Bibiana Chávez-Munguía

Entamoeba invadens, encystation process and enolase.

The reptilian parasite Entamoeba invadens is accepted as a model for the study of the Entamoeba encystation process. Here we describe the production and characterization of a mAb (B4F2), generated against a component of the E. invadens cyst wall. This mAb specifically recognizes a 48-kDa protein present in cytoplasmic vesicles of cells encysting for 24 h. In mature cysts (96 h), the antigen was detected on the cyst surface. By two-dimensional electrophoresis and mass spectrometry analysis, the B4F2 specific antigen was identified as enolase. Levels of enolase mRNA were increased in encysting cells and the B4F2 mAb was found to inhibit cyst formation. Therefore, these results strongly suggest a new role for enolase in E. invadens encystation, and the B4F2 mAb will be useful tool to study its role in the differentiation process.

Actin, RhoA, and Rab11 participation during encystment in Entamoeba invadens.

In the genus Entamoeba, actin reorganization is necessary for cyst differentiation; however, its role is still unknown. The aim of this work was to investigate the role of actin and encystation-related proteins during Entamoeba invadens encystation. Studied proteins were actin, RhoA, a small GTPase involved through its effectors in the rearrangement of the actin cytoskeleton; Rab11, a protein involved in the transport of encystation vesicles; and enolase, as an encystment vesicles marker. Results showed a high level of polymerized actin accompanied by increased levels of RhoA-GTP during cell rounding and loss of vacuoles. Cytochalasin D, an actin polymerization inhibitor, and Y27632, an inhibitor of RhoA activity, reduced encystment in 80%. These inhibitors also blocked cell rounding, disposal of vacuoles, and the proper formation of the cysts wall. At later times, F-actin and Rab11 colocalized with enolase, suggesting that Rab11 could participate in the transport of the cyst wall components through the F-actin cytoskeleton. These results suggest that actin cytoskeleton rearrangement is playing a decisive role in determining cell morphology changes and helping with the transport of cell wall components to the cell surface during encystment of E. invadens.

Antiamoebic Activity of Adenophyllum aurantium (L.) Strother and Its Effect on the Actin Cytoskeleton of Entamoeba histolytica

In Mexico, the Adenophyllum aurantium (L.) Strother plant is consumed as an infusion to treat intestinal diseases such as amoebiasis, which is an endemic health problem in Mexico and other countries. However, the effect of A. aurantium on Entamoeba histolytica, the causative agent of amoebiasis, is unknown. An aerial part methanolic extract (AaMeA), a root methanolic extract (AaMeR) and a root ethyl acetate extract (AaEaR) were tested on E. histolytica trophozoites. AaMeA and AaMeR did not show antiproliferative activity; however, AaEaR exhibited an in vitro GI50 of 230 μg/ml, and it was able to inhibit the differentiation of Entamoeba invadens trophozoites into cysts. The intraperitoneal administration of AaEaR (2.5 or 5 mg) to hamsters that were infected with E. histolytica inhibited the development of amoebic liver abscesses in 48.5 or 89.0% of the animals, respectively. Adhesion to fibronectin and erythrophagocytosis were 28.7 and 37.5% inhibited by AaEaR, respectively. An ultrastructure analysis of AaEaR-treated trophozoites shows a decrease in the number of vacuoles but no apparent cell damage. Moreover, this extract affected the actin cytoskeleton structuration, and it prevented the formation of contractile rings by mechanism(s) that were independent of reactive oxygen species and RhoA activation pathways. 13C NMR data showed that the major compounds in the AaEaR extract are thiophenes. Our results suggest that AaEaR may be effective in treatments against amoebiasis, nevertheless, detailed toxicity studies on thiophenes, contained in AaEaR, are required to avoid misuse of this vegetal species.

Heterodimerization of the Entamoeba histolytica EhCPADH virulence complex through molecular dynamics and protein–protein docking

