Page 101 - Resúmen - XXV Congreso Latinoamericano de Parasitología - FLAP

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Characterization of antigenic -Gal- containing extracellular vesicles (EVs)

from Trypanosoma cruzi

[Caracterización de vesículas extracelulares alfa-galactosiladas
antigénicas de Trypanosoma cruzi]

Nasim Hosseini Karimi, Maria Tays Mendes, Susana Portillo, Brian Grajeda, Cameron
Ellis, and Igor C. Almeida*
Border Biomedical Research Center, Department of Biological Sciences, University of Texas at El Paso, El
Paso, 79968, U.S.A. * Corresponding author: icalmeida@utep.edu

Chagas disease (CD), caused by Trypanosoma cruzi, is chronically affects 6-8 million people worldwide.
Chemotherapy is toxic and partially effective in the chronic stage, and there is no reliable biomarkers
(BMKs) for early assessment of therapeutic outcomes in chronic adult patients. The surface of mammalian
cell-derived infective T. cruzi trypomastigotes (TCTs) is covered by highly abundant glycosylphos-
phatidylinositol (GPI)-anchored glycoproteins, such as mucins, trans-sialidases (TS), and mucin-associate
surface proteins (MASPs). These glycoproteins are known virulence factors, involved in host-cell invasion
and immune evasion, and are released by two major populations of extracellular vesicles (EVs) (i.e.,
exosomes and ectosomes). The total secretome of T. cruzi trypomastigotes have recently been analyzed
by LC-MS/MS and revealed major surface proteins such as TS, MASPs, mucins, GP63, and DGF-1. Highly
immunogenic -Gal-containing epitopes found on trypomastigote GPI-anchored mucins (tGPI-mucins or
TcMUC II) are strongly recognized by patients with CD. These -Gal glycotopes have been used as BMKs
for follow-up of chemotherapy in children and lately in adults with CD, and as prophylactic vaccine targets.
The objective of this study is to identify potential BMKs in TCT-secretome (TCT-Secr) and TCT-EVs that
could be used for CD diagnosis, early assessment of therapeutic outcomes, and vaccine candidates. We
performed proteomic analysis and immunoassays of EVs purified from trypomastigotes from the three major
T. cruzi genotypes (DTU TcI, II, and VI). Methodology: Colombiana (Col, DTU TcI) and Y (TcII) strains, and
CL-Brener clone (CLB, TcVI) were used. TCTSecr was obtained from the supernatant of rhesus LLC-MK2
cells 6-8 days following infection with Y, Col, or CLB tissue culture-derived trypomastigotes (TCTs). TCT-
Secr (10-100 µg protein/sample) was digested with trypsin and subjected to high-resolution LC-MS/MS
proteomic analysis using an Ultimate 3000 nanoUPLC coupled to a QE Plus Orbitrap. Results: We
observed that the TCT-Secr was highlly enriched with proteins that were strongly and specifically reactive
with sera from chronic CD patients. TCT-Secr reactivity with CD sera was reduced ~40-60% following
treatment of antigens with -galactosidase, thus corroborating the presence of the abundant -Gal
glycotopes in the parasite secretome. These glycotopes are highly immunogenic to humans because they
are nonself and trigger high levels of trypanolytic protective Ch anti--Gal antibodies. A subset of highly
enriched -Gal-positive glycoproteins, particularly TcMUC II mucins, was obtained from both Y strain and
CLB clone, in the eluate fraction of an immobilized Bandeiraea simplicifolia isolectin B4 (IB4) column, which
binds to terminal -Gal glycotopes. The -Gal-containing TCT-EVs obtained by IB4-lectin affinity
purification seemed to be devoid of other major GPI-anchored glycoproteins such as MASP, TS, and GP63,
indicating that the -Gal-enriched TCT-EVs have a distinct biogenesis and, most likely, are not located on
the same plasma membrane regions. Funding: National Institutes of Health.

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