A Bioinformatical Approach to Study the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery in Protozoan Parasites: The Entamoeba histolytica Case

López-Reyes, Israel; Bañuelos, Cecilia; Betanzos, Abigail; Orozco, Esther

doi:10.5772/19480

Cited by 6 publications

(14 citation statements)

References 58 publications

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“…Earlier, we identified and partially characterized the Ehvps2, Ehvps24 , and Ehvps32 genes and transcripts, as well as the EhVps32 protein, members of the ESCRT-III complex (López-Reyes et al, 2011 ; Avalos-Padilla et al, 2015 ). However, there are no studies regarding the Ehvps20 gene and its product and we have not dissected the proteins function.…”

Section: Resultsmentioning

confidence: 99%

“…E. histolytica possesses the majority of the ESCRT machinery genes (López-Reyes et al, 2011 ), but their role in phagocytosis is poorly understood. To further elucidate the phagocytosis puzzle, we performed in vivo and in vitro studies using mutant trophozoites, the E. histolytica ESCRT-III purified recombinant proteins (rEhVps20, rEhVps32, rEhVps24, and rEhVps2) and giant unilamellar vesicles (GUVs).…”

Section: Introductionmentioning

confidence: 99%

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The Conserved ESCRT-III Machinery Participates in the Phagocytosis of Entamoeba histolytica

Avalos-Padilla

Knorr

Javier-Reyna

et al. 2018

Front. Cell. Infect. Microbiol.

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The endosomal sorting complex required for transport (ESCRT) orchestrates cell membrane-remodeling mechanisms in eukaryotes, including endocytosis. However, ESCRT functions in phagocytosis (ingestion of ≥250 nm particles), has been poorly studied. In macrophages and amoebae, phagocytosis is required for cell nutrition and attack to other microorganisms and cells. In Entamoeba histolytica, the voracious protozoan responsible for human amoebiasis, phagocytosis is a land mark of virulence. Here, we have investigated the role of ESCRT-III in the phagocytosis of E. histolytica, using mutant trophozoites, recombinant proteins (rEhVps20, rEhVps32, rEhVps24, and rEhVps2) and giant unilamellar vesicles (GUVs). Confocal images displayed the four proteins located around the ingested erythrocytes, in erythrocytes-containing phagosomes and in multivesicular bodies. EhVps32 and EhVps2 proteins co-localized at the phagocytic cups. Protein association increased during phagocytosis. Immunoprecipitation and flow cytometry assays substantiated these associations. GUVs revealed that the protein assembly sequence is essential to form intraluminal vesicles (ILVs). First, the active rEhVps20 bound to membranes and recruited rEhVps32, promoting membrane invaginations. rEhVps24 allowed the detachment of nascent vesicles, forming ILVs; and rEhVps2 modulated their size. The knock down of Ehvps20 and Ehvps24genes diminished the rate of erythrophagocytosis demonstrating the importance of ESCRT-III in this event. In conclusion, we present here evidence of the ESCRT-III participation in phagocytosis and delimitate the putative function of proteins, according to the in vitro reconstruction of their assembling.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The Conserved ESCRT-III Machinery Participates in the Phagocytosis of Entamoeba histolytica

Avalos-Padilla

Knorr

Javier-Reyna

et al. 2018

Front. Cell. Infect. Microbiol.

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“…E . histolytica possesses the genes encoding ESCRT proteins [ 34 ] and those encoding EhVps4 AAA ATPase and EhADH, both ESCRT associated proteins [ 13 , 35 ]. Here, we show the participation of EhVps32 in both receptor-mediated and non-specific phagocytosis as well as in pinocytosis; we also revealed its co-localization with EhADH, Gal/GalNac lectin and actin during erythrophagocytosis.…”

Section: Introductionmentioning

confidence: 99%

EhVps32 Is a Vacuole-Associated Protein Involved in Pinocytosis and Phagocytosis of Entamoeaba histolytica

