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

Sofía Moyano,María C Touz,Juliana Musso,Nahuel Zamponi,Marco Lalle,Constanza Feliziani,Adriana Lanfredi-Rangel,Andrea Silvana Ropolo,Lorena Soledad Frontera

doi:10.3390/cells8121600

Abstract

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.

Highlights

We found that the endosomal sorting complex required for transport (ESCRT)-associated protein Vps4a, Rab11, and ceramide play a significant role in the generation of intraluminal vesicles (ILVs) inside the peripheral vacuoles (PVs) and the production of exosome-like vesicles (ElV) in G. lamblia
The results showed a population of vesicles with different sizes and shapes, including a subgroup with an exosome cup-like shape and a diameter of 50–100 nm (Figure 2A), which we termed exosome-like vesicles (ElV), together with abundant smaller vesicles
In search of the participation of the ESCRT machinery in the biogenesis of ILVs and ElVs in Giardia trophozoites, we found that transgenic cells overexpressing the ATP hydrolysis-deficient mutant GlVps4aE228Q or the antisense of glvps4a mRNA showed a reduction of ILV formation on the PVs, which was correlated with the absence of ElV release when compared to wild-type cells

Summary

Introduction

G. duodenalis; G. intestinalis) is a globally distributed protozoan parasite that can inhabit the small intestine of mammals including humans, causing a diarrheal disease known as. Ingestion of G. lamblia cysts present in contaminated water or food or following contact with feces of infected hosts are the common routes of infection [1]. Human giardiasis is the most common cause of diarrheal disease not associated with viruses or bacteria and can affect people with normal or altered immune systems [1]. The cysts release trophozoites that, following active replication, colonize the upper part of the small intestine and cause the symptoms. G. lamblia is an extracellular parasite that adheres to the apical surface of intestinal epithelial cells (IEC) and, by coating the host intestine, impairs the absorption of nutrients and micronutrients, leading to weight loss and to malnutrition, especially in children living in highly endemic areas [2]. The pathogenic effect of trophozoites has been associated with the secretion of several proteins [3,4]

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Conclusion