Current status of research in rare genetic disorders and drug discovery in India.

The major health agenda of India so far has prioritized infectious diseases and public health. Given the socioeconomic conditions and poverty, a large fraction of the Indian population is exposed to infections from different pathogens, most notably enteric, parasitic, mycobacterial, and viral. In recent years, however, there has been a decline in the spread of these diseases with better surveillance, availability of therapy, improvement of socioeconomic conditions, and education. It is now being realized that non-communicable diseases are reaching epidemic proportions in India and there is a greater emphasis on the diagnosis and management of these diseases. The proportion of deaths due to non-communicable diseases has gone up substantially and was found to be about 61.8% of all deaths in 2016 (https://www.wbhealth.gov.in/NCD/).

Episomal and chromosomal DNA replication and recombination in Entamoeba histolytica.

Entamoeba histolytica is the causative agent of amoebiasis. DNA replication studies in E. histolytica first started with the ribosomal RNA genes located on episomal circles. Unlike most plasmids, Entamoeba histolytica rDNA circles lacked a fixed origin. Replication initiated from multiple sites on the episome, and these were preferentially used under different growth conditions. In synchronized cells the early origins mapped within the rDNA transcription unit, while at later times an origin in the promoter-proximal upstream intergenic spacer was activated. This is reminiscent of eukaryotic chromosomal replication where multiple potential origins are used. Biochemical studies on replication and recombination proteins in Entamoeba histolytica picked up momentum once the genome sequence was available. Sequence search revealed homologs of DNA replication and recombination proteins, including meiotic genes. The replicative DNA polymerases identified included the α, δ, ε of polymerase family B; lesion repair polymerases Rev1 and Rev3; a translesion repair polymerase of family A, and five families of polymerases related to family B2. Biochemical analysis of EhDNApolA confirmed its polymerase activity with expected kinetic constants. It could perform strand displacement, and translesion synthesis. The purified EhDNApolB2 had polymerase and exonuclease activities, and could efficiently bypass some types of DNA lesions. The single DNA ligase (EhDNAligI) was similar to eukaryotic DNA ligase I. It was a high-fidelity DNA ligase, likely involved in both replication and repair. Its interaction with EhPCNA was also demonstrated. The recombination-related proteins biochemically characterized were EhRad51 and EhDmc1. Both shared the canonical properties of a recombinase and could catalyse strand exchange over long DNA stretches. Presence of Dmc1 indicates the likelihood of meiosis in this parasite. Direct evidence of recombination in Entamoeba histolytica was provided by use of inverted repeat sequences located on plasmids or chromosomes. In response to a variety of stress conditions, and during encystation in Entamoeba invadens, recombination-related genes were upregulated and homologous recombination was enhanced. These data suggest that homologous recombination could have critical roles in trophozoite growth and stage conversion. Availability of biochemically characterized replication and recombination proteins is an important resource for exploration of novel anti-amoebic drug targets.

Multi-omics analysis to characterize molecular adaptation of Entamoeba histolytica during serum stress.

Entamoeba histolytica is responsible for dysentery and extraintestinal disease in humans. To establish successful infection, it must generate adaptive response against stress due to host defense mechanisms. We have developed a robust proteomics workflow by combining miniaturized sample preparation, low flow-rate chromatography, and ultra-high sensitivity mass spectrometry, achieving increased proteome coverage, and further integrated proteomics and RNA-seq data to decipher regulation at translational and transcriptional levels. Label-free quantitative proteomics led to identification of 2344 proteins, an improvement over the maximum number identified in E. histolytica proteomic studies. In serum-starved cells, 127 proteins were differentially abundant and were associated with functions including antioxidant activity, cytoskeleton, translation, catalysis, and transport. The virulence factor, Gal/GalNAc-inhibitable lectin subunits, was significantly altered. Integration of transcriptomic and proteomic data revealed that only 30% genes were coordinately regulated at both transcriptional and translational levels. Some highly expressed transcripts did not change in protein abundance. Conversely, genes with no transcriptional change showed enhanced protein abundance, indicating post-transcriptional regulation. This multi-omics approach enables more refined gene expression analysis to understand the adaptive response of E. histolytica during growth stress.

Transcriptomic analysis of ribosome biogenesis and pre-rRNA processing during growth stress in Entamoeba histolytica.

