The minimal ESCRT machinery of Giardia lamblia has altered inter-subunit interactions within the ESCRT-II and ESCRT-III complexes

Saha, Nabanita; Dutta, Somnath; Datta, Shankari Prasad; Sarkar, Soumalya

doi:10.1016/j.ejcb.2017.11.004

Cited by 23 publications

(28 citation statements)

References 70 publications

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“…Moreover, it was shown that the expression of an ATP hydrolysis-deficient Vps4 (VPS4A-E228Q and VPS4B-E235Q) mutant affected MVB formation [67,68] and EV biogenesis [66] in mammalian cells. Besides the reduced set of ESCRT subunits, recent data show that the remaining subunits present in the Giardia genome are expressed in trophozoites and cysts and that at least GlVps25, GlVps2, GlVps4a, GlVps4b, and GlVps4c are functional in yeast, thus arguing in favor of a functional ESCRT machinery in this parasite [22,23]. To investigate the role of the ESCRT complex in exosomal vesicle biogenesis in G. lamblia, we analyzed the participation of the AAA+-ATPase GlVps4a (GlVps4a) using the transgenic trophozoites glvps4a-ha and glvps4a E228Q -ha overexpressing GlVps4a-HA or the ATP hydrolysis-deficient mutant GlVps4a E228Q (GlVps4a E228Q -HA), respectively.…”

Section: The Giardial Glvps4a Protein Is Involved In Elv and Ilv Biogmentioning

confidence: 99%

“…Although the presence of ILVs inside the PVs has been reported [5,[19][20][21], it was not addressed whether the ESCRT machinery is involved. In fact, Giardia harbors a reduced ESCRT machinery with only putative orthologs for the Vps22 and Vps25 (ESCRT-II), the Vps2 and Vps24 (ESCRT-III), and the Vps46(a-b) and the AAA-ATPase Vps4(a-c) identified in its genome [22,23]. The same group had identified a Vps27 putative protein that contains the FYVE domain, which preferentially binds PI3P and of which the expression showed a selective localization in endosomes enriched in PI3P in S. cerevisiae [24] ( Figure 1D).…”

Section: Introductionmentioning

confidence: 99%

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

Moyano

Musso

Feliziani

et al. 2019

Cells

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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.

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Section: The Giardial Glvps4a Protein Is Involved In Elv and Ilv Biogmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Moyano

Musso

Feliziani

et al. 2019

Cells

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“…Each of the three giardial α-SNAPs was expressed and purified from BL21 (DE3) as previously described, except 0.2 mM IPTG was used [ 17 ]. The N-terminal region of GlNSF was also induced with the same concentration of IPTG but was purified from the pellet fraction, as previously described [ 35 ].…”

Section: Methodsmentioning

confidence: 99%

“…Only eight Rab GTPases have been identified in this protist, in contrast to the large repertoire of Rab members in other parasitic protists such as Trichomonas and Entamoeba [ 12 – 15 ]. Even the ESCRT machinery for endosomal sorting is composed of fewer components, with either entire complexes, such as ESCRT-I, being absent, or complexes being composed of fewer subunits, as in the case of ESCRT-II and ESCRT-III [ 16 , 17 ].…”

Section: Introductionmentioning

confidence: 99%

Multiple paralogues of α-SNAP in Giardia lamblia exhibit independent subcellular localization and redistribution during encystation and stress