et al. 2015

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Here, we investigated the role of EhVps32 protein (a member of the endosomal-sorting complex required for transport) in endocytosis of Entamoeba histolytica, a professional phagocyte. Confocal microscopy, TEM and cell fractionation revealed EhVps32 in cytoplasmic vesicles and also located adjacent to the plasma membrane. Between 5 to 30 min of phagocytosis, EhVps32 was detected on some erythrocytes-containing phagosomes of acidic nature, and at 60 min it returned to cytoplasmic vesicles and also appeared adjacent to the plasma membrane. TEM images revealed it in membranous structures in the vicinity of ingested erythrocytes. EhVps32, EhADH (an ALIX family member), Gal/GalNac lectin and actin co-localized in the phagocytic cup and in some erythrocytes-containing phagosomes, but EhVps32 was scarcely detected in late phagosomes. During dextran uptake, EhVps32, EhADH and Gal/GalNac lectin, but not actin, co-localized in pinosomes. EhVps32 recombinant protein formed oligomers composed by rings and filaments. Antibodies against EhVps32 monomers stained cytoplasmic vesicles but not erythrocytes-containing phagosomes, suggesting that in vivo oligomers are formed on phagosome membranes. The involvement of EhVps32 in phagocytosis was further study in pNeoEhvps32-HA-transfected trophozoites, which augmented almost twice their rate of erythrophagocytosis as well as the membranous concentric arrays built by filaments, spirals and tunnel-like structures. Some of these structures apparently connected phagosomes with the phagocytic cup. In concordance, the EhVps32-silenced G3 trophozoites ingested 80% less erythrocytes than the G3 strain. Our results suggest that EhVps32 participates in E. histolytica phagocytosis and pinocytosis. It forms oligomers on erythrocytes-containing phagosomes, probably as a part of the scission machinery involved in membrane invagination and intraluminal vesicles formation.

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“…This dissociation of the ESCRT components is carried out by the AAA ATPase Vps4 [3,25]. The membrane association and ATPase activity of Vps4 is controlled by ESCRT components Vta1, Ist1 and Did2/Vps46, which together constitute the Vps4 complex (also known as ESCRT-III-associated complex) [2,14]. BLAST analyses of the G. lamblia genome indicated the presence of Vps4 and Did2/Vps46 orthologues only [12,14].…”

Section: Introductionmentioning

confidence: 97%

“…The membrane association and ATPase activity of Vps4 is controlled by ESCRT components Vta1, Ist1 and Did2/Vps46, which together constitute the Vps4 complex (also known as ESCRT-III-associated complex) [2,14]. BLAST analyses of the G. lamblia genome indicated the presence of Vps4 and Did2/Vps46 orthologues only [12,14]. The absence of putative orthologues of Vta1 and Ist1 raises the interesting possibility that even if a functional MVB sorting pathway is present in G. lamblia, the machinery for ESCRT disassembly is likely to be minimal.…”

Section: Introductionmentioning

confidence: 99%

Significantly Diverged Did2/Vps46 Orthologues from the Protozoan Parasite Giardia lamblia

et al. 2015

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The endosomal compartment performs extensive sorting functions in most eukaryotes, some of which are accomplished with the help of the multivesicular body (MVB) sorting pathway. This pathway depends on the sequential action of complexes, termed the endosomal sorting complex required for transport (ESCRT). After successful sorting, the crucial step of recycling of the ESCRT complex components requires the activation of the AAA ATPase Vps4, and Did2/Vps46 plays an important role in this activation event. The endolysosomal system of the protozoan parasite Giardia lamblia appears to lack complexity, for instead of having distinct early endosomes, late endosomes and lysosomes, there are only peripheral vesicles (PVs) that are located close to the cell periphery. Additionally, comparative genomics studies predict the presence of only a subset of the ESCRT components in G. lamblia. Thus, it is possible that the MVB pathway is not functional in G. lamblia. To address this issue, the present study focused on the two putative orthologues of Did2/Vps46 of G. lamblia as their function is likely to be pivotal for a functional MVB sorting pathway. In spite of considerable sequence divergence, compared to other eukaryotic orthologues, the proteins encoded by both these genes have the ability to function as Did2/Vps46 in the context of the yeast ESCRT pathway. Furthermore, they also localized to the cellular periphery, where PVs are also located. Thus, this report is the first to provide experimental evidence indicating the presence of a functional ESCRT component in G. lamblia by characterizing the putative Did2/Vps46 orthologues.

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A Bioinformatical Approach to Study the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery in Protozoan Parasites: The Entamoeba histolytica Case

Cited by 6 publications

References 58 publications

The Conserved ESCRT-III Machinery Participates in the Phagocytosis of Entamoeba histolytica

The Conserved ESCRT-III Machinery Participates in the Phagocytosis of Entamoeba histolytica

EhVps32 Is a Vacuole-Associated Protein Involved in Pinocytosis and Phagocytosis of Entamoeaba histolytica

Significantly Diverged Did2/Vps46 Orthologues from the Protozoan Parasite Giardia lamblia

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