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Alok Bhattacharya

Publications and source records attributed to Alok Bhattacharya.

At least 19 recordsLinked to original sources
PubMed2006

Identification of insertion hot spots for non-LTR retrotransposons: computational and biochemical application to Entamoeba histolytica.

The genome of the human pathogen Entamoeba histolytica contains non-long terminal repeat (LTR) retrotransposons, the EhLINEs and EhSINEs, which lack targeted insertion. We investigated the importance of local DNA structure, and sequence preference of the element-encoded endonuclease (EN) in selecting target sites for retrotransposon insertion. Pre-insertion loci were tested computationally to detect unique features based on DNA structure, thermodynamic considerations and protein interaction measures. Target sites could readily be distinguished from other genomic sites based on these criteria. The contribution of the EhLINE1-encoded EN in target site selection was investigated biochemically. The sequence-specificity of the EN was tested in vitro with a variety of mutated substrates. It was possible to assign a consensus sequence, 5'-GCATT-3', which was efficiently nicked between A-T and T-T. The upstream G residue enhanced EN activity, possibly serving to limit retrotransposition in the A+T-rich E.histolytica genome. Mutated substrates with poor EN activity showed structural differences compared with normal substrates. Analysis of retrotransposon insertion sites from a variety of organisms showed that, in general, regions of favorable DNA structure were recognized for retrotransposition. A combination of favorable DNA structure and preferred EN nicking sequence in the vicinity of this structure may determine the genomic hotspots for retrotransposition.

Animals↗
PubMed2006

A phagocytosis mutant of Entamoeba histolytica is less virulent due to deficient proteinase expression and release.

Cysteine proteinases are key virulence factors of Entamoeba histolytica that are released during the process of invasion. We used a chemical mutant of E. histolytica strain HM-1:IMSS, clone L6, which is deficient in virulence, phagocytosis, and cysteine proteinase activity to help define the mechanisms of cysteine proteinase release. All cysteine proteinase genes of wild type HM-1 were present in the L6 mutant genome, but three of the major expressed proteinases, ehcp1, ehcp2, and ehcp5 were both transcribed, translated, and released at lower levels in L6. We hypothesized that a central protein such as the calcium binding protein 1, EhCaBP1, which is required for both phagocytosis and exocytosis might be deficient in this mutant. We found that both mRNA and proteinase levels of EhCaBP1 were decreased in L6. These findings provide an important link between phagocytosis, passive release of multiple cysteine proteinases, and attenuated virulence of this E. histolytica mutant.

Animals↗
PubMed2006

Calcium-binding proteins of Entamoeba histolytica.

Calcium plays an essential role in many fundamental processes in almost all eukaryotic cells including protozoan parasite Entamoeba histolytica. Many of the calcium-mediated processes are carried out through the help of calcium-binding proteins (CaBPs). A few of these E. histolytica CaBPs have been described before. These proteins are unique to this organism and are thought to be essential. Availability of genome sequence has opened up the possibility of studying CaBPs at the whole genome level. In this preliminary report, we describe the complement of CaBPs present in E. histolytica. A large fraction of these genes are expressed in the trophozoites and are likely to be functional. The results suggest a number of pathways that are involved in calcium signaling and may be unique for this organism.

Animals↗
PubMed2006

Expression and function of a family of transmembrane kinases from the protozoan parasite Entamoeba histolytica.

The signaling proteome of Entamoeba histolytica is made of transmembrane kinases (TMKs) that are rarely found in unicellular eukaryotes. There are 90 TMK genes reported for E. histolytica, and these have been grouped into nine distinct families based on motifs present on both extracellular and kinase domains. Of these, the B1 family was chosen for further analysis. Genomic sequencing revealed the presence of 28 members belonging to this family. Genes corresponding to the majority of these were truncated and not considered for further analysis. Only five members were full length and contained both extracellular and cytosolic kinase domains. BLAST analysis revealed the presence of homologs of these B1 TMKs in the nonpathogenic Entamoeba dispar. However, the ligand binding domains of the orthologous B1 TMKs of the two species showed considerable divergence, indicating the possibility of a correlation with the pathogenic potential of the organism. Only two of the five full-length copies (B1.I.1 and B1.I.2) were expressed in E. histolytica under the culture conditions used. Antisera generated against the extracellular domain of B1.I.1 stained the cell surface, particularly the areas of contact between the trophozoites. Staining was also seen in the frontal and posterior regions of the motile amoeba. An amoebic cell line expressing a truncated version of the B1.I.1 that lacked the kinase domain was generated. Inducible expression of the truncated TMK resulted in a decrease in cellular proliferation and an increase in sensitivity to serum starvation. Our data indicate that the B1.I class of TMKs is involved in parasite proliferation.

