Publications by authors named "Alok Bhattacharya"

128 Publications

Coordinated activity of amoebic formin and profilin are essential for phagocytosis.

Mol Microbiol 2021 Jul 19. Epub 2021 Jul 19.

Multidisciplinary Centre for Advanced Research and Studies, Jamia Millia Islamia, New Delhi, India.

For the protist parasite Entamoeba histolytica, endocytic processes, such as phagocytosis, are essential for its survival in the human gut. The actin cytoskeleton is involved in the formation of pseudopods and phagosomal vesicles by incorporating a number of actin-binding and modulating proteins along with actin in a temporal manner. The actin dynamics, which comprises polymerization, branching, and depolymerization is very tightly regulated and takes place directionally at the sites of initiation of phagocytosis. Formin and profilin are two actin-binding proteins that are known to regulate actin cytoskeleton dynamics and thereby, endocytic processes. In this article, we report the participation of formin and profilin in E. histolytica phagocytosis and propose that these two proteins interact with each other and their sequential recruitment at the site is required for the successful completion of phagocytosis. The evidence is based on detailed microscopic, live imaging, interaction studies, and expression downregulation. The cells downregulated for expression of formin show absence of profilin at the site of phagocytosis, whereas downregulation of profilin does not affect formin localization.
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http://dx.doi.org/10.1111/mmi.14787DOI Listing
July 2021

Pattern of genomic variation in SARS-CoV-2 (COVID-19) suggests restricted nonrandom changes: Analysis using Shewhart control charts.

J Biosci 2021 ;46

School of Computational and Integrative Sciences, Jawaharlal Nehru University, New Delhi, India.

SARS-CoV-2 is a member of the Coronavirus family which recently originated from the Wuhan province of China and spread very rapidly through the world infecting more than 4 million people. In the past, other Coronaviruses have also been found to cause human infection, but not as widespread as COVID-19. Since Coronavirus sequences constantly change due to mutation and recombination, it is important to understand the pattern of changes and likely path the virus can take in the future. In this study, we have used the Shewhart control chart to identify and analyze hypervariable (hotspots) and hypovariable (coldspots) regions of the virus. Our analysis shows that SARS-CoV-2 has changed in a few regions of the genome. Analysis of SARS-CoV-1 and MERS sequences suggests that over time, mutations start accumulating in different regions and most likely SARS-CoV-2 may also follow a similar path. The results suggest a possible emergence of modified viruses over some time.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7856336PMC
March 2021

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

Plasmid 2021 03 20;114:102560. Epub 2021 Jan 20.

School of Environmental Sciences, Jawaharlal Nehru University, India. Electronic address:

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.
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http://dx.doi.org/10.1016/j.plasmid.2021.102560DOI Listing
March 2021

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

J Muscle Res Cell Motil 2021 03 7;42(1):99-116. Epub 2020 Oct 7.

Ashoka University, Plot No. 2, Rajiv Gandhi Education City, P.O.Rai, Sonepat, Haryana, 131029, India.

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.
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http://dx.doi.org/10.1007/s10974-020-09590-7DOI Listing
March 2021

Calcium modulates the domain flexibility and function of an α-actinin similar to the ancestral α-actinin.

Proc Natl Acad Sci U S A 2020 09 26;117(36):22101-22112. Epub 2020 Aug 26.

Department of Structural and Computational Biology, Max Perutz Labs, University of Vienna, A-1030 Vienna, Austria;

The actin cytoskeleton, a dynamic network of actin filaments and associated F-actin-binding proteins, is fundamentally important in eukaryotes. α-Actinins are major F-actin bundlers that are inhibited by Ca in nonmuscle cells. Here we report the mechanism of Ca-mediated regulation of α-actinin-2 (Actn2) with features expected for the common ancestor of and higher eukaryotic α-actinins. Crystal structures of Ca-free and Ca-bound Actn2 reveal a calmodulin-like domain (CaMD) uniquely inserted within the rod domain. Integrative studies reveal an exceptionally high affinity of the Actn2 CaMD for Ca, binding of which can only be regulated in the presence of physiological concentrations of Mg Ca binding triggers an increase in protein multidomain rigidity, reducing conformational flexibility of F-actin-binding domains via interdomain cross-talk and consequently inhibiting F-actin bundling. In vivo studies uncover that Actn2 plays an important role in phagocytic cup formation and might constitute a new drug target for amoebic dysentery.
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http://dx.doi.org/10.1073/pnas.1917269117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7486759PMC
September 2020

Ca-binding protein from Entamoeba histolytica (EhCaBP6) is a novel GTPase.

