Hydrogenosomes and mitosomes are mitochondrion-related organelles (MROs) that have highly reduced and divergent functions in anaerobic/microaerophilic eukaryotes. Entamoeba histolytica, a microaerophilic, parasitic amoebozoan species, which causes intestinal and extraintestinal amoebiasis in humans, possesses mitosomes, the existence and biological functions of which have been a longstanding enigma in the evolution of mitochondria. We previously demonstrated that sulfate activation, which is not generally compartmentalized to mitochondria, is a major function of E. histolytica mitosomes. However, because the final metabolites of sulfate activation remain unknown, the overall scheme of this metabolism and the role of mitosomes in Entamoeba have not been elucidated. In this study we purified and identified cholesteryl sulfate (CS) as a final metabolite of sulfate activation. We then identified the gene encoding the cholesteryl sulfotransferase responsible for synthesizing CS. Addition of CS to culture media increased the number of cysts, the dormant form that differentiates from proliferative trophozoites. Conversely, chlorate, a selective inhibitor of the first enzyme in the sulfate-activation pathway, inhibited cyst formation in a dose-dependent manner. These results indicate that CS plays an important role in differentiation, an essential process for the transmission of Entamoeba between hosts. Furthermore, we show that Mastigamoeba balamuthi, an anaerobic, free-living amoebozoan species, which is a close relative of E. histolytica, also has the sulfate-activation pathway in MROs but does not possess the capacity for CS production. Hence, we propose that a unique function of MROs in Entamoeba contributes to its adaptation to its parasitic life cycle. mitochondrion-related organelles | protist | sulfolipids | differentiation M itochondrion-related organelles (MROs) are derived from canonical mitochondria and are found in a wide range of anaerobic/microaerophilic eukaryotes (1, 2). During the course of evolution MROs have undergone secondary loss of mitochondrial functions; this loss has occurred independently multiple times, resulting in the broad phylogenetic distribution of organisms possessing MROs (2). Furthermore, MROs occasionally acquire novel functions from other organisms by lateral gene transfer (LGT) (1, 3). Hence, MROs are not simply remnants of mitochondria but rather are organelles that display a variety of unique features (1-5).Unique features have been demonstrated in different types of MRO (2-4). Some anaerobic lineages of eukaryotes possess MROs (hydrogenosomes or hydrogen-producing mitochondria) that have remodeled their mitochondria drastically to couple ATP generation with hydrogen production (2-4). Mitosomes, another type of MRO maintained in some organisms that inhabit anaerobic/microaerophilic environments, do not produce ATP or hydrogen and have lost typical mitochondrial functions, such as the tricarboxylic acid (TCA) cycle, electron transport, oxidative phosphorylation, and β-oxidation...

Encystation is an essential differentiation process for the completion of the life cycle of a group of intestinal protozoa including Entamoeba histolytica, the causative agent of intestinal and extraintestinal amebiasis. However, regulation of gene expression during encystation is poorly understood. To comprehensively understand the process at the molecular level, the transcriptomic profiles of E . invadens , which is a related reptilian species that causes an invasive disease similar to that of E. histolytica, was investigated during encystation. Using a custom-generated Affymetrix platform microarray, we performed time course (0.5, 2, 8, 24, 48, and 120 h) gene expression analysis of encysting E . invadens . ANOVA analysis revealed that a total of 1,528 genes showed ≥3 fold up-regulation at one or more time points, relative to the trophozoite stage. Of these modulated genes, 8% (116 genes) were up-regulated at the early time points (0.5, 2 and 8h), while 63% (962 genes) were up-regulated at the later time points (24, 48, and 120 h). Twenty nine percent (450 genes) are either up-regulated at 2 to 5 time points or constitutively up-regulated in both early and late stages. Among the up-regulated genes are the genes encoding transporters, cytoskeletal proteins, proteins involved in vesicular trafficking (small GTPases), Myb transcription factors, cysteine proteases, components of the proteasome, and enzymes for chitin biosynthesis. This study represents the first kinetic analysis of gene expression during differentiation from the invasive trophozoite to the dormant, infective cyst stage in Entamoeba . Functional analysis on individual genes and their encoded products that are modulated during encystation may lead to the discovery of targets for the development of new chemotherapeutics that interfere with stage conversion of the parasite.

Encystation, which is cellular differentiation from the motile, proliferative, labile trophozoite form to the dormant, resistant cyst form, is a crucial process found in parasitic and free-living protozoa such as Entamoeba, Giardia, Acanthamoeba, and Balamuthia. Since encystation is an essential process to deal with the adverse external environmental changes during the life cycle, and often integral to the transmission of the diseases, biochemical understanding of the process potentially provides useful measures against the infections caused by this group of protozoa. In this study, we investigated metabolic and transcriptomic changes that occur during encystation in Entamoeba invadens, the reptilian sibling of mammal-infecting E. histolytica, using capillary electrophoresis-tandem mass spectrometry-based metabolite profiling and DNA microarray-based expression profiling. As the encystation progressed, the levels of majority of metabolites involved in glycolysis and nucleotides drastically decreased, indicating energy generation is ceased. Furthermore, the flux of glycolysis was redirected toward chitin wall biosynthesis. We found remarkable temporal increases in biogenic amines such as isoamylamine, isobutylamine, and cadaverine, during the early period of encystation, when the trophozoites form large multicellular aggregates (precyst). We also found remarkable induction of γ-aminobutyric acid (GABA) during encystation. This study has unveiled for the first time the dynamics of the transcriptional and metabolic regulatory networks during encystation, and should help in better understanding of the process in pathogenic eukaryotes, and further development of measures controlling infections they cause.

[1] Snowmelt and icemelt are believed to be important regulators of seasonal discharge of Himalayan rivers. To analyze the long term contribution of snowmelt and glacier/icemelt to river hydrology we apply a water budget model to simulate hydrology of the Liddar watershed in the western Himalaya, India for the 20th century and future IPCC A1B climate change scenario. Long term temperature and precipitation data in this region show a warming trend (0.08 C yr À1 ) and an increase in precipitation (0.28 mm yr À1 ), with a significant variability in seasonal trends. In particular, winter months have undergone the most warming, along with a decrease in precipitation rates; precipitation has increased throughout the spring. These trends have accelerated the melting and rapid disappearance of snow, causing a seasonal redistribution in the availability of water. Our model results show that about 60% of the annual runoff of the Liddar watershed is contributed from the snowmelt, while only 2% is contributed from glacier ice. The climate trend observed from the 1901 to 2010 time period and its impact on the availability of water will become significantly worse under the IPCC climate change scenarios. Our results suggest that there is a significant shift in the timing and quantity of water runoff in this region of the Himalayas due to snow distribution and melt. With greatly increased spring runoff and its reductions in summer potentially leading to reduced water availability for irrigation agriculture in summer.Citation: Jeelani, G., J. J. Feddema, C. J. van der Veen, and L. Stearns (2012), Role of snow and glacier melt in controlling river hydrology in Liddar watershed (western Himalaya) under current and future climate, Water Resour. Res., 48, W12508,