Genome-Wide Classification of Myb Domain-Containing Protein Families in Entamoeba invadens.

Entamoeba histolytica, the causative agent of amebiasis, is the third leading cause of death among parasitic diseases globally. Its life cycle includes encystation, which has been mostly studied in Entamoeba invadens, responsible for reptilian amebiasis. However, the molecular mechanisms underlying this process are not fully understood. Therefore, we focused on the identification and characterization of Myb proteins, which regulate the expression of encystation-related genes in various protozoan parasites. Through bioinformatic analysis, we identified 48 genes in E. invadens encoding MYB-domain-containing proteins. These were classified into single-repeat 1R (20), 2R-MYB proteins (27), and one 4R-MYB protein. The in-silico analysis suggests that these proteins are multifunctional, participating in transcriptional regulation, chromatin remodeling, telomere maintenance, and splicing. Transcriptomic data analysis revealed expression signatures of eimyb genes, suggesting a potential orchestration in the regulation of early and late encystation-excystation genes. Furthermore, we identified probable target genes associated with reproduction, the meiotic cell cycle, ubiquitin-dependent protein catabolism, and endosomal transport. In conclusion, our findings suggest that E. invadens Myb proteins regulate stage-specific proteins and a wide array of cellular processes. This study provides a foundation for further exploration of the molecular mechanisms governing encystation and unveils potential targets for therapeutic intervention in amebiasis.

Epithelial Cells Expressing EhADH, An Entamoeba histolytica Adhesin, Exhibit Increased Tight Junction Proteins.

In Entamoeba histolytica, the EhADH adhesin together with the EhCP112 cysteine protease, form a 124 kDa complex named EhCPADH. This complex participates in trophozoite adherence, phagocytosis and cytolysis of target cells. EhCPADH and EhCP112 are both involved on epithelium damage, by opening tight junctions (TJ) and reaching other intercellular junctions. EhADH is a scaffold protein belonging to the ALIX family that contains a Bro1 domain, expresses at plasma membrane, endosomes and cytoplasm of trophozoites, and is also secreted to the medium. Contribution of EhADH to TJ opening still remains unknown. In this paper, to elucidate the role of EhADH on epithelium injury, we followed two strategies: producing a recombinant protein (rEhADH) and transfecting the ehadh gene in MDCK cells. Results from the first strategy revealed that rEhADH reached the intercellular space of epithelial cells and co-localized with claudin-1 and occludin at TJ region; later, rEhADH was mainly internalized by clathrin-coated vesicles. In the second strategy, MDCK cells expressing EhADH (MDCK-EhADH) showed the adhesin at plasma membrane. In addition, MDCK-EHADH cells exhibited adhesive features, producing epithelial aggregation and adherence to erythrocytes, as described in trophozoites. Surprisingly, the adhesin expression produced an increase of claudin-1, occludin, ZO-1 and ZO-2 at TJ, and also the transepithelial electric resistance (TEER), which is a measure of TJ gate function. Moreover, MDCK-EhADH cells resulted more susceptible to trophozoites attack, as showed by TEER and cytopathic experiments. Overall, our results indicated that EhADH disturbed TJ from the extracellular space and also intracellularly, suggesting that EhADH affects by itself TJ proteins, and possibly synergizes the action of other parasite molecules during epithelial invasion.

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.

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 ß-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 ß-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 ß-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.

Determinación y análisis de costos reales de tratamientos intensivos por paciente y día cama / Analysis of real costs of intensive treatments per patient and bed/day

El objetivo del trabajo es medir los costos reales asociados a las patologías tratadas en las Unidades de Cuidados Intensivos Adulto de los Hospitales Públicos de la Región del Maule, y compararlos con el costo asignado por Fonasa al día cama para el año 2011. Materiales y métodos: Se trata de un estudio prospectivo, aplicando el Sistema de Costos Basado en Actividades (ABC). Se incluyó 469 pacientes, 222 pacientes de la Unidad de Cuidados Intensivos del Hospital de Curicó y 247 pacientes la Unidad de Cuidados Intensivos del Hospital de Talca entre el 01 de enero y 30 de agosto de 2011, los que de acuerdo a edad y APACHE, representan los niveles de complejidad de pacientes de Unidades de Cuidados Intensivos a nivel nacional. Los pacientes se clasificaron según las patologías: sepsis, cardiovascular, respiratorias, neurológicas, trauma, digestivos, renales y otros. Resultados: Las patologías que presentan mayor mediana de costos por día cama son: sepsis ($362.115), respiratorias ($352.793), trauma ($348.442), renales ($341.928) y cardiovascular ($291.061). La estructura de costos del día cama está conformada principalmente por el costo asociado al recurso humano, cuyo valor máximo asciende a 64 por ciento, seguido del costo asociado a los medicamentos con un valor máximo de 15 por ciento. Los pacientes con sepsis y trauma absorben la mayor proporción de recursos en la Unidad de Cuidados Intensivos en estudio; 35 por ciento y 19 por ciento respectivamente y una proporción significativa de dichos costos es utilizada por pacientes que fallecen (34 por ciento y 19 por ciento) respectivamente. Conclusión: Todas las patologías en estudio tienen desviación desfavorable de costos, con respecto al arancel fijado por Fonasa, que sólo asciende a $192.160, para el año 2011.

The aim of the study is measures the real costs associated with the pathologies treated in the of Intensive Care Unid (ICU) Adults of the Public Hospitals of the region of the Maule, and to compare them with the cost assigned by Fonasa to the bed/day for the year 2011. Materials and methods: it is a question of a market study, applying the Activity-based costing (ABC). There was included 469 patients, 222 patients of the ICU of the Curicó’s Hospital and 247 patients the ICU of the Talca’s Hospital between January 01 and August 30, 2011, which in agreement to age and APACHE II, represent the levels of patients’ complexity of ICU to national level. The patients qualified according to the pathologies: sepsis, cardiovascular, respiratory, neurological, trauma, digestive, renal and others. Results: The pathologies that present major median of costs per day bed are: sepsis ($362.115), respiratory ($352.793),trauma ($348.442), renal ($341.928) and cardiovascular ($291.061). The structure of costs of the day bed is shaped principally by the cost associated with the human resource, which maximum value promotes 64 percent, followed by the cost associated with the medicines with a maximum value of 15 percent. The patients with sepsis and trauma absorbed the major proportion of resources in the ICU in our study; 35 percent and 19 percent respectively and a significant proportion of the abovementioned costs is used by patients who die (34 percent and 19 percent)respectively. Conclusion: All the pathologies in study have unfavorable diversion of costs, with regard to the duty fixed by Fonasa, which only promotes to $192.160, for the year2011.