et al. 2018

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BackgroundThe differently-diverged parasitic protist Giardia lamblia is known to have minimal machinery for vesicular transport. Yet, it has three paralogues of SNAP, a crucial component that together with NSF brings about disassembly of the cis-SNARE complex formed following vesicle fusion to target membranes. Given that most opisthokont hosts of this gut parasite express only one α-SNAP, this study was undertaken to determine whether these giardial SNAP proteins have undergone functional divergence.ResultsAll three SNAP paralogues are expressed in trophozoites, encysting trophozoites and cysts. Even though one of them clusters with γ-SNAP sequences in a phylogenetic tree, functional complementation analysis in yeast indicates that all the three proteins are functionally orthologous to α-SNAP. Localization studies showed a mostly non-overlapping distribution of these α-SNAPs in trophozoites, encysting cells and cysts. In addition, two of the paralogues exhibit substantial subcellular redistribution during encystation, which was also seen following exposure to oxidative stress. However, the expression of the three genes remained unchanged during this redistribution process. There is also a difference in the affinity of each of these α-SNAP paralogues for GlNSF.ConclusionsNone of the genes encoding the three α-SNAPs are pseudogenes and the encoded proteins are likely to discharge non-redundant functions in the different morphological states of G. lamblia. Based on the difference in the interaction of individual α-SNAPs with GlNSF and their non-overlapping pattern of subcellular redistribution during encystation and under stress conditions, it may be concluded that the three giardial α-SNAP paralogues have undergone functional divergence. Presence of one of the giardial α-SNAPs at the PDRs of flagella, where neither GlNSF nor any of the SNAREs localize, indicates that this α-SNAP discharges a SNARE-independent role in this gut pathogen.Electronic supplementary materialThe online version of this article (10.1186/s13071-018-3112-1) contains supplementary material, which is available to authorized users.

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“…The life cycle of this flagellated parasite consists of two evolutionary stages: (i) the trophozoite, which adheres to the intestinal epithelium and multiplies by binary fission, and (ii) the infectious stage, the cyst, that is released through feces and is acquired by ingestion of food or water. Giardia has a simple cell biology, lacking organelles and a typical endosomal sorting complex required for transport, the ESCRT (Saha et al, 2018 ). The parasite accesses nutrients through specialized structures called peripheral vesicles (PV), and the secretion of cyst wall proteins is operated by the encystation-specific vesicles (ESVs) which are absent in non-encysting trophozoites (Gottig et al, 2006 ).…”

Section: Introductionmentioning

confidence: 99%

Peptidylarginine Deiminase Inhibition Abolishes the Production of Large Extracellular Vesicles From Giardia intestinalis, Affecting Host-Pathogen Interactions by Hindering Adhesion to Host Cells

Gavinho

Sabatke

Feijoli

et al. 2020

Front. Cell. Infect. Microbiol.

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Giardia intestinalis is a microaerophilic protozoan that is an important etiologic agent of diarrhea worldwide. There is evidence that under diverse conditions, the parasite is capable of shedding extracellular vesicles (EVs) which modulate the physiopathology of giardiasis. Here we describe new features of G. intestinalis EV production, revealing its capacity to shed two different enriched EV populations: large (LEV) and small extracellular vesicles (SEV) and identified relevant adhesion functions associated with the larger population. Proteomic analysis revealed differences in proteins relevant for virulence and host-pathogen interactions between the two EV subsets, such as cytoskeletal and anti-oxidative stress response proteins in LEVS. We assessed the effect of two recently identified inhibitors of EV release in mammalian cells, namely peptidylarginine deiminase (PAD) inhibitor and cannabidiol (CBD), on EV release from Giardia. The compounds were both able to effectively reduce EV shedding, the PAD-inhibitor specifically affecting the release of LEVs and reducing parasite attachment to host cells in vitro. Our results suggest that LEVs and SEVs have a different role in host-pathogen interaction, and that treatment with EV-inhibitors may be a novel treatment strategy for recurrent giardiasis.

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The minimal ESCRT machinery of Giardia lamblia has altered inter-subunit interactions within the ESCRT-II and ESCRT-III complexes

Cited by 23 publications

References 70 publications

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

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

Multiple paralogues of α-SNAP in Giardia lamblia exhibit independent subcellular localization and redistribution during encystation and stress

Peptidylarginine Deiminase Inhibition Abolishes the Production of Large Extracellular Vesicles From Giardia intestinalis, Affecting Host-Pathogen Interactions by Hindering Adhesion to Host Cells

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