Evidence of nuclear transport mechanisms in the protozoan parasite Giardia lamblia.

Nuclear-cytoplasmic trafficking of proteins is a highly regulated process that modulates multiple biological processes in eukaryotic cells. In Giardia lamblia, shuttling has been described from the cytoplasm to nuclei of proteins during the biological cell cycle of the parasite. This suggests that a mechanism of nucleocytoplasmic transport is present and functional in G. lamblia. By means of computational biology analyses, we found that there are only two genes for nuclear transport in this parasite, named Importin α and Importin ß. When these transporters were overexpressed, both localized close to the nuclear envelope, and no change was observed in trophozoite growth rate. However, during the encystation process, both transporters induced an increase in the number of cysts produced. Importazole and Ivermectin, two known specific inhibitors of importins, separately influenced the encysting process by inducing an arrest in the trophozoite stage that prevents the production of cysts. This effect was more noticeable when Ivermectin, an anti-parasitic drug, was used. Finally, we tested whether the enzyme arginine deiminase, which shuttles from the cytoplasm to the nuclei during encystation, was influenced by these transporters. We found that treatment with each of the inhibitors abrogates arginine deiminase nuclear translocation and favors perinuclear localization. This suggests that Importin α and Importin ß are key transporters during the encystation process and are involved, at least, in the transport of arginine deiminase into the nuclei. Considering the effect produced by Ivermectin during growth and encystation, we postulate that this drug could be used to treat giardiasis.

Exosome Biogenesis in the Protozoa Parasite Giardia lamblia: A Model of Reduced Interorganellar Crosstalk.

: Extracellular vesicles (EVs) facilitate intercellular communication and are considered a promising therapeutic tool for the treatment of infectious diseases. These vesicles involve microvesicles (MVs) and exosomes and selectively transfer proteins, lipids, mRNAs, and microRNAs from one cell to another. While MVs are formed by extrusion of the plasma membrane, exosomes are a population of vesicles of endosomal origin that are stored inside the multivesicular bodies (MVBs) as intraluminal vesicles (ILVs) and are released when the MVBs fuse with the plasma membrane. Biogenesis of exosomes may be driven by the endosomal sorting complex required for transport (ESCRT) machinery or may be ESCRT independent, and it is still debated whether these are entirely separate pathways. In this manuscript, we report that the protozoan parasite, Giardia lamblia, although lacking a classical endo-lysosomal pathway, is able to produce and release exosome-like vesicles (ElV). By using a combination of biochemical and cell biology analyses, we found that the ElVs have the same size, shape, and protein and lipid composition as exosomes described for other eukaryotic cells. Moreover, we established that some endosome/lysosome peripheral vacuoles (PVs) contain ILV during the stationary phase. Our results indicate that ILV formation and ElV release depend on the ESCRT-associated AAA+-ATPase Vps4a, Rab11, and ceramide in this parasite. Interestingly, EIV biogenesis and release seems to occur in Giardia despite the fact that this parasite has lost most of the ESCRT machinery components during evolution and is unable to produce ceramide de novo. The differences in protozoa parasite EV composition, origin, and release may reveal functional and structural properties of EVs and, thus, may provide information on cell-to-cell communication and on survival mechanisms.

Arginine deiminase has multiple regulatory roles in the biology of Giardia lamblia.

The protozoan parasite Giardia lamblia uses arginine deiminase (ADI) to produce energy from free L-arginine under anaerobic conditions. In this work, we demonstrate that, in addition to its known role as a metabolic enzyme, it also functions as a peptidylarginine deiminase, converting protein-bound arginine into citrulline. G. lamblia ADI specifically binds to and citrullinates the arginine in the conserved CRGKA tail of variant-specific surface proteins (VSPs), affecting both antigenic switching and antibody-mediated cell death. During encystation, ADI translocates from the cytoplasm to the nuclei and appears to play a regulatory role in the expression of encystation-specific genes. ADI is also sumoylated, which might modulate its activity. Our findings reveal a dual role played by ADI and define novel regulatory pathways used by Giardia for survival.