Biochem Biophys Res Commun 2020 06 16;527(3):631-637. Epub 2020 May 16.

Tata Institute of Fundamental Research, Mumbai, 400005, India; Tata Institute of Fundamental Research, Centre for Interdisciplinary Sciences, 500075, Hyderabad, India; Indian Institute of Science Education and Research Berhampur, 760010, India. Electronic address:

GTPases are molecular switches, which regulate a variety of cellular processes such as cell polarity, gene transcription, microtubule dynamics, cell-cycle etc. In this paper, we characterize a Ca-binding protein from Entamoeba histolytica (EhCaBP6) as a novel GTPase. We locate the active site for GTP hydrolysis within the C-terminal domain of EhCaBP6, although it requires full length protein for its complete range of activity. Using NMR studies, we observe that GTP binding induces conformational change in EhCaBP6. The identification of this novel and unusual Ca-dependent GTPase is important to elucidate the unconventional cell cycle of E. histolytica.
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http://dx.doi.org/10.1016/j.bbrc.2020.04.141DOI Listing
June 2020

Entamoeba histolytica and pathogenesis: A calcium connection.

PLoS Pathog 2020 05 7;16(5):e1008214. Epub 2020 May 7.

Department of Biology, Ashoka University, Sonepat, Haryana, India.

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.
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http://dx.doi.org/10.1371/journal.ppat.1008214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7205191PMC
May 2020

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

Neurol India 2019 Sep-Oct;67(5):1213-1219

World without GNE Myopathy, Jawaharlal Nehru University; School of Environmental Sciences, Jawaharlal Nehru University, New Delhi, India.

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.
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http://dx.doi.org/10.4103/0028-3886.271259DOI Listing
April 2020

Advances in Biology Through Transcriptomic Analysis.

Front Microbiol 2019 20;10:1921. Epub 2019 Aug 20.

School of Environmental Sciences, Jawaharlal Nehru University, New Delhi, India.

A large number of transcriptome-level studies in , 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 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 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 .
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http://dx.doi.org/10.3389/fmicb.2019.01921DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6710346PMC
August 2019

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

Cell Microbiol 2019 Oct 30;21(10):e13087. Epub 2019 Jul 30.

Department of Biological Sciences, Ashoka University, India.

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)P 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)P , 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)P in trophozoites. In summary, EhPIPKI regulates initiation of phagocytosis by regulating actin dynamics.
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http://dx.doi.org/10.1111/cmi.13087DOI Listing
October 2019

Patient-driven initiatives for prioritizing drug discovery for rare diseases.

Indian J Med Res 2019 03;149(3):326-328

School of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110 067, India.

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http://dx.doi.org/10.4103/ijmr.IJMR_499_19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6607820PMC
March 2019

EhP3, a homolog of 14-3-3 family of protein participates in actin reorganization and phagocytosis in Entamoeba histolytica.

PLoS Pathog 2019 05 16;15(5):e1007789. Epub 2019 May 16.

School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.

The highly conserved proteins of the 14-3-3 family are universal adaptors known to regulate an enormous range of cellular processes in eukaryotes. However, their biological functions remain largely uncharacterized in pathogenic protists comprising of several 14-3-3 protein isoforms. In this study, we report the role of 14-3-3 in coordinating cytoskeletal dynamics during phagocytosis in a professional phagocytic protist Entamoeba histolytica, the etiological agent of human amebiasis. There are three isoforms of 14-3-3 protein in amoeba and here we have investigated Eh14-3-3 Protein 3 (EhP3). Live and fixed cell imaging studies revealed the presence of this protein throughout the parasite phagocytosis process, with high rate of accumulation at the phagocytic cups and closed phagosomes. Conditional suppression of EhP3 expression caused significant defects in phagocytosis accompanied by extensive diminution of F-actin at the site of cup formation. Downregulated cells also exhibited defective recruitment of an F-actin stabilizing protein, EhCoactosin at the phagocytic cups. In addition, mass spectrometry based analysis further revealed a large group of EhP3-associated proteins, many of these proteins are known to regulate cytoskeletal architecture in E histolytica. The dynamics of these proteins may also be controlled by EhP3. Taken together, our findings strongly suggest that EhP3 is a novel and a key regulatory element of actin dynamics and phagocytosis in E. histolytica.
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http://dx.doi.org/10.1371/journal.ppat.1007789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6541287PMC
May 2019

Structural and functional characterisation of phosphoserine phosphatase, that plays critical role in the oxidative stress response in the parasite Entamoeba histolytica.

