Rab11B small GTPase regulates secretion of cysteine proteases in the enteric protozoan parasiteEntamoeba histolytica

Abstract

SummaryVesicular trafficking plays a pivotal role in the virulence of the enteric protozoan parasite Entamoeba histolytica. In the present study, we showed that one isotype of the small GTPase Rab11, EhRab11B, plays a central role in the secretion of a major virulence factor, cysteine proteases. EhRab11B did not colocalize with markers for the endoplasmic reticulum, early endosomes and lysosomes, but was partially associated with non-acidified vesicles in the endocytic pathway, likely recycling endosomes. Overexpression of EhRab11B resulted in a remarkable increase in both intracellular and secreted cysteine protease activity, concomitant with an augmentation of cytolytic activity as demonstrated by an increased ability to destroy mammalian cells. The oversecretion of cysteine proteases with EhRab11B overexpression was neither sensitive to brefeldin A nor specific to a certain cysteine protease species (e.g. CP1, 2 or 5), suggesting that these three major cysteine proteases are trafficked via an EhRab11B-associated secretory pathway, which is distinct from the classical brefeldinsensitive pathway. Overexpression of EhRab11B also enhanced exocytosis of the incorporated fluid-phase marker, supporting the notion that it is involved in recycling. This is the first report demonstrating that Rab11 plays a central role in the transport and secretion of pathogenic factors.

A Novel Entamoeba histolytica Cysteine Proteinase, EhCP4, Is Key for Invasive Amebiasis and a Therapeutic Target

“…Although the protein was not expressed in a soluble form, a relatively high level of expression (average 47 mg/liter of culture) was achieved in BL21 Codon Plus (DE3) RIPL (Stratagene) cells as inclusion bodies. Although existing refolding methods of EhCPs (16,17,38) failed to solubilize and recover active EhCP4, a refolding system that was modified from conditions used to refold Falcipain-2 (22) successfully produced active enzyme. Like other papain family cysteine proteinases (39), EhCP4 underwent autocatalytic activation in buffers containing 5 mM reducing reagent (e.g.…”

Use of RecombinantEntamoeba histolyticaCysteine Proteinase 1 To Identify a Potent Inhibitor of Amebic Invasion in a Human Colonic Model

“…We had previously shown that EhCP1, EhCP2, and EhCP5 are passively released during phagocytosis (17). This release is likely to be part of the newly described EhRab11B-associated secretory pathway, as overexpression of EhRab11B led to increased release of all three proteinases (30).…”

Phagocytosis in Entamoeba histolytica

“…Overexpression of this gene led to increased cysteine protease [ 76 ], Majumder and Lohia [ 77 ] and Bosch et al [ 78 ] and cytolytic activities. Overexpression of Rab11B is also associated with enhanced exocytosis, which indicates its involvement in recycling of endosomes [ 81 ]. Moreover, Rab7 has nine members, of which Rab7A and Rab7B display distinct localization in trophozoites.…”