Ribosome biogenesis, a multi-step process involving transcription, modification, folding and processing of rRNA, is the major consumer of cellular energy. It involves sequential assembly of ribosomal proteins (RP)s via more than 200 ribogenesis factors. Unlike model organisms where transcription of rRNA and RP genes slows down during stress, in Entamoeba histolytica, pre-rRNA synthesis continues, and unprocessed pre-rRNA accumulates. Northern hybridization from different spacer regions depicted the accumulation of unprocessed intermediates during stress. To gain insight into the vast repertoire of ribosome biogenesis factors and understand the major components playing role during stress we computationally identified ribosome biogenesis factors in E. histolytica. Of the ∼279 Saccharomyces cerevisiae proteins, we could only find 188 proteins in E. histolytica. Some of the proteins missing in E. histolytica were also missing in humans. A number of proteins represented by multiple genes in S. cerevisiae had a single copy in E. histolytica. Interestingly E. histolytica lacked mitochondrial ribosome biogenesis factors and had far less RNase components compared to S. cerevisiae. Transcriptomic studies revealed the differential regulation of ribosomal factors both in serum starved and RRP6 down-regulation conditions. These included the NEP1 and TSR3 proteins that chemically modify 18S-rRNA. Pre-rRNA precursors accumulate upon downregulation of the latter proteins in S. cerevisiae and humans. These data reveal the major factors that regulate pre-rRNA processing during stress in E. histolytica and provide the first complete repertoire of ribosome biogenesis factors in this early-branching protist.

Tissue-specific isoform expression of GNE gene in human tissues.

Mutations in the sialic acid biosynthesis enzyme GNE lead to a late-onset, debilitating neuromuscular disorder, GNE myopathy, characterized by progressive skeletal muscle weakness. The mechanisms responsible for skeletal muscle specificity, late-onset, and disease progression are unknown. Our main aim is to understand the reason for skeletal muscle-specific phenotype. To answer this question, we have analyzed the expression profile of the GNE gene and its multiple mRNA variants in different human tissues. A combinatorial approach encompassing bioinformatics tools and molecular biology techniques was used. NCBI, Ensembl, and GTEx were used for data mining. The expression analysis of GNE and its variants was performed with cDNA tissue panel using PCR and targeted RNA-seq. Among nine different GNE isoforms reported in this study, transcript variants 1, X1, and X2 were not tissue specific. Transcript variants 1, 6, X1, and X2, were found in skeletal muscles suggesting their possible role in GNE myopathy. In the current study, we present new data about GNE expression patterns in human tissues. Our results suggest that there may be a link between tissue-specific pathology and isoform pattern in skeletal muscles, which could provide clues for the development of new treatment strategies for GNE myopathy.

Functional characterization of GNE mutations prevalent in Asian subjects with GNE myopathy, an ultra-rare neuromuscular disorder.

UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) is a bifunctional enzyme (N-terminal epimerase and C-terminal Kinase domain) that catalyses the rate limiting step in sialic acid biosynthesis. More than 200 homozygous missense or compound heterozygous mutations in GNE have been reported worldwide to cause a rare neuromuscular disorder, GNE myopathy. It is characterized by a slowly progressive defect in proximal and distal skeletal muscles with patients becoming wheel-chair-bound. There are no current approved therapies available for GNE myopathy. ManNAc therapy is currently in advanced clinical trials and has shown signs of slowing the disease progression in a phase 2 trial. The present study aims to understand the effect of GNE mutation on its enzymatic activity and identification of potential small effector molecules. We characterized different GNE mutations (p.Asp207Val, p.Val603Leu, p.Val727Met, p.Ile618Thr and p.Arg193Cys) prevalent in Asian population that were cloned, expressed and purified from Escherichia coli as full-length recombinant proteins. Our study demonstrates that full length GNE can be expressed in E. coli in its active form and analysed for the functional activity. Each mutation showed variation in epimerase and kinase activity and responded to the small effector molecules (metformin, BGP-15 kaempferol, catechin, quercetin) in a differential manner. Our study opens an area for futuristic structural determination of full length GNE and identification of potential therapeutic molecules.

The non-LTR retrotransposons of Entamoeba histolytica: genomic organization and biology.