J Struct Biol 2019 05 29;206(2):254-266. Epub 2019 Mar 29.

School of Life Sciences, Jawaharlal Nehru University, New Delhi, India. Electronic address:

Amoebiasis is a common parasitic infection in the developing world and is caused by the protist Entameoba histolytica. The proliferation of E. histolytica and its ability to invade epithelial tissues have been shown in several studies to be greatly decreased during oxidative stress. It is therefore not surprising that this amoeba has evolved several mechanisms to evade oxidative stress. Cysteine is thought to be one of the crucial molecules that help in redox defence, and a de novo cysteine biosynthetic pathway involving serine as one of the substrates has been partially elucidated in E. histolytica. Though most of the enzymes of this pathway in E. histolytica have been characterized, phosphoserine phosphatase (EhPSP), a key regulatory enzyme of the serine biosynthetic pathway, has not yet even been identified. In the current work, we identified and characterized EhPSP using various molecular, structural and functional approaches. The crystal structures of native and substrate-bound EhPSP were determined and showed the residues that play a crucial role in its phosphatase activity and substrate binding. Structural and biochemical studies indicated that EhPSP belongs to the histidine phosphatase superfamily. EhPSP-overexpressing amoebic cells were found to be more tolerant to oxidative stress. However, protection during oxidative stress was not seen when a functionally defective mutant was overexpressed. Our results clearly showed that E. histolytica has a functional PSP and that this protein participates in protecting the organism against oxidative stress.
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http://dx.doi.org/10.1016/j.jsb.2019.03.012DOI Listing
May 2019

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

BMC Genomics 2019 Mar 12;20(1):206. Epub 2019 Mar 12.

School of Environmental Sciences, Jawaharlal Nehru University, New Delhi, India.

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.
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http://dx.doi.org/10.1186/s12864-019-5570-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6416950PMC
March 2019

Phagocytosis of Gut Bacteria by .

Front Cell Infect Microbiol 2019 26;9:34. Epub 2019 Feb 26.

School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.

The protist parasite causes amoebiasis, a major public health problem in developing countries. Only a small fraction of patients infected with the parasite display invasive disease involving colon or extra intestinal tissues such as liver. exists as two distinct forms, cysts, the infective form, and trophozoites, that are responsible for disease pathology. The latter multiply in the large intestine occasionally causing disease. The large intestine in humans is populated by a number of different bacterial communities and amoebic cells grow in their midst using some as food material. Several studies have shown relationship between bacteria and growth and virulence. However, an understanding of this relationship in human gut environment is not clear. We have investigated the possibility that there may be specific interaction of amoeba with different bacteria present in the gut environment by using a metagenomic pipe line. This was done by incubating bacteria isolated from human fecal material with and then identifying the bacterial population isolated from amoebic cells using a rRNA based metagenomic approach. Our results show that the parasite prefers a few bacterial species. One of these species is which has never shown to be associated with .
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http://dx.doi.org/10.3389/fcimb.2019.00034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6399400PMC
November 2019

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

FEBS J 2019 06 25;286(11):2216-2234. Epub 2019 Mar 25.

School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.

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)P ), 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.
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http://dx.doi.org/10.1111/febs.14804DOI Listing
June 2019

EhFP10: A FYVE family GEF interacts with myosin IB to regulate cytoskeletal dynamics during endocytosis in Entamoeba histolytica.

PLoS Pathog 2019 02 19;15(2):e1007573. Epub 2019 Feb 19.

School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.

