomban7,8,
Bowler Chris6, Burger Gertraud9, Horák Aleš1,2
1
- Institute of Parasitology, Biology Centre, Czech
Academy of Sciences
2
- Faculty of Science, University of South Bohemia,
České Budějovice
3
- Faculty of Science, University of Ostrava, Ostrava,
Czech Republic
4
- Faculty of Science, Charles University, Prague,
Czech Republic
5
- Japan Agency for Marine-Earth Science and
Technology, Kanagawa, Japan
6
- Ecole Normale Supérieure, PSL Research University, Institut de Biologie de l’Ecole Normale
Supérieure (IBENS), Paris, France
7
- Station Biologique de Roscoff, Roscoff, France
8
- Sorbonne Universités, Paris, France
9
- Université de Montréal, Montreal, Canada
jula@paru.cas.cz
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interplay, using, immunolocalization, antibodies, system, anti, toxoplasma, human, disulfide, vitro, pdi, assays, isomerase, monoclonal, protein, role, culture, mabs, host
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