Animals↗
PubMed2005

Comparison of theoretical proteomes: identification of COGs with conserved and variable pI within the multimodal pI distribution.

BACKGROUND: Theoretical proteome analysis, generated by plotting theoretical isoelectric points (pI) against molecular masses of all proteins encoded by the genome show a multimodal distribution for pI. This multimodal distribution is an effect of allowed combinations of the charged amino acids, and not due to evolutionary causes. The variation in this distribution can be correlated to the organisms ecological niche. Contributions to this variation maybe mapped to individual proteins by studying the variation in pI of orthologs across microorganism genomes. RESULTS: The distribution of ortholog pI values showed trimodal distributions for all prokaryotic genomes analyzed, similar to whole proteome plots. Pairwise analysis of pI variation show that a few COGs are conserved within, but most vary between, the acidic and basic regions of the distribution, while molecular mass is more highly conserved. At the level of functional grouping of orthologs, five groups vary significantly from the population of orthologs, which is attributed to either conservation at the level of sequences or a bias for either positively or negatively charged residues contributing to the function. Individual COGs conserved in both the acidic and basic regions of the trimodal distribution are identified, and orthologs that best represent the variation in levels of the acidic and basic regions are listed. CONCLUSION: The analysis of pI distribution by using orthologs provides a basis for resolution of theoretical proteome comparison at the level of individual proteins. Orthologs identified that significantly vary between the major acidic and basic regions maybe used as representative of the variation of the entire proteome.

Bacterial Proteins↗
PubMed2005

Crystallization and preliminary crystallographic analysis of calcium-binding protein-2 from Entamoeba histolytica and its complexes with strontium and the IQ1 motif of myosin V.

Calcium plays a pivotal role in the pathogenesis of amoebiasis, a major disease caused by Entamoeba histolytica. Two domains with four canonical EF-hand-containing calcium-binding proteins (CaBPs) have been identified from E. histolytica. Even though they have very high sequence similarity, these bind to different target proteins in a Ca2+-dependent manner, leading to different functional pathways. Calcium-binding protein-2 (EhCaBP2) crystals were grown using MPD as a precipitant. The crystals belong to space group P2(1), with unit-cell parameters a = 111.74, b = 68.83, c = 113.25 A, beta = 116.7 degrees. EhCaBP2 also crystallized in complex with strontium (replacing calcium) at similar conditions. The crystals belong to space group P2(1), with unit-cell parameters a = 69.18, b = 112.03, c = 93.42 A, beta = 92.8 degrees. Preliminary data for EhCaBP2 crystals in complex with an IQ motif are also reported. This complex was crystallized with MPD and ethanol as precipitating agents. These crystals belong to space group P2(1), with unit-cell parameters a = 60.5, b = 69.86, c = 86.5 A, beta = 97.9 degrees.

Amino Acid Motifs↗
PubMed2005

The LINEs and SINEs of Entamoeba histolytica: comparative analysis and genomic distribution.