Motility and phagocytosis are key processes that are involved in invasive amoebiasis disease caused by intestinal parasite Entamoeba histolytica. Previous studies have reported unconventional myosins to play significant role in membrane based motility as well as endocytic processes. EhMyosin IB is the only unconventional myosin present in E. histolytica, is thought to be involved in both of these processes. Here, we report an interaction between the SH3 domain of EhMyosin IB and c-terminal domain of EhFP10, a Rho guanine nucleotide exchange factor. EhFP10 was found to be confined to Entamoeba species only, and to contain a c-terminal domain that binds and bundles actin filaments. EhFP10 was observed to localize in the membrane ruffles, phagocytic and macropinocytic cups of E. histolytica trophozoites. It was also found in early pinosomes but not early phagosomes. A crystal structure of the c-terminal SH3 domain of EhMyosin IB (EhMySH3) in complex with an EhFP10 peptide and co-localization studies established the interaction of EhMySH3 with EhFP10. This interaction was shown to lead to inhibition of actin bundling activity and to thereby regulate actin dynamics during endocytosis. We hypothesize that unique domain architecture of EhFP10 might be compensating the absence of Wasp and related proteins in Entamoeba, which are known partners of myosin SH3 domains in other eukaryotes. Our findings also highlights the role of actin bundling during endocytosis.
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http://dx.doi.org/10.1371/journal.ppat.1007573DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6396940PMC
February 2019

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

J Biol Chem 2018 10 31;293(42):16242-16260. Epub 2018 Aug 31.

From the School of Environmental Sciences,

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 , 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 mutant. Of note, no Rrp47 homologue was detected in Immunolocalization studies revealed that EhRrp6 is present both in the nucleus and cytosol of normal cells. However, growth stress induced its complete loss from the nuclei, reversed by proteasome inhibitors. EhRrp6-depleted 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 This reduction was due to a specific decrease in transcript levels of some phagocytosis-related genes ( and ), whereas expression of other genes (, , , and ) was unaffected. This is the first report of the role of Rrp6 in cell growth and stress responses in a protozoan parasite.
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http://dx.doi.org/10.1074/jbc.RA118.004632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6200944PMC
October 2018

Calcium-binding protein EhCaBP3 is recruited to the phagocytic complex of Entamoeba histolytica by interacting with Arp2/3 complex subunit 2.

Cell Microbiol 2018 Dec 7;20(12):e12942. Epub 2018 Sep 7.

School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.

Phagocytosis is involved in invasive disease of the parasite Entamoeba histolytica. Upon binding of red blood cells, there is a sequential recruitment of EhC2PK, EhCaBP1, EhAK1, and Arp2/3 complex during the initiation phase. In addition, EhCaBP3 is also recruited to the site and, along with myosin 1B, is thought to be involved in progression of phagocytic cups from initiation to phagosome formation. However, it is not clear how EhCaBP3 gets recruited to the rest of the phagocytic machinery. Here, we show that EhARPC2, a subunit of Arp2/3 complex, interacts with EhCaBP3 in a Ca -dependent manner both in vivo and in vitro. Imaging and pull down experiments suggest that interaction with EhARPC2 is required for the closure of cups and formation of phagosomes. Moreover, downregulation of EhARPC2 prevents localisation of EhCaBP3 to phagocytic cups, suggesting that EhCaBP3 is part of EhC2PK-EhCaBP1-EhAK1-Arp2/3 complex (EhARPC1) pathway. In conclusion, these results suggest that the EhCaBP3-EhARPC2 interaction helps to recruit EhCaBP3 along with myosin 1B to the phagocytic machinery that plays an indispensable role in E. histolytica phagocytosis.
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http://dx.doi.org/10.1111/cmi.12942DOI Listing
December 2018

EhRho1 regulates phagocytosis by modulating actin dynamics through EhFormin1 and EhProfilin1 in Entamoeba histolytica.

Cell Microbiol 2018 09 7;20(9):e12851. Epub 2018 May 7.

School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.