Genome sequence analysis of Entamoeba species revealed various classes of transposable elements. While E. histolytica and E. dispar are rich in non-long terminal repeat (LTR) retrotransposons, E. invadens contains predominantly DNA transposons. Non-LTR retrotransposons of E. histolytica constitute three families of long interspersed nuclear elements (LINEs), and their short, nonautonomous partners, SINEs. They occupy ~ 11% of the genome. The EhLINE1/EhSINE1 family is the most abundant and best studied. EhLINE1 is 4.8 kb, with two ORFs that encode functions needed for retrotransposition. ORF1 codes for the nucleic acid-binding protein, and ORF2 has domains for reverse transcriptase (RT) and endonuclease (EN). Most copies of EhLINEs lack complete ORFs. ORF1p is expressed constitutively, but ORF2p is not detected. Retrotransposition could be demonstrated upon ectopic over expression of ORF2p, showing that retrotransposition machinery is functional. The newly retrotransposed sequences showed a high degree of recombination. In transcriptomic analysis, RNA-Seq reads were mapped to individual EhLINE1 copies. Although full-length copies were transcribed, no full-length 4.8 kb transcripts were seen. Rather, sense transcripts mapped to ORF1, RT and EN domains. Intriguingly, there was strong antisense transcription almost exclusively from the RT domain. These unique features of EhLINE1 could serve to attenuate retrotransposition in E. histolytica.

Ultra-structural analysis and morphological changes during the differentiation of trophozoite to cyst in Entamoeba invadens.

Entamoeba histolytica, a pathogenic parasite, is the causative organism of amoebiasis and uses human colon to complete its life cycle. It destroys intestinal tissue leading to invasive disease. Since it does not form cyst in culture medium, a reptilian parasite Entamoeba invadens serves as the model system to study encystation. Detailed investigation on the mechanism of cyst formation, information on ultra-structural changes and cyst wall formation during encystation are still lacking in E. invadens. Here, we used electron microscopy to study the ultrastructural changes during cyst formation and showed that the increase in heterochromatin patches and deformation of nuclear shape were early events in encystation. These changes peaked at ∼20 h post induction, and normal nuclear morphology was restored by 72 h. Two types of cellular structures were visible by 16 h. One was densely stained and consisted of the cytoplasmic mass with clearly visible nucleus. The other consisted of membranous shells with large vacuoles and scant cytoplasm. The former structure developed into the mature cyst while the latter structure was lost after 20 h, This study of ultra-structural changes during encystation in E. invadens opens up the possibilities for further investigation into the mechanisms involved in this novel process.

Transcriptomic analysis of Entamoeba histolytica reveals domain-specific sense strand expression of LINE-encoded ORFs with massive antisense expression of RT domain.

LINEs are retrotransposable elements found in diverse organisms. Their activity is kept in check by several mechanisms, including transcriptional silencing. Here we have analyzed the transcription status of LINE1 copies in the early-branching parasitic protist Entamoeba histolytica. Full-length EhLINE1 encodes ORF1, and ORF2 with reverse transcriptase (RT) and endonuclease (EN) domains. RNA-Seq analysis of EhLINE1 copies (both truncated and full-length) showed unique features. Firstly, although 20/41 transcribed copies were full-length, we failed to detect any full-length transcripts. Rather, sense-strand transcripts mapped to the functional domains- ORF1, RT and EN. Secondly, there was strong antisense transcription specifically from RT domain. No antisense transcripts were seen from ORF1. Antisense RT transcripts did not encode known functional peptides. They could possibly be involved in attenuating translation of RT domain, as we failed to detect ORF2p, whereas ORF1p was detectable. Lack of full-length transcripts and strong antisense RT expression may serve to limit EhLINE1 retrotransposition.

Tissue specific expression of sialic acid metabolic pathway: role in GNE myopathy.

GNE myopathy is an adult-onset degenerative muscle disease that leads to extreme disability in patients. Biallelic mutations in the rate-limiting enzyme UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine-kinase (GNE) of sialic acid (SA) biosynthetic pathway, was shown to be the cause of this disease. Other genetic disorders with muscle pathology where defects in glycosylation are known. It is yet not clear why a defect in SA biosynthesis and glycosylation affect muscle cells selectively even though they are ubiquitously present in all tissues. Here we have comprehensively examined the complete SA metabolic pathway involving biosynthesis, sialylation, salvage, and catabolism. To understand the reason for tissue-specific phenotype caused by mutations in genes of this pathway, we analysed the expression of different SA pathway genes in various tissues, during the muscle tissue development and in muscle tissues from GNE myopathy patients (p.Met743Thr) using publicly available databases. We have also analysed gene co-expression networks with GNE in different tissues as well as gene interactions that are unique to muscle tissues only. The results do show a few muscle specific interactions involving ANLN, MYO16 and PRAMEF25 that could be involved in specific phenotype. Overall, our results suggest that SA biosynthetic and catabolic genes are expressed at a very low level in skeletal muscles that also display a unique gene interaction network.