Autonomous non-long terminal repeat retrotransposons are commonly referred to as long interspersed elements (LINEs). Short non-autonomous elements that borrow the LINE machinery are called SINES. The Entamoeba histolytica genome contains three classes of LINEs and SINEs. Together the EhLINEs/SINEs account for about 6% of the genome. The recognizable functional domains in all three EhLINEs included reverse transcriptase and endonuclease. A novel feature was the presence of two types of members-some with a single long ORF (less frequent) and some with two ORFs (more frequent) in both EhLINE1 and 2. The two ORFs were generated by conserved changes leading to stop codon. Computational analysis of the immediate flanking sequences for each element showed that they inserted in AT-rich sequences, with a preponderance of Ts in the upstream site. The elements were very frequently located close to protein-coding genes and other EhLINEs/SINEs. The possible influence of these elements on expression of neighboring genes needs to be determined.

Amino Acid Sequence↗
PubMed2005

The genome of the protist parasite Entamoeba histolytica.

Entamoeba histolytica is an intestinal parasite and the causative agent of amoebiasis, which is a significant source of morbidity and mortality in developing countries. Here we present the genome of E. histolytica, which reveals a variety of metabolic adaptations shared with two other amitochondrial protist pathogens: Giardia lamblia and Trichomonas vaginalis. These adaptations include reduction or elimination of most mitochondrial metabolic pathways and the use of oxidative stress enzymes generally associated with anaerobic prokaryotes. Phylogenomic analysis identifies evidence for lateral gene transfer of bacterial genes into the E. histolytica genome, the effects of which centre on expanding aspects of E. histolytica's metabolic repertoire. The presence of these genes and the potential for novel metabolic pathways in E. histolytica may allow for the development of new chemotherapeutic agents. The genome encodes a large number of novel receptor kinases and contains expansions of a variety of gene families, including those associated with virulence. Additional genome features include an abundance of tandemly repeated transfer-RNA-containing arrays, which may have a structural function in the genome. Analysis of the genome provides new insights into the workings and genome evolution of a major human pathogen.

Animals↗
PubMed2005

Reduction of cell surface glycosylphosphatidylinositol conjugates in Entamoeba histolytica by antisense blocking of E. histolytica GlcNAc-phosphatidylinositol deacetylase expression: effect on cell proliferation, endocytosis, and adhesion to target cells.

Glycosylphosphatidylinositol (GPI)-anchored molecules such as cell surface Gal/GalNAc lectin and proteophosphoglycans of the protozoan parasite Entamoeba histolytica are thought to be involved in pathogenesis. Here, we report the identification of genes that may be involved in the GPI biosynthetic pathway of E. histolytica by use of bioinformatic tools applied to the recently published genome sequence. Of the genes identified, one of the early genes, GlcNAc-phosphatidylinositol deacetylase (PIG-L), was partially characterized. Cell lines deficient in E. histolytica PIG-L (EhPL-AS) or overproducing it (EhPL-S) were generated by expressing the gene in the antisense or sense orientation, respectively, in a tetracycline-inducible system. The overexpressing cells showed higher EhPIG-L activity and increased production of GlcN-PI. Conversely, cells expressing the antisense RNA displayed reduced GlcN-PI production. The total number of GPI-containing molecules was also reduced in these cells, as demonstrated by Alexa 488 fluorescently labeled proaerolysin labeling. The distribution of GPI-linked PPG and Gal/GalNAc lectin was altered in the tetracycline-induced EhPL-AS cell lines. Further, the antisense-blocked cells showed 36% suppression of cell growth, 50 to 60% inhibition of fluid phase endocytosis, and about 50% inhibition of adhesion to target cells. Therefore, our data suggest the importance of GPI anchors in regulating some of the events in amoebic pathogenesis. They also demonstrated the use of antisense RNA-mediated inhibition of GPI biosynthetic enzymes as an approach to decrease the amount of GPI conjugates in E. histolytica.

Amidohydrolases↗
PubMed2004

Calcium binding protein 1 of the protozoan parasite Entamoeba histolytica interacts with actin and is involved in cytoskeleton dynamics.