The protist parasite Entamoeba histolytica causes amoebiasis, a major public health problem in developing countries and a major cause of morbidity and mortality. Invasive infection in amoebiasis mostly affects intestinal epithelial cell lining but can also involve other organs, such as liver, lungs, or brain. Phagocytosis is an essential mode of nutrition in amoeba and has often been associated with virulence behaviour of E. histolytica. E. histolytica possesses a highly dynamic and actin-rich cytoskeleton that is thought to be involved in many processes, such as motility, pseudopod formation, and pathogenesis. Rho GTPases are known to be key regulators of the actin cytoskeleton and consequently influence the shape and movement of cells. Our study is mainly focused to understand the role of EhRho1 in the phagocytosis process of E. histolytica. EhRho1 got enriched in the phagocytic cups along with EhActin and remains attached with phagosomal membrane. However, there was no direct binding of EhRho1 with G- or F-actin, though binding was observed with the actin nucleating proteins EhFormin1 and EhProfilin1. Overexpression of dominant negative mutant or lowering the expression by antisense RNA of EhRho1 in trophozoites caused delocalisation of EhFormin1 and EhProfilin1 from phagocytic cups, which results in impairment of phagocytic process and decrease in F-actin content. The overall results show that EhRho1 regulates phagocytosis by modulating actin dynamics through recruitment of EhFormin1 and EhProfilin1 at the phagocytosis nucleation site in E. histolytica.
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http://dx.doi.org/10.1111/cmi.12851DOI Listing
September 2018

Mutation Spectrum of GNE Myopathy in the Indian Sub-Continent.

J Neuromuscul Dis 2018;5(1):85-92

School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.

Background: GNE myopathy is an adult onset recessive genetic disorder that affects distal muscles sparing the quadriceps. GNE gene mutations have been identified in GNE myopathy patients all over the world. Homozygosity is a common feature in GNE myopathy patients worldwide.

Objectives: The major objective of this study was to investigate the mutation spectrum of GNE myopathy in India in relation to the population diversity in the country.

Materials And Methods: We have collated GNE mutation data of Indian GNE myopathy patients from published literature and from recently identified patients. We also used data of people of Indian subcontinent from 1000 genomes database, South Asian Genome database and Strand Life Science database to determine frequency of GNE mutations in the general population.

Results: A total of 67 GNE myopathy patients were studied, of whom 21% were homozygous for GNE variants, while the rest were compound heterozygous. Thirty-five different mutations in the GNE gene were recorded, of which 5 have not been reported earlier. The most frequent mutation was p.Val727Met (65%) found mainly in the heterozygous form. Another mutation, p.Ile618Thr was also common (16%) but was found mainly in patients from Rajasthan, while p.Val727Met was more widely distributed. The latter was also seen at a high frequency in general population of Indian subcontinent in all the databases. It was also present in Thailand but was absent in general population elsewhere in the world.

Conclusion: p.Val727Met is likely to be a founder mutation of Indian subcontinent.
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http://dx.doi.org/10.3233/JND-170270DOI Listing
June 2018

Classification of pathogenic microbes using a minimal set of single nucleotide polymorphisms derived from whole genome sequences.

Genomics 2019 03 10;111(2):205-211. Epub 2018 Feb 10.

School of Computational & Integrative Sciences, Jawaharlal Nehru University, New Delhi, India; School of Life Sciences, Jawaharlal Nehru University, New Delhi, India. Electronic address:

In a context specific manner, Intra-species genomic variation plays an important role in phenotypic diversity observed among pathogenic microbes. Efficient classification of these pathogens is important for diagnosis and treatment of several infectious diseases. NGS technologies have provided access to wealth of data that can be utilized to discover important markers for pathogen classification. In this paper, we described three different approaches (Jensen-Shannon divergence, random forest and Shewhart control chart) for identification of a minimal set of SNPs that can be used for classification of organisms. These methods are generic and can be implemented for analysis of any organism. We have shown usefulness of these approaches for analysis of Mycobacterium tuberculosis and Escherichia coli isolates. We were able to identify a minimal set of 18 SNPs that can be used as molecular markers for phylogroup based classification and 8 SNPs for pathogroup based classification of E. coli.
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http://dx.doi.org/10.1016/j.ygeno.2018.02.004DOI Listing
March 2019

Application of SHAPE reveals in vivo RNA folding under normal and growth-stressed conditions in the human parasite Entamoeba histolytica.

Mol Biochem Parasitol 2018 01 23;219:42-51. Epub 2017 Nov 23.