Manipulación del mecanismo de variación antigénica para el desarrollo de una vacuna contra el protozoario intestinal Giardia lamblia

Conclusiones: El silenciamiento de genes correspondientes a enzimas que participan del silenciamiento post-transcripcional lleva a la expresión en superficie y en citoplasma de un amplio repertorio de proteínas variables de superficie. Estos trofozoítos utilizados en esquemas de inmunización y con la ayuda de un adyuvante como CpG-ODN son capaces de generar un tipo de respuesta TH1, observándose altos niveles de IgG específica para Giardia en periferia. Mediante inmunización oral, observamos mucha dispersión en los valores de inmunoglobulinas en cada uno de los animales inmunizados, lo cual no la posiciona como una vía de elección para la administración de vacunas. Sin embargo estos resultados deben repertirse utilizando un mayor número de animales. Con la inmunización nasal no encontramos niveles de IgA considerables ni significativamente diferentes entre los distintos grupos de animales inmunizados, lo cual indica que no sería la vía de inmunización de elección cuando se pretende lograr inmunidad hacia patógenos que ingresan al organismo por la vía oral. No se pueden utilizar proteínas recombinantes como un parámetro de respuesta inmune en este caso en particular.

CpG oligodeoxinucleotides functions as an effective adjuvant in aged BALB/c mice.

During aging, there is an increased rise in susceptibility to infectious diseases. However, it is still unresolved whether standard vaccine adjuvants are efficient in the elderly. We report that immunization with OVA plus synthetic oligodeoxinucleotides containing immunostimulatory CpG motifs (CpG-ODN) stimulates specific Th1 response in aged mice. The immunization with OVA/CpG-ODN induced an increase in CD19+ cells. These cells from aged mice in vivo captured OVA-FITC as efficiently as those from young mice. Interestingly, naïve aged mice, which showed Th2 polarization and up-regulation of the expression of GATA-3, with immunization with OVA/CpG-ODN inducing Th1-specific response but maintaining a Th2 pattern in response to a non-specific stimulator of T cells. Our data suggest that the response elicited by CpG-ODN in aged mice have similar properties to the response developed in young mice, emphasizing the importance of the use of this adjuvant during aging.

Manipulación del mecanismo de variación antigénica para el desarrollo de una vacuna contra el protozoario intestinal Giardia lamblia

Conclusiones: El silenciamiento de genes correspondientes a enzimas que participan del silenciamiento post-transcripcional lleva a la expresión en superficie y en citoplasma de un amplio repertorio de proteínas variables de superficie. Estos trofozoítos utilizados en esquemas de inmunización y con la ayuda de un adyuvante como CpG-ODN son capaces de generar un tipo de respuesta TH1, observándose altos niveles de IgG específica para Giardia en periferia. Mediante inmunización oral, observamos mucha dispersión en los valores de inmunoglobulinas en cada uno de los animales inmunizados, lo cual no la posiciona como una vía de elección para la administración de vacunas. Sin embargo estos resultados deben repertirse utilizando un mayor número de animales. Con la inmunización nasal no encontramos niveles de IgA considerables ni significativamente diferentes entre los distintos grupos de animales inmunizados, lo cual indica que no sería la vía de inmunización de elección cuando se pretende lograr inmunidad hacia patógenos que ingresan al organismo por la vía oral. No se pueden utilizar proteínas recombinantes como un parámetro de respuesta inmune en este caso en particular.

Manipulación del mecanismo de variación antigénica para el desarrollo de una vacuna contra el protozoario intestinal Giardia lamblia

Conclusiones: El silenciamiento de genes correspondientes a enzimas que participan del silenciamiento post-transcripcional lleva a la expresión en superficie y en citoplasma de un amplio repertorio de proteínas variables de superficie. Estos trofozoítos utilizados en esquemas de inmunización y con la ayuda de un adyuvante como CpG-ODN son capaces de generar un tipo de respuesta TH1, observándose altos niveles de IgG específica para Giardia en periferia. Mediante inmunización oral, observamos mucha dispersión en los valores de inmunoglobulinas en cada uno de los animales inmunizados, lo cual no la posiciona como una vía de elección para la administración de vacunas. Sin embargo estos resultados deben repertirse utilizando un mayor número de animales. Con la inmunización nasal no encontramos niveles de IgA considerables ni significativamente diferentes entre los distintos grupos de animales inmunizados, lo cual indica que no sería la vía de inmunización de elección cuando se pretende lograr inmunidad hacia patógenos que ingresan al organismo por la vía oral. No se pueden utilizar proteínas recombinantes como un parámetro de respuesta inmune en este caso en particular.