Blocking expression of EhCaBP1, a calmodulin-like, four EF-hand protein from the protozoan parasite Entamoeba histolytica, resulted in inhibition of cellular proliferation. In this paper we report that EhCaBP1 is involved in dynamic changes of the actin cytoskeleton. Both endocytosis and phagocytosis were severely impaired in cells where EhCaBP1 expression was blocked by inducible expression of the antisense RNA. In wild-type cells both actin and EhCaBP1 were found to co-localize in phagocytic cups and in pseudopods. However, in antisense-blocked cells the phagocytic cup formation is affected. Analysis of the staining patterns in the presence and absence of actin dynamics inhibitors, jasplakinolide and cytochalasin D suggested that EhCaBP1 and polymerized F-actin co-localize on membrane protrusions. Direct interaction between soluble EhCaBP1 and F-actin was further demonstrated by a co-sedimentation assay. A variant of EhCaBP1 did not bind F-actin showing the specificity of the interaction between EhCaBP1 and actin. There is no significant change in the kinetics of in vitro polymerization of actin in presence of EhCaBP1, indicating that EhCaBP1 does not affect filament treadmilling. In addition, using atomic force microscopy; it was found that filaments of F-actin, polymerized in presence of EhCaBP1, were thinner. These results indicate that EhCaBP1 may be involved in dynamic membrane restructuring at the time of cell pseudopod formation, phagocytosis and endocytosis in a process mediated by direct binding of EhCaBP1 to actin, affecting the bundling of actin filaments.

Actins↗
PubMed2004

Identification and characterization of EhCaBP2. A second member of the calcium-binding protein family of the protozoan parasite Entamoeba histolytica.

Entamoeba histolytica, an early branching eukaryote, is the etiologic agent of amebiasis. Calcium plays a pivotal role in the pathogenesis of amebiasis by modulating the cytopathic properties of the parasite. However, the mechanistic role of Ca(2+) and calcium-binding proteins in the pathogenesis of E. histolytica remains poorly understood. We had previously characterized a novel calcium-binding protein (EhCaBP1) from E. histolytica. Here, we report the identification and partial characterization of an isoform of this protein, EhCaBP2. Both EhCaBPs have four canonical EF-hand Ca(2+) binding domains. The two isoforms are encoded by genes of the same size (402 bp). Comparison between the two genes showed an overall identity of 79% at the nucleotide sequence level. This identity dropped to 40% in the 75-nucleotide central linker region between the second and third Ca(2+) binding domains. Both of these genes are single copy, as revealed by Southern hybridization. Analysis of the available E. histolytica genome sequence data suggested that the two genes are non-allelic. Homology-based structural modeling showed that the major differences between the two EhCaBPs lie in the central linker region, normally involved in binding target molecules. A number of studies indicated that EhCaBP1 and EhCaBP2 are functionally different. They bind different sets of E. histolytica proteins in a Ca(2+)-dependent manner. Activation of endogenous kinase was also found to be unique for the two proteins and the Ca(2+) concentration required for their optimal functionality was also different. In addition, a 12-mer peptide was identified from a random peptide library that could differentially bind the two proteins. Our data suggest that EhCaBP2 is a new member of a class of E. histolytica calcium-binding proteins involved in a novel calcium signal transduction pathway.

Alleles↗
PubMed2004

An Entamoeba histolytica LINE/SINE pair inserts at common target sites cleaved by the restriction enzyme-like LINE-encoded endonuclease.

The non-long-terminal-repeat (non-LTR) retrotransposons (also called long interspersed repetitive elements [LINEs]) are among the oldest retroelements. Here we describe the properties of such an element from a primitive protozoan parasite, Entamoeba histolytica, that infects the human gut. This 4.8-kb element, called EhLINE1, is present in about 140 copies dispersed throughout the genome. The element belongs to the R4 clade of non-LTR elements. It has a centrally located reverse transcriptase domain and a restriction enzyme-like endonuclease (EN) domain at the carboxy terminus. We have cloned and expressed a 794-bp fragment containing the EN domain in Escherichia coli. The purified protein could nick supercoiled pBluescript DNA to yield open circular and linear DNAs. The conserved PDX(12-14)D motif was required for activity. Genomic sequences flanking the sites of insertion of EhLINE1 and the putative partner short interspersed repetitive element (SINE), EhSINE1, were analyzed. Both elements resulted in short target site duplications (TSD) upon insertion. A common feature was the presence of a short T-rich stretch just upstream of the TSD in most insertion sites. By sequence analysis an empty target site in the E. histolytica genome, known to be occupied by EhSINE1, was identified. When a 176-bp fragment containing the empty site was used as a substrate for EN, it was prominently nicked on the bottom strand at the precise point of insertion of EhSINE1, showing that this SINE could use the LINE-encoded endonuclease for its insertion. The nick on the bottom strand was toward the right of the TSD, which is uncommon. The lack of strict target site-specificity of the restriction enzyme-like EN encoded by EhLINE1 is also exceptional. A model for retrotransposition of EhLINE1/SINE1 is presented.