School of Environmental Sciences, Jawaharlal Nehru University, New Delhi, India. Electronic address:

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) is a versatile sequence independent method to probe RNA structure in vivo and in vitro. It has so far been tried mainly with model organisms. We show that cells of Entamoeba histolytica, a protozoan parasite of humans are hyper-sensitive to the in vivo SHAPE reagent, NAI, and show rapid loss of viability and RNA integrity. We optimized treatment conditions with 5.8S rRNA and Eh_U3 snoRNA to obtain NAI-modification while retaining RNA integrity. The modification patterns were highly reproducible. The in vivo folding was different from in vitro and correlated well with known interactions of 5.8S rRNA with proteins in vivo. The Eh_U3 snoRNA also showed many differences in its in vivo versus in vitro folding, which correlated with conserved interactions of this RNA with 18S rRNA and 5'-ETS. Further, Eh_U3 snoRNA obtained from serum-starved cells showed an open 3'-hinge structure, indicating disruption of 5'-ETS interaction. This could contribute to the observed slow processing of pre-rRNA in starved cells. Our work shows the applicability of SHAPE to study in vivo RNA folding in a parasite and will encourage the use of this reagent for RNA structure analysis in other such organisms.
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http://dx.doi.org/10.1016/j.molbiopara.2017.11.003DOI Listing
January 2018

IsomiR processing during differentiation of myelogenous leukemic cell line K562 by phorbol ester PMA.

Gene 2018 Jan 17;641:172-179. Epub 2017 Oct 17.

School of Life Sciences, Jawaharlal Nehru University, New Delhi, India; School of Computational and Integrative Sciences, Jawaharlal Nehru University, New Delhi, India. Electronic address:

Chronic myelocytic leukemia cell line K562 undergoes differentiation by phorbol esters to megakaryocytes and we have used this system to understand miRNA processing leading to isomiR generation. PMA treatment significantly altered the production of miRNA in K562 cells. Expression of 24.4% of miRNAs were found to be stimulated whereas expression of 10% miRNAs were inhibited by PMA treatment. Our results suggest that miRNA precursors are processed into isomiRs in a deterministic manner. The relative levels of different isomiRs of a miRNA remained mainly unchanged even after PMA treatment irrespective of overall changes in expression (either up-regulation or down-regulation). However, not all miRNAs behave in the same way, about 7% showed a variation of isomiR profiles after PMA treatment. Most of the later class of miRNAs were found to be oncogenic miRNAs. Further, it was also found that number of isomiRs was independent of abundance of a miRNA. Functional importance of different isomiRs was demonstrated using three different isomiRs of miR-22. Our results showed that different isomiRs could inhibit expression of targets genes with different efficiencies. Our study suggests that the heterogeneity of a miRNA population generated during processing is in general regulated and that variation in the generation of an isomiR can be a functionally important regulatory feature.
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http://dx.doi.org/10.1016/j.gene.2017.10.025DOI Listing
January 2018

Structure of Ca2+-binding protein-6 from Entamoeba histolytica and its involvement in trophozoite proliferation regulation.

PLoS Pathog 2017 May 15;13(5):e1006332. Epub 2017 May 15.

Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai, India.

Cell cycle of Entamoeba histolytica, the etiological agent of amoebiasis, follows a novel pathway, which includes nuclear division without the nuclear membrane disassembly. We report a nuclear localized Ca2+-binding protein from E. histolytica (abbreviated hereafter as EhCaBP6), which is associated with microtubules. We determined the 3D solution NMR structure of EhCaBP6, and identified one unusual, one canonical and two non-canonical cryptic EF-hand motifs. The cryptic EF-II and EF-IV pair with the Ca2+-binding EF-I and EF-III, respectively, to form a two-domain structure similar to Calmodulin and Centrin proteins. Downregulation of EhCaBP6 affects cell proliferation by causing delays in transition from G1 to S phase, and inhibition of DNA synthesis and cytokinesis. We also demonstrate that EhCaBP6 modulates microtubule dynamics by increasing the rate of tubulin polymerization. Our results, including structural inferences, suggest that EhCaBP6 is an unusual CaBP involved in regulating cell proliferation in E. histolytica similar to nuclear Calmodulin.
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http://dx.doi.org/10.1371/journal.ppat.1006332DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5444848PMC
May 2017

EhRho1 regulates plasma membrane blebbing through PI3 kinase in Entamoeba histolytica.

Cell Microbiol 2017 10 22;19(10). Epub 2017 Jun 22.

School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.