EhCPADH is a protein complex involved in the virulence of Entamoeba histolytica, the protozoan responsible for human amebiasis. It is formed by the EhCP112 cysteine protease and the EhADH adhesin. To explore the molecular basis of the complex formation, three-dimensional models were built for both proteins and molecular dynamics simulations (MDS) and docking calculations were performed. Results predicted that the pEhCP112 proenzyme and the mEhCP112 mature enzyme were globular and peripheral membrane proteins. Interestingly, in pEhCP112, the propeptide appeared hiding the catalytic site (C167, H329, N348); while in mEhCP112, this site was exposed and its residues were found structurally closer than in pEhCP112. EhADH emerged as an extended peripheral membrane protein with high fluctuation in Bro1 and V shape domains. 500 ns-long MDS and protein–protein docking predictions evidenced different heterodimeric complexes with the lowest free energy. pEhCP112 interacted with EhADH by the propeptide and C-terminal regions and mEhCP112 by the C-terminal through hydrogen bonds. In contrast, EhADH bound to mEhCP112 by 442–479 residues, adjacent to the target cell-adherence region (480–600 residues), and by the Bro1 domain (9–349 residues). Calculations of the effective binding free energy and per residue free energy decomposition showed that EhADH binds to mEhCP112 with a higher binding energy than to pEhCP112, mainly through van der Waals interactions and the nonpolar part of solvation energy. The EhADH and EhCP112 structural relationship was validated in trophozoites by immunofluorescence, TEM, and immunoprecipitation assays. Experimental findings fair agreed with in silico results.

Adherens junctions and desmosomes are damaged by Entamoeba histolytica: Participation of EhCPADH complex and EhCP112 protease.

Entamoeba histolytica trophozoites adhere to epithelium at the cell–cell contact and perturb tight junctions disturbing the transepithelial electrical resistance. Behind tight junctions are the adherens junctions (AJs) that reinforce them and the desmosomes (DSMs) that maintain the epithelium integrity. The damage produced to AJs and DMSs by this parasite is unknown. Here, we studied the effect of the trophozoites, the EhCPADH complex, and the EhCP112 recombinant enzyme (rEhCP112) on AJ and DSM proteins. We found that trophozoites degraded β‐cat, E‐cad, Dsp l/ll, and Dsg‐2 with the participation of EhCPADH and EhCP112. After contact of epithelial cells with trophozoites, immunofluorescence and transmission electron microscopy assays revealed EhCPADH and rEhCP112 at the intercellular space where they colocalised with β‐cat, E‐cad, Dsp l/ll, and Dsg‐2. Moreover, our results suggested that rEhCP112 could be internalised by caveolae and clathrin‐coated vesicles. Immunoprecipitation assays showed the interaction of EhCPADH with β‐cat and Dsp l/ll. Besides, in vivo assays demonstrated that rEhCP112 concentrates at the cellular borders of the mouse intestine degrading E‐cad and Dsp I/II. Our research gives the first clues on the trophozoite attack to AJs and DSMs and point out the role of the EhCPADH and EhCP112 in the multifactorial event of trophozoites virulence.

Exploring the Possible Role of Lysine Acetylation on Entamoeba histolytica Virulence: A Focus on the Dynamics of the Actin Cytoskeleton

Cytoskeleton remodeling can be regulated, among other mechanisms, by lysine acetylation. The role of acetylation on cytoskeletal and other proteins of Entamoeba histolytica has been poorly studied. Dynamic rearrangements of the actin cytoskeleton are crucial for amebic motility and capping formation, processes that may be effective means of evading the host immune response. Here we report the possible effect of acetylation on the actin cytoskeleton dynamics and in vivo virulence of E. histolytica. Using western blot, immunoprecipitation, microscopy assays, and in silico analysis, we show results that strongly suggest that the increase in Aspirin-induced cytoplasm proteins acetylation reduced cell movement and capping formation, likely as a consequence of alterations in the structuration of the actin cytoskeleton. Additionally, intrahepatic inoculation of Aspirin-treated trophozoites in hamsters resulted in severe impairment of the amebic virulence. Taken together, these results suggest an important role for lysine acetylation in amebic invasiveness and virulence.

Entamoeba histolytica EhCP112 Dislocates and Degrades Claudin-1 and Claudin-2 at Tight Junctions of the Intestinal Epithelium

During intestinal invasion, Entamoeba histolytica opens tight junctions (TJs) reflected by transepithelial electrical resistance (TEER) dropping. To explore the molecular mechanisms underlying this, we studied in vitro and in vivo the damage produced by the recombinant E. histolytica cysteine protease (rEhCP112) on TJ functions and proteins. rEhCP112 reduced TEER in Caco-2 cells in a dose- and time-dependent manner; and EhCP112-overexpressing trophozoites provoked major epithelial injury compared to control trophozoites. rEhCP112 penetrated through the intercellular space, and consequently the ion flux increased and the TJs fence function was disturbed. However, macromolecular flux was not altered. Functional in vitro assays revealed specific association of rEhCP112 with claudin-1 and claudin-2, that are both involved in regulating ion flux and fence function. Of note, rEhCP112 did not interact with occludin that is responsible for regulating macromolecular flux. Moreover, rEhCP112 degraded and delocalized claudin-1, thus affecting interepithelial adhesion. Concomitantly, expression of the leaky claudin-2 at TJ, first increased and then it was degraded. In vivo, rEhCP112 increased intestinal epithelial permeability in the mouse colon, likely due to apical erosion and claudin-1 and claudin-2 degradation. In conclusion, we provide evidence that EhCP112 causes epithelial dysfunction by specifically altering claudins at TJ. Thus, EhCP112 could be a potential target for therapeutic approaches against amoebiasis.

