Entamoeba histolytica calreticulin: an endoplasmic reticulum protein expressed by trophozoites into experimentally induced amoebic liver abscesses

Abstract

Entamoeba histolytica calreticulin (EhCRT) is remarkably immunogenic in humans (90-100% of invasive amoebiasis patients). Nevertheless, the study of calreticulin in this protozoan is still in its early stages. The exact location, biological functions, and its role in pathogenesis are yet to be fully understood. The aim of the present work is to determine the location of EhCRT in virulent trophozoites in vivo and the expression of the Ehcrt gene during the development of experimentally induced amoebic liver abscesses (ALA) in hamsters. Antibodies against recombinant EhCRT were used for the immunolocalization of EhCRT in trophozoites through confocal microscopy; immunohistochemical assays were also performed on tissue sections of ALAs at different times after intrahepatic inoculation. The expression of the Ehcrt gene during the development of ALA was estimated through both in situ RT-PCR and real-time RT-PCR. Confocal assays of virulent trophozoites showed a distribution of EhCRT in the cytoplasmic vesicles of different sizes. Apparently, EhCRT is not exported into the hepatic tissue. Real-time RT-PCR demonstrated an over-expression of the Ehcrt gene at 30 min after trophozoite inoculation, reaching a peak at 1-2 h; thereafter, the expression fell sharply to its original levels. These results demonstrate for the first time in an in vivo model of ALA, the expression of Ehcrt gene in E. histolytica trophozoites and add evidence that support CRT as a resident protein of the ER in E. histolytica species. The in vivo experiments suggest that CRT may play an important role during the early stages of the host-parasite relationship, when the parasite is adapting to a new environment, although the protein seems to be constitutively synthesized. Moreover, trophozoites apparently do not export EhCRT into the hepatic tissue in ALA.

Entamoeba histolytica Cell Surface Calreticulin Binds Human C1q and Functions in Amebic Phagocytosis of Host Cells

“…Its predicted amino acid sequence is 53% identical and 70% similar to human calreticulin (25). In addition to identification in amebic phagosome preparations (9,36), E. histolytica calreticulin is known to be immunogenic during human infection and has been shown to colocalize with a resident ER protein, protein disulfide isomerase (PDI) (24,25). Calreticulin has also been demonstrated within the uroid (i.e., tail region) on the E. histolytica cell surface following capping of surface receptors with the lectin concanavalin A (23).…”

Entamoeba histolyticaandE. disparCalreticulin: Inhibition of Classical Complement Pathway and Differences in the Level of Expression in Amoebic Liver Abscess

“…This evasion mechanism has been described in schistosomiasis, oncocercosis, trypanosomiasis [3, 5, 14], and now in amoebiasis. The interaction of CRT from T. cruzi ( Tc CRT) with human C1q is one of the most studied systems.…”

“…In a similar assay, trophozoites were incubated with NHS (1 : 10) or human peripheral mononuclear blood cells (PMBC) (1 : 6, trophozoites/lymphocytes ratio) for 60 min before incubation with 4  μ g of C1q and finally processed for immunohistochemical assay as previously described [5]. Samples were examined by confocal microscopy (DM1RE-2, Leica Mikrosysteme, Wetzlar, Germany) using appropriate fluorescence emission filters.…”

“…The detection of CRT mRNA was carried out using a two-step in situ RT-PCR procedure as previously reported with some modifications [5]. Previously selected hamster liver tissue sections (3 sections after intraportal inoculation) were pretreated with 0.5  μ g/ μ L proteinase K (Sigma Aldrich, St. Louis, MO, USA) and with 1 U/sample of DNase I, RNase-free (Roche Diagnostics GmbH, Mannheim, Germany).…”

Cell-Surface Molecules as Virulence Determinants in Entamoeba histolytica

“…Calreticulin was found in the proteome of early phagosomes [ 35 , 53 ] and localized to the uropod [ 55 ]. A function in early stages of ALA was inferred from the increase in calreticulin levels shortly after inoculation [ 54 ].…”