The protozoan parasite Entamoeba histolytica causes amoebiasis, a major public health problem in developing countries. Motility of E. histolytica is important for its pathogenesis. Blebbing is an essential process contributing to cellular motility in many systems. In mammalian cells, formation of plasma membrane blebs is regulated by Rho-GTPases through its effectors, such as Rho kinase, mDia1, and acto-myosin proteins. In this study, we have illuminated the role of EhRho1 in bleb formation and motility of E. histolytica. EhRho1 was found at the site of bleb formation in plasma membrane of trophozoites. Overexpression of mutant EhRho1 defective for Guanosine triphosphate (GTP)-binding or down-regulating EhRho1 by antisense RNA resulted in reduced blebbing and motility. Moreover, serum-starvation reduced blebbing that was restored on serum-replenishment. Lysophosphatidic acid treatment induced bleb formation, whereas wortmannin inhibited the process. In all these cases, concentration of GTP-EhRho1 (active) and Phosphatidylinositol 4,5-bisphosphate (PIP2) inversely correlated with the level of plasma membrane blebbing. Our study suggests the role of EhRho1 in blebbing and bleb-based motility through PI3 kinase pathway in E. histolytica.
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http://dx.doi.org/10.1111/cmi.12751DOI Listing
October 2017

Complex multifractal nature in Mycobacterium tuberculosis genome.

Sci Rep 2017 04 25;7:46395. Epub 2017 Apr 25.

School of Computational and Integrative Sciences, Jawaharlal Nehru University, New Delhi-110067, India.

The mutifractal and long range correlation (C(r)) properties of strings, such as nucleotide sequence can be a useful parameter for identification of underlying patterns and variations. In this study C(r) and multifractal singularity function f(α) have been used to study variations in the genomes of a pathogenic bacteria Mycobacterium tuberculosis. Genomic sequences of M. tuberculosis isolates displayed significant variations in C(r) and f(α) reflecting inherent differences in sequences among isolates. M. tuberculosis isolates can be categorised into different subgroups based on sensitivity to drugs, these are DS (drug sensitive isolates), MDR (multi-drug resistant isolates) and XDR (extremely drug resistant isolates). C(r) follows significantly different scaling rules in different subgroups of isolates, but all the isolates follow one parameter scaling law. The richness in complexity of each subgroup can be quantified by the measures of multifractal parameters displaying a pattern in which XDR isolates have highest value and lowest for drug sensitive isolates. Therefore C(r) and multifractal functions can be useful parameters for analysis of genomic sequences.
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http://dx.doi.org/10.1038/srep46395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5404331PMC
April 2017

Cytosine DNA methylation at promoter of non LTR retrotransposon and heat shock protein gene (HSP70) of Entamoeba histolytica and lack of correlation with transcription status.

Mol Biochem Parasitol 2017 03 9;212:21-27. Epub 2017 Jan 9.

School of Environmental Sciences, Jawaharlal Nehru University, New Delhi, India. Electronic address:

Non LTR retrotransposons (EhLINEs and EhSINEs) occupy 11% of the Entamoeba histolytica genome. Since promoter DNA methylation at cytosines has been correlated with transcriptional silencing of transposable elements in model organisms we checked whether this was the case in EhLINE1. We located promoter activity in a 841bp fragment at 5'-end of this element by luciferase reporter assay. From RNAseq and RT-PCR analyses we selected a transcriptionally active and silent copy to study cytosine DNA methylation of the promoter region by bisulfite sequencing. None of the cytosines were methylated in either copy. Further, we looked at methylation status of a few selected cytosines in all 5'-intact EhLINE1 copies by single nucleotide incorporation opposite cytosine in bisulfite-treated DNA, where dGTP would be incorporated if the cytosine was methylated. Again we did not find evidence of cytosine methylation, indicating that expression status of this element was not correlated with promoter DNA methylation. To test for any role of cytosine methylation in transcriptional regulation of the E. histolytica Hsp70 gene in which the promoter is fully methylated under normal growth conditions, we checked methylation status and found that the promoter remained fully methylated during heat-shock as well, although transcription was greatly enhanced by heat-shock, showing that cytosine methylation is not a repressive mark for EhHsp70. Our data present direct evidence that promoter methylation, a common mode of transposon silencing, is unlikely to be involved in transcriptional regulation of EhLINE1, and reinforce the conclusion that promoter DNA methylation may not be a major contributor to transcriptional regulation in E. histolytica.
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http://dx.doi.org/10.1016/j.molbiopara.2017.01.001DOI Listing
March 2017
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