Characterization of a novel endogenous cysteine proteinase inhibitor, trichocystatin-3 (TC-3), localized on the surface of Trichomonas vaginalis

Trichomonas vaginalis is a flagellated protist responsible for human trichomoniasis. T. vaginalis has three genes encoding for endogenous cysteine proteinase (CP) inhibitors, known as trichocystatin-1 through trichocystatin-3 (TC-1, TC-2, and TC-3). These inhibitors belong to the cystatin family. In this study, we characterized trichocystatin-3 (TC-3), an endogenous cysteine proteinase (CP) inhibitor of T. vaginalis. TC-3 possesses a signal peptide in the N-terminus and two putative glycosylation sites (typical of family 2, cystatins) but lacks the PW motif and cysteine residues (typical of family 1, stefins). Native TC-3 was recognized as an ∼18u2009kDa protein band in a T. vaginalis protein extract. By confocal microscopy, endogenous TC-3 was found in the Golgi complex, cytoplasm, large vesicles, and the plasma membrane. These localizations are consistent with an in silico prediction. In addition, the purified recombinant protein (TC-3r) functions as an inhibitor of cathepsin L CPs, such as human liver cathepsin L and trichomonad CPs, present in a proteinase-resistant extract (PRE). Via a pull-down assay using TC-3r as bait and PRE, we identified several trichomonad CPs targeted by TC-3, primarily TvCP3. These CP-TC-3 interactions occur in vesicles, in the cytoplasm, and on the parasite surface. In addition, TC-3r showed a protective effect on HeLa cell monolayers by inhibiting trichomonad surface CPs involved in cellular damage. Our results show that the endogenous inhibitor TC-3 plays a key role in the regulation of endogenous CP proteolytic activity.

Influence of Micropatterned Grill Lines on Entamoeba histolytica Trophozoites Morphology and Migration

Entamoeba histolytica, the causal agent of human amoebiasis, has two morphologically different phases: a resistant cyst and a trophozoite responsible for the invasion of the host tissues such as the colonic mucosa and the intestinal epithelium. During in vitro migration, trophozoites usually produce protuberances such as pseudopods and rarely filopodia, structures that have been observed in the interaction of trophozoites with human colonic epithelial tissue. To study the different membrane projections produced by the trophozoites, including pseudopods, filopodia, uropods, blebs, and others, we designed an induction system using erythrocyte extract or fibronectin (FN) in micropatterned grill lines (each micro-line containing multiple micro-portions of FN or erythrocyte extract) on which the trophozoites were placed in culture for migration assays. Using light, confocal, and scanning electron microscopy, we established that E. histolytica trophozoites frequently produce short and long filopodia, large retractile uropods in the rear, pseudopods, blebs, and others structures, also showing continuous migration periods. The present study provides a simple migration method to induce trophozoites to generate abundant membrane protrusion structures that are rarely obtained in normal or induced cultures, such as long filopodia; this method will allow a–better understanding of the interactions of trophozoites with FN and cell debris. E. histolytica trophozoites motility plays an important role in invasive amoebiasis. It has been proposed that both physical forces and chemical signals are involved in the trophozoite motility and migration. However, the in vivo molecules that drive the chemotactic migration remain to be determined. We propose the present assay to study host molecules that guide chemotactic behavior because the method is highly reproducible, and a live image of cell movement and migration can be quantified.

Giardia lamblia: Identification of peroxisomal-like proteins

The protozoan parasite Giardia lamblia has traditionally been reported as lacking peroxisomes, organelles involved in fatty acid metabolism and detoxification of reactive oxygen species. We here report the finding with transmission electron microscopy of an oxidase activity in cytoplasmic vesicles of trophozoites and cysts of G. lamblia. These vesicles were positive to 3,3-diaminobenzidine and to cerium chloride staining. In addition, using bioinformatic tools, two peroxisomal proteins were identified in the G. lamblia proteome: acyl-CoA synthetase long chain family member 4 (ACSL-4) and peroxin-4 (PEX-4). With confocal and immunoelectron microscopy using polyclonal antibodies both proteins were identified in cytoplasmic vesicles of trophozoites. Altogether, our results suggest for the first time the presence of peroxisomal-like proteins in the cytoplasm of G. lamblia.