Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and liver abscess. E. histolytica relies on motility, phagocytosis, host cell adhesion, and proteolysis of extracellular matrix for virulence. In eukaryotic cells, these processes are mediated in part by phosphatidylinositol 3-kinase (PI3K) signaling. Thus, PI3K may be critical for virulence. We utilized a functional genomics approach to identify genes whose products may operate in the PI3K pathway in E. histolytica . We treated a population of trophozoites that were overexpressing genes from a cDNA library with a near-lethal dose of the PI3K inhibitor wortmannin. This screen was based on the rationale that survivors would be overexpressing gene products that directly or indirectly function in the PI3K pathway. We sequenced the overexpressed genes in survivors and identified a cDNA encoding a Rap GTPase, a protein previously shown to participate in the PI3K pathway. This supports the validity of our approach. Genes encoding a coactosin-like protein, EhCoactosin, and a serine-rich E. histolytica protein (SREHP) were also identified. Cells overexpressing EhCoactosin or SREHP were also less sensitive to a second PI3K inhibitor, LY294002. This corroborates the link between these proteins and PI3K. Finally, a mutant cell line with an increased level of phosphatidylinositol (3,4,5)-triphosphate, the product of PI3K activity, exhibited increased expression of SREHP and EhCoactosin. This further supports the functional connection between these proteins and PI3K in E. histolytica . To our knowledge, this is the first forward-genetics screen adapted to reveal genes participating in a signal transduction pathway in this pathogen.

Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and liver abscess. Vesicle trafficking events, such as phagocytosis and delivery of plasma membrane proteins, have been implicated in pathogenicity. Rab GTPases are proteins whose primary function is to regulate vesicle trafficking; therefore, understanding the function of Rabs in this organism may provide insight into virulence. E. histolytica possesses a number of unique Rabs that exhibit limited homology to host Rabs. In this study we examined the function of one such Rab, EhRabA, by characterizing a mutant overexpressing a constitutively GTP-bound version of the protein. Overexpression of mutant EhRabA resulted in decreased adhesion to and phagocytosis of human red blood cells and in the appearance of large tubular organelles that could be stained with endoplasmic reticulum (ER)-specific but not Golgi complex-specific antibodies. Consistent with the adhesion defect, two subunits of a cell surface adhesin, the galactose/ N -acetylgalactosamine lectin, were mislocalized to the novel organelle. A cysteine protease, EhCP2, was also localized to the ER-like compartment in the mutant; however, the localization of two additional cell surface proteins, Igl and SREHP, remained unchanged in the mutant. The phenotype of the mutant could be recapitulated by treatment with brefeldin A, a cellular toxin that disrupts ER-to-Golgi apparatus vesicle traffic. This suggests that EhRabA influences vesicle trafficking pathways that are also sensitive to brefeldin A. Together, the data indicate that EhRabA directly or indirectly influences the morphology of secretory organelles and regulates trafficking of a subset of secretory proteins in E. histolytica .

Background: Diarrheal disease (DD)-associated mortality has declined since 1990; however, the incidence of DD has experienced a less-pronounced decrease. Thus, it is important to track progress in managing DD by following loss of healthy years. A disability-adjusted life-year (DALY), which combines data on years-of-life lost (YLL) and years-lived with-disability (YLD), is a metric that can track such a burden. Methods and findings: Using all 28 years of data in the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2017, we compared DD DALYs among different demographic subsets including sex, age, country, and World Bank (WB) income level. We also evaluated DD DALYs as a function of the socio-demographic index (SDI), a measure of a region’s socio-demographic development. On a global level, DD DALYs have decreased by approximately 85.43% from 1990 to 2017. Incidence and prevalence have decreased by 1.53% and 4.45%, respectively. A dramatic decrease in DD DALYs were observed for WB low-income countries, but not for WB high-income constituents. The temporal decrease in DD DALY rates in WB low-income countries was likely driven by a decrease in YLL. Alternatively, temporal increases in both YLL and YLD may have contributed to the apparent lack of progress in WB high-income countries. Regardless of WB income classification, children under the age of five and the elderly were the most vulnerable to DD. In nearly every year from 1990 to 2017, DD DALYs for females were higher than those for males in WB high-income regions, but lower than those for males in WB low-income constituents. The reason for these differences is not known. We also observed that the rate of DD DALYs was highly correlated to SDI regardless of WB income classification. Conclusions: To the best of our knowledge, this is the only temporal study of DD DALYs that encompasses all 28 years of data available from the GBD. Overall, our analyses show that temporal reductions in DD DALYs are not equivalent across regions, sexes and age groups. Therefore, careful attention to local and demography-specific risk factors will be necessary to tailor solutions in region- and demography-specific manners.