scholarly journals Rab7D small GTPase is involved in phago‐, trogocytosis and cytoskeletal reorganization in the enteric protozoan Entamoeba histolytica

2020 ◽  
Vol 23 (1) ◽  
Author(s):  
Yumiko Saito‐Nakano ◽  
Ratna Wahyuni ◽  
Kumiko Nakada‐Tsukui ◽  
Kentaro Tomii ◽  
Tomoyoshi Nozaki
Keyword(s):  
Entamoeba Histolytica ◽  
Small Gtpase ◽  
Cytoskeletal Reorganization ◽  
Enteric Protozoan

scholarly journals Human hololactoferrin: endocytosis and use as an iron source by the parasite Entamoeba histolytica

Microbiology ◽  
2005 ◽  
Vol 151 (12) ◽  
pp. 3859-3871 ◽  
Author(s):  
Nidia León-Sicairos ◽  
Magda Reyes-López ◽  
Adrián Canizalez-Román ◽  
Rosa María Bermúdez-Cruz ◽  
Jesús Serrano-Luna ◽  
...  
Keyword(s):  
Entamoeba Histolytica ◽  
Cysteine Proteases ◽  
Human Beings ◽  
Minimum Concentration ◽  
Iron Source ◽  
Protein Levels ◽  
Metabolic Functions ◽  
Mucosal Surfaces ◽  
Enteric Protozoan

Entamoeba histolytica is an enteric protozoan that exclusively infects human beings. This parasite requires iron for its metabolic functions. Lactoferrin is a mammalian glycoprotein that chelates extracellular iron on mucosal surfaces, including the surface of the large intestine, where E. histolytica initiates infection. This work examined the interaction in vitro of E. histolytica trophozoites with human hololactoferrin (iron-saturated lactoferrin). A minimum concentration of 50 μM Fe from hololactoferrin supported growth of the amoeba. Amoebic binding sites for hololactoferrin were different from those for human apolactoferrin, holotransferrin and haemoglobin. One amoebic hololactoferrrin-binding polypeptide of 90 kDa was found, which was not observed after treatment of trophozoites with trypsin. Hololactoferrin-binding-protein levels increased in amoebas starved of iron, or grown in hololactoferrin. Internalization of hololactoferrin was inhibited by filipin. Endocytosed hololactoferrin colocalized with an anti-chick embryo caveolin mAb in amoebic vesicles, and lactoferrin was further detected in acidic vesicles; amoebic caveolin of 22 kDa was detected by Western blotting using this antibody. Cysteine proteases from amoebic extracts were able to cleave hololactoferrin. Together, these data indicate that E. histolytica trophozoites bind to hololactoferrin through specific membrane lactoferrin-binding proteins. This ferric protein might be internalized via caveolae-like microdomains, then used as an iron source, and degraded.

scholarly journals Identification and Characterization of the Entamoeba Histolytica Rab8a Binding Protein: A Cdc50 Homolog

2018 ◽  
Vol 19 (12) ◽  
pp. 3831 ◽  
Author(s):  
Yuki Hanadate ◽  
Yumiko Saito-Nakano ◽  
Kumiko Nakada-Tsukui ◽  
Tomoyoshi Nozaki
Keyword(s):  
Plasma Membrane ◽  
Entamoeba Histolytica ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Membrane Traffic ◽  
Membrane Lipid ◽  
Small Gtpase ◽  
Triton X100 ◽  
Lipid Flippase

Membrane traffic plays a pivotal role in virulence in the enteric protozoan parasite Entamoeba histolytica. EhRab8A small GTPase is a key regulator of membrane traffic at the endoplasmic reticulum (ER) of this protist and is involved in the transport of plasma membrane proteins. Here we identified the binding proteins of EhRab8A. The Cdc50 homolog, a non-catalytic subunit of lipid flippase, was identified as an EhRab8A binding protein candidate by affinity coimmunoprecipitation. Binding of EhRab8A to EhCdc50 was also confirmed by reciprocal immunoprecipitation and blue-native polyacrylamide gel electrophoresis, the latter of which revealed an 87 kDa complex. Indirect immunofluorescence imaging with and without Triton X100 showed that endogenous EhCdc50 localized on the surface in the absence of permeabilizing agent but was observed on the intracellular structures and overlapped with the ER marker Bip when Triton X100 was used. Overexpression of N-terminal HA-tagged EhCdc50 impaired its translocation to the plasma membrane and caused its accumulation in the ER. As reported previously in other organisms, overexpression and accumulation of Cdc50 in the ER likely inhibited surface transport and function of the plasma membrane lipid flippase P4-ATPase. Interestingly, HA-EhCdc50-expressing trophozoites gained resistance to miltefosine, which is consistent with the prediction that HA-EhCdc50 overexpression caused its accumulation in the ER and mislocalization of the unidentified lipid flippase. Similarly, EhRab8A gene silenced trophozoites showed increased resistance to miltefosine, supporting EhRab8A-dependent transport of EhCdc50. This study demonstrated for the first time that EhRab8A mediates the transport of EhCdc50 and lipid flippase P4-ATPase from the ER to the plasma membrane.

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