Entamoeba histolytica and pathogenesis: A calcium connection.

Calcium signaling plays a key role in many essential processes in almost all eukaryotic systems. It is believed that it may also be an important signaling system of the protist parasite Entamoeba histolytica. Motility, adhesion, cytolysis, and phagocytosis/trogocytosis are important steps in invasion and pathogenesis of E. histolytica, and Ca2+ signaling is thought to be associated with these processes leading to tissue invasion. There are a large number of Ca2+-binding proteins (CaBPs) in E. histolytica, and a number of these proteins appear to be associated with different steps in pathogenesis. The genome encodes 27 EF-hand-containing CaBPs in addition to a number of other Ca2+-binding domain/motif-containing proteins, which suggest intricate calcium signaling network in this parasite. Unlike other eukaryotes, a typical calmodulin-like protein has not been seen in E. histolytica. Though none of the CaBPs display sequence similarity with a typical calmodulin, extensive structural similarity has been seen in spite of lack of significant functional overlap with that of typical calmodulins. One of the unique features observed in E. histolytica is the identification of CaBPs (EhCaBP1, EhCaBP3) that have the ability to directly bind actin and modulate actin dynamics. Direct interaction of CaBPs with actin has not been seen in any other system. Pseudopod formation and phagocytosis are some of the processes that require actin dynamics, and some of the amoebic CaBPs (EhC2Pk, EhCaBP1, EhCaBP3, EhCaBP5) participate in this process. None of these E. histolytica CaBPs have any homolog in organisms other than different species of Entamoeba, suggesting a novel Ca2+ signaling pathway that has evolved in this genus.

The Inherited Neuromuscular Disorder GNE Myopathy: Research to Patient Care.

Inherited neuromuscular diseases are a heterogeneous group of rare diseases for which the low general awareness leads to frequent misdiagnosis. Advances in DNA sequencing technologies are changing this situation, and it is apparent that these diseases are not as rare as previously thought. Knowledge of the pathogenic variants in patients is helping in research efforts to develop new therapies. Here we present a review of current knowledge in GNE myopathy, a rare neuromuscular disorder caused by mutations in the GNE gene that catalyzes the biosynthesis of sialic acid. The most common initial symptom is foot drop caused by anterior tibialis muscle weakness. There is a progressive wasting of distal skeletal muscles in the lower and upper extremities as well. The quadriceps is relatively spared, which is a distinguishing feature of this disease. The characteristic histological features include autophagic rimmed vacuoles with inclusion bodies. GNE variant analysis of Indian patients has revealed a founder mutation (p.Val727Met) common within the normal Indian populations, especially in the state of Gujurat. We discuss therapeutic options, including metabolite supplementation, pharmacological chaperones, and gene therapy. Initiatives that bring together patients, researchers, and physicians are necessary to improve knowledge and treatment for these rare disorders.

Advances in Entamoeba histolytica Biology Through Transcriptomic Analysis.

A large number of transcriptome-level studies in Entamoeba histolytica, the protozoan parasite that causes amoebiasis, have investigated gene expression patterns to help understand the pathology and biology of the organism. They have compared virulent and avirulent strains in lab culture and after tissue invasion, cells grown under different stress conditions, response to anti-amoebic drug treatments, and gene expression changes during the process of encystation. These studies have revealed interesting molecules/pathways that will help increase our mechanistic understanding of differentially expressed genes during growth perturbations and tissue invasion. Some of the important insights obtained from transcriptome studies include the observations that regulation of carbohydrate metabolism may be an important determinant for tissue invasion, while the novel up-regulated genes during encystation include phospholipase D, and meiotic genes, suggesting the possibility of meiosis during the process. Classification of genes according to expression levels showed that amongst the highly transcribed genes in cultured E. histolytica trophozoites were some virulence factors, raising the question of the role of these factors in normal parasite growth. Promoter motifs associated with differential gene expression and regulation were identified. Some of these motifs associated with high gene expression were located downstream of start codon, and were required for efficient transcription. The listing of E. histolytica genes according to transcript expression levels will help us determine the scale of post-transcriptional regulation, and the possible roles of predicted promoter motifs. The small RNA transcriptome is a valuable resource for detailed structural and functional analysis of these molecules and their regulatory roles. These studies provide new drug targets and enhance our understanding of gene regulation in E. histolytica.