Amino Acid Motifs↗
PubMed2003

Differential use of multiple replication origins in the ribosomal DNA episome of the protozoan parasite Entamoeba histolytica.

The factors that control the initiation of eukaryotic DNA replication from defined origins (oris) on the chromosome remain incompletely resolved. Here we show that the circular rDNA episome of the human pathogen Entamoeba histolytica contains multiple potential oris, which are utilized in a differential manner. The primary ori in exponentially growing cells was mapped close to the promoter of rRNA genes in the upstream intergenic spacer (IGS) by two-dimensional gel electrophoresis. Replication initiated predominantly from the upstream IGS and terminated in the downstream IGS. However, when serum-starved cells were allowed to resume growth, the early oris which became activated were located in other parts of the molecule. Later the ori in the upstream IGS became activated, with concomitant silencing of the early oris. When the upstream IGS was located ectopically in an artificial plasmid, it again lost ori activity, while other parts of the rDNA episome could function as oris in this system. Therefore, the activation or silencing of the ori in this episome is context dependent, as is also the case with many eukaryotic replicons. This is the first replication origin to be mapped in this primitive protozoan and will provide an opportunity to define the factors involved in differential ori activity, and their comparison with metazoans.

Animals↗
PubMed2003

Biosynthesis of Entamoeba histolytica proteophosphoglycan in vitro.

A complex glycoconjugate proteophosphoglycan (PPG) is present on the surface of the pathogenic protozoan parasite Entamoeba histolytica but not in the non-pathogenic Entamoeba dispar. It is thought to be an important molecule involved in pathogenesis. In order to study its biosynthesis, an in vitro cell-free system was developed. The specificity of the system was demonstrated by various criteria including immunoprecipitation by a specific monoclonal antibody. The in vitro synthesized molecule was found to be susceptible to mild acid hydrolysis, digestion by phosphoinositol-specific phospholipase C and nitrous acid deamination, the salient features for a PPG-like molecule. The in vitro product was not synthesized when heat-treated cellular-extract was used in the assay or when the cell extract was prepared from Entamoeba invadens, a species that lacks these glycoconjugates. Analysis of the glycan side chains of the in vitro synthesized product by thin layer chromatography revealed side chains of variable sizes including a fraction greater than six glycan units. The crude membranes used in the cell-free system were further fractionated by sucrose density gradient centrifugation. The fraction containing the PPG synthesizing activity when used in the assay resulted in a 10-fold increase in specific activity. Development of this cell-free system will facilitate further studies on the nature of intracellular organelles and the pathways that are involved in PPG biosynthesis.

Animals↗
PubMed2003

Nontranslated polyadenylated RNAs from Entamoeba histolytica.

Protozoan parasites display a range of unusual molecular mechanisms that could be helpful for their survival in Nature. Among these parasites, Entamoeba histolytica is one of the most prevalent in developing countries such as India. Entamoeba histolytica produces at least four different unusual transcripts, IE, Tr, ehapt1 and UEE1, that are polyadenylated, but do not have significant open reading frames. Availability of large-scale sequence information has helped us to understand the nature of these sequences and their possible role. Entamoeba histolytica also encodes at least three classes of non-long-terminal-repeats containing retrotransposons, similar to mammalian long retrotransposable elements. This article describes the current status of our understanding of these transcripts and suggests a relationship between some of these transcripts and short retrotransposable element-like retro-elements present in many eukaryotes.

Animals↗