This document discusses the parasite Toxoplasma gondii and its dense granule protein 3 (GRA3). It was first discovered in Tunisia in a rodent called gundi. T. gondii can infect mammals including humans and is mainly spread by cats. Central nervous system infection can lead to toxoplasmosis encephalitis. The document investigates how GRA3 promotes endoplasmic reticulum stress-induced apoptosis by activating the PERK pathway. Methods used include real time PCR, immunoblotting and assays to detect apoptosis, viability and associated proteins. Results show GRA3 induces greater cell death and apoptosis through the PERK-CHOP pathway. The conclusions are that molecular biology tests demonstrated

1. Mariana Holguín Bustamante
Tercer semestre

2. TOXOPLASMA GONDII
This parasite of the family Sarcocystidae was first
discovered in Tunisia in a rodent called gundi
Toxoplasma gondii is a neurotropic single-celled
parasite that can infect mammals, including humans.
The main reservoir of Toxoplasma gondii is the
domestic cat (Felis catus), which by means of the
oocysts it removes with feces, spreads the infection
in the environment and can transmit it to many
animals
Central nervous system infection with T.
gondii infection can lead to Toxoplasma
encephalitis.
Severely affects immunosuppressed people
and pregnant women

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SIGNALLING
PATHWAY
PERK
When the expression of proteins exceeds the folding
capacity in the endoplasmic reticulum, causing stress
The UPR transduction signal cascade is activated, which
decreases protein synthesis due to the activation of the
PERK trans-membrane kinase.
These pathways are inhibited by being bound to chaperone
Grp78, however an accumulation of misfolded proteins
causes Grp78 dissociation
PERK dimerizes and autophosphorylates, activating and
phosphorylating to the elongation factor eIF2α
eIF2 inhibits protein synthesis by decreasing the amount
of proteins within ER.
In the case of toxoplasmosis it can induce cell death of neurons
by favoring transcription factors such as CHOP and Noxa, and
activating pro-apoptotic proteins

4. GENERAL OBJECTIVE
Investigate how the Toxoplasma gondii
dense granule protein 3
promotes endoplasmic reticulum
stress‑induced
apoptosis by activating the PERK pathway

5. MÉTODOS
PCR en tiempo real Inmunoblot
GRA3
phospho-PERK (p-PERK)
Proteína regulada por glucosa
(GRP)-78
Escindida caspasa-12, caspasa-3
C/EBP homóloga
Reacción entre un antígeno y un anticuerpo
Detección por electroforesis
Calcular densidades ópticas relativas de
cada banda:
La expresión del gen GRA3 en cepas RH,
ME49, Wh3 y Wh6 se determinó utilizando
reacción en cadena de la polimerasa en
tiempo real
Es utilizada para amplificar y
simultáneamente cuantificar el producto
de un ácido desoxirribonucleico (ADN)
con inmunofluorescencia

6. MÉTODOS
Detección de apoptosis Viabilidad celular
Muestreo de la ausencia de material
celular posterior a inducción de factores
causantes de apoptosis
Se evidencia apoptosis de las células N2a
(neuroblastoma cerebral del ratón) al
usar un citómetro de flujo
Viabilidad celular=
Estudio de las células N2a para ver la
respuesta de sus marcadores y grado de
funcionalidad

7. RESULTADOS
Fluorescencia*
Control: Practicamente negro
pEGFP: Se ve poca muerte celular
pEGFP-GRA3: Mayor muerte celular
Análisis de peso de proteínas*
Control: no proteínas
pEGFP: 28 KD
pEGFP-GRA3: 58 KD

8. RESULTADOS
Proteínas asociadas a apoptosis y a estrés
del RE* Efectos de inhibidores de PERK y caspasa 12
sobre la pérdida de viabilidad celular y
apoptosis*

9. DISCUSSION
AUTHOR
WHAT THE
AUTHOR SAID
AGREE OR
DISAGREE
Craver
and Knoll sought to investigate the importance
of GRA3
in the virulence of T. gondii
Craver MP, Knoll LJ.
Toxoplasmosis is known to be one of the most common
parasitic infections that infect warm-blooded animals,
including humans. Nearly a third of the human population
have been chronically infected with T. gondii
Halonen SK, Weiss
LM.
PERKA TF4-CHOP pathway activation during prolonged
UPR
induces apoptosis
Walter F, Schmid J,
Düssmann H, Concannon
CG, Prehn JHM.

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CONCLUSIONS
It was possible to scientifically
demonstrate how there is activation
of specific signaling pathways that
generate a response in the
formation of proteins thanks to the
use of molecular biology tests like
the immunobloting
The mechanism of action of
Toxoplasma gondii by causing
damage and even neuronal death
could be verified by recognizing the
apoptosis factors activated and tested
in molecular tests, thanks to this, the
medicine can advance in the
treatment for Toxicoplasmosis

11. THANKS!
THANKS!
THANKS!