Novel regulatory roles of PtdIns(4,5)P2 generating enzyme EhPIPKI in actin dynamics and phagocytosis of Entamoeba histolytica.

Motility and phagocytosis are the two important processes that are intricately linked to survival and virulence potential of the protist parasite Entamoeba histolytica. These processes primarily rely on actin-dependent pathways, and regulation of these pathways is critical for understanding the pathology of E. histolytica. Generally, phosphoinositides dynamics have not been explored in amoebic actin dynamics and particularly during phagocytosis in E. histolytica. We have explored the roles of PtdIns(4,5)P2 as well as the enzyme that produces this metabolite, EhPIPKI during phagocytosis. Immunofluorescence and live cell images showed enrichment of EhPIPKI in different stages of phagocytosis from initiation till the cups progressed towards closure. However, the enzyme was absent after phagosomes are pinched off from the membrane. Overexpression of a dominant negative mutant revealed a reduction in the formation of phagocytic cups and inhibition in the rate of engulfment of erythrocytes. Moreover, EhPIPKI binds directly to F and G-actin unlike PIPKs from other organisms. PtdIns(4,5)P2 , the product of the enzyme, also followed a similar distribution pattern during phagocytosis as determined by a GFP-tagged PH-domain from PLCδ, which specifically binds PtdIns(4,5)P2 in trophozoites. In summary, EhPIPKI regulates initiation of phagocytosis by regulating actin dynamics.

Myg1 exonuclease couples the nuclear and mitochondrial translational programs through RNA processing.

Semi-autonomous functioning of mitochondria in eukaryotic cell necessitates coordination with nucleus. Several RNA species fine-tune mitochondrial processes by synchronizing with the nuclear program, however the involved components remain enigmatic. In this study, we identify a widely conserved dually localized protein Myg1, and establish its role as a 3'-5' RNA exonuclease. We employ mouse melanoma cells, and knockout of the Myg1 ortholog in Saccharomyces cerevisiae with complementation using human Myg1 to decipher the conserved role of Myg1 in selective RNA processing. Localization of Myg1 to nucleolus and mitochondrial matrix was studied through imaging and confirmed by sub-cellular fractionation studies. We developed Silexoseqencing, a methodology to map the RNAse trail at single-nucleotide resolution, and identified in situ cleavage by Myg1 on specific transcripts in the two organelles. In nucleolus, Myg1 processes pre-ribosomal RNA involved in ribosome assembly and alters cytoplasmic translation. In mitochondrial matrix, Myg1 processes 3'-termini of the mito-ribosomal and messenger RNAs and controls translation of mitochondrial proteins. We provide a molecular link to the possible involvement of Myg1 in chronic depigmenting disorder vitiligo. Our study identifies a key component involved in regulating spatially segregated organellar RNA processing and establishes the evolutionarily conserved ribonuclease as a coordinator of nucleo-mitochondrial crosstalk.

Transcriptomic analysis reveals novel downstream regulatory motifs and highly transcribed virulence factor genes of Entamoeba histolytica.

BACKGROUND: Promoter motifs in Entamoeba histolytica were earlier analysed using microarray data with lower dynamic range of gene expression. Additionally, previous transcriptomic studies did not provide information on the nature of highly transcribed genes, and downstream promoter motifs important for gene expression. To address these issues we generated RNA-Seq data and identified the high and low expressing genes, especially with respect to virulence potential. We analysed sequences both upstream and downstream of start site for important motifs. RESULTS: We used RNA-Seq data to classify genes according to expression levels, which ranged six orders of magnitude. Data were validated by reporter gene expression. Virulence-related genes (except AIG1) were amongst the highly expressed, while some kinases and BspA family genes were poorly expressed. We looked for conserved motifs in sequences upstream and downstream of the initiation codon. Following enrichment by AME we found seven motifs significantly enriched in high expression- and three in low expression-classes. Two of these motifs (M4 and M6) were located downstream of AUG, were exclusively enriched in high expression class, and were mostly found in ribosomal protein, and translation-related genes. Motif deletion resulted in drastic down regulation of reporter gene expression, showing functional relevance. Distribution of core promoter motifs (TATA, GAAC, and Inr) in all genes revealed that genes with downstream motifs were not preferentially associated with TATA-less promoters. We looked at gene expression changes in cells subjected to growth stress by serum starvation, and experimentally validated the data. Genes showing maximum up regulation belonged to the low or medium expression class, and included genes in signalling pathways, lipid metabolism, DNA repair, Myb transcription factors, BspA, and heat shock. Genes showing maximum down regulation belonged to the high or medium expression class. They included genes for signalling factors, actin, Ariel family, and ribosome biogenesis factors. CONCLUSION: Our analysis has added important new information about the E. histolytica transcriptome. We report for the first time two downstream motifs required for gene expression, which could be used for over expression of E. histolytica genes. Most of the virulence-related genes in this parasite are highly expressed in culture.

PtdIns(4,5)P2 is generated by a novel phosphatidylinositol 4-phosphate 5-kinase in the protist parasite Entamoeba histolytica.

Entamoeba histolytica is an intestinal protist parasite that causes amoebiasis, a major source of morbidity and mortality in developing countries. Phosphoinositides are involved in signalling systems that have a role in invasion and pathogenesis of this parasite. Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) catalyses the generation of phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P2 ), a key species of phosphoinositide that regulates various cellular processes. However, phosphatidylinositol phosphate kinase (PIPK) family of enzymes have not been characterized in E. histolytica. Here, we report the identification and characterization of type I PIPK (EhPIPKI) of E. histolytica. Computational analysis revealed homologs of type I and III PIPK family in E. histolytica and the absence of type II PIPK. In spite of low overall sequence identity, the kinase domain was found to be highly conserved. Interestingly, a unique insertion of a tandem repeat motif was observed in EhPIPKI distinguishing it from existing PIPKs of other organisms. Substrate profiling showed that EhPIPKI could phosphorylate at third and fifth hydroxyl positions of phosphatidylinositols, though the predominant substrate was phosphatidylinositol 4-phosphate (PtdIns(4)P). Furthermore, EhPIPKI underwent intracellular cleavage close to the amino-terminal, generating two distinct fragments Nter-EhPIPKI (27p) and Cter-EhPIPKI (47p). Immunofluorescence and cellular fractionation revealed that the full-length EhPIPKI and the Cter-EhPIPKI containing carboxyl-terminal activation loop were present in the plasma membrane while the Nter-EhPIPKI was observed in the cytosolic region. In conclusion, E. histolytica has a single EhPIPKI gene that displays novel properties of post-translational processing, the presence of a repeat domain and substrate specificity not observed in any PIPK enzyme so far.

Stress-induced nuclear depletion of Entamoeba histolytica 3'-5' exoribonuclease EhRrp6 and its role in growth and erythrophagocytosis.

The 3'-5' exoribonuclease Rrp6 is a key enzyme in RNA homeostasis involved in processing and degradation of many stable RNA precursors, aberrant transcripts, and noncoding RNAs. We previously have shown that in the protozoan parasite Entamoeba histolytica, the 5'-external transcribed spacer fragment of pre-rRNA accumulates under serum starvation-induced growth stress. This fragment is a known target of degradation by Rrp6. Here, we computationally and biochemically characterized EhRrp6 and found that it contains the catalytically important EXO and HRDC domains and exhibits exoribonuclease activity with both unstructured and structured RNA substrates, which required the conserved DEDD-Y catalytic-site residues. It lacked the N-terminal PMC2NT domain for binding of the cofactor Rrp47, but could functionally complement the growth defect of a yeast rrp6 mutant. Of note, no Rrp47 homologue was detected in E. histolytica Immunolocalization studies revealed that EhRrp6 is present both in the nucleus and cytosol of normal E. histolytica cells. However, growth stress induced its complete loss from the nuclei, reversed by proteasome inhibitors. EhRrp6-depleted E. histolytica cells were severely growth restricted, and EhRrp6 overexpression protected the cells against stress, suggesting that EhRrp6 functions as a stress sensor. Importantly EhRrp6 depletion reduced erythrophagocytosis, an important virulence determinant of E. histolytica This reduction was due to a specific decrease in transcript levels of some phagocytosis-related genes (Ehcabp3 and Ehrho1), whereas expression of other genes (Ehcabp1, Ehcabp6, Ehc2pk, and Eharp2/3) was unaffected. This is the first report of the role of Rrp6 in cell growth and stress responses in a protozoan parasite.