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Identification of Genomic Responses to Collagen Binding by Trophozoites of Entamoeba histolytica
Abstract
Attachment of Entamoeba histolytica trophozoites to collagen is a known stimulus for parasite activation, leading to subsequent tissue destruction and invasion.
To identify cellular mechanisms of trophozoite activation, we assessed global variations in gene expression during collagen
interaction with E. histolytica. A shotgun DNA microarray was constructed by use of 9600 random inserts from an E. histolytica genomic DNA library. Through differential hybridization, key differences between gene expression in collagen-activated trophozoites
and that in nonactivated trophozoites were identified. Fourteen differentially regulated clones were reproducibly identified
and selected for sequencing. Among the genes identified were those coding for (1) components of a signaling cascade that had
been previously hypothesized to transmit responses to cell attachment, (2) adapter proteins for vesicle formation, and (3)
proteins that are implicated in cytoskeletal reorganization and locomotion. Two known virulence-factor genes—those for cysteine
proteinases and amebapore—also were up-regulated in response to collagen stimulation. These results provide important new
clues about how a pathogen orchestrates responses to the host environment as well as a new tool for the analysis of other
aspects of Entamoeba species infection and pathogenicity.
... Total RNA was isolated from HM-1 and L6 trophozoites using TRIZOL (Invitrogen). cDNA synthesis, microarray hybridization, and microarray scanning were performed as described (Debnath et al., 2004). BrieXy, cDNA was synthesized from total RNA extracted from HM-1:IMSS or L6, coupled to CyScribe Cy3 or Cy5 (Amersham), and hybridized to the shotgun DNA microarray constructed by use of 6144 random inserts from an E. histolytica genomic DNA library (Debnath et al., 2004). ...
... cDNA synthesis, microarray hybridization, and microarray scanning were performed as described (Debnath et al., 2004). BrieXy, cDNA was synthesized from total RNA extracted from HM-1:IMSS or L6, coupled to CyScribe Cy3 or Cy5 (Amersham), and hybridized to the shotgun DNA microarray constructed by use of 6144 random inserts from an E. histolytica genomic DNA library (Debnath et al., 2004). Four hybridizations were performed with HM-1 trophozoite RNA labeled with Cy3 and L6 trophozoite RNA labeled with Cy5 (EhCS1p1-095, EhCS1p1-096, EhCS1p1-102, and EhCS1p1-103); one hybridization with HM-1 trophozoite RNA labeled with Cy5 and L6 trophozoite RNA labeled with Cy3 (EhCS1p1-099). ...
... Four hybridizations were performed with HM-1 trophozoite RNA labeled with Cy3 and L6 trophozoite RNA labeled with Cy5 (EhCS1p1-095, EhCS1p1-096, EhCS1p1-102, and EhCS1p1-103); one hybridization with HM-1 trophozoite RNA labeled with Cy5 and L6 trophozoite RNA labeled with Cy3 (EhCS1p1-099). Scanning and data analysis were performed as previously described (Debnath et al., 2004). Only spots in which almost more than 70% of the pixels had a signal of at least twice the standard deviation (SD) of the local background were considered in subsequent data analysis. ...
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.
... E. histolytica has emerged as an important opportunistic pathogen in chronically immunosuppressed patients, such as people suffering from HIV-AIDS. Such patients are also at a higher risk of developing invasive amoebiasis, in comparison to immune-competent subjects [5]. ...
... Several lines of evidence suggest that adhesion of E. histolytica to ECM may be likened to focal adhesions of higher eukaryotes [2,3]. This interaction is also believed to alter signal transduction pathways and enhance parasitic virulence [2,4,5]. For example, ...
... This, in turn, may regulate changes in gene expression. In support of this, collagen exposure results in increased expression of an amoebapore and a cysteine protease, two secreted proteins which aid in host tissue destruction [5]. Likewise, binding of E. histolytica trophozoites to fibronectin induces phosphorylation of FAK, association of an integrin like receptor with paxillin, and activation of protein kinase A (PKA) [8,9], a protein involved in Gprotein coupled receptor signaling. ...
Adhesion is an important virulence function for Entamoeba histolytica, the causative agent of amoebic dysentery. Lipid rafts, cholesterol-rich domains, function in compartmentalization of cellular processes. In E. histolytica, rafts participate in parasite-host cell interactions; however, their role in parasite-host extracellular matrix (ECM) interactions has not been explored. Disruption of rafts with a cholesterol extracting agent, methyl-beta-cyclodextrin (MbetaCD), resulted in inhibition of adhesion to collagen, and to a lesser extent, to fibronectin. Replenishment of cholesterol in MbetaCD-treated cells, using a lipoprotein-cholesterol concentrate, restored adhesion to collagen. Confocal microscopy revealed enrichment of rafts at parasite-ECM interfaces. A raft-resident adhesin, the galactose/N-acetylgalactosamine-inhibitable lectin, mediates interaction to host cells by binding to galactose or N-acetylgalactosamine moieties on host glycoproteins. In this study, galactose inhibited adhesion to collagen, but not to fibronectin. Together these data suggest that rafts participate in E. histolytica-ECM interactions and that raft-associated Gal/GalNAc lectin may serve as a collagen receptor.
... HM-1 exhibited the highest cytopathic, cytotoxic, and hemolytic activities, complement resistance, and liver abscesses formation in hamsters (Table 1), consistent with previous reports. No remarkable differences between the strains were found for erythrophagocytosis and digestion of erythrocytes, except Rahman, which is known for having lower phagocytic activity [57]. The cysteine protease activity remained unchanged among the strains, except for the G3 strain, which showed higher proteolytic activity (Table 1). ...
... E. histolytica HM-1 strain produced considerable liver abscesses, low sensitivity to human complement, and high cell-damaging activities, while G3, Rahman, and UG10 were unable to produce any liver abscesses and exhibited increased sensitivity to human complement and low cell-damaging activities. Phagocytosis was reduced only in the Rahman strain as reported previously [57]. The virulence of E. histolytica strains was not correlated to cysteine proteinase activity as previously reported [60]. ...
Entamoeba histolytica virulence results from complex host–parasite interactions implicating multiple amoebic components (e.g., Gal/GalNAc lectin, cysteine proteinases, and amoebapores) and host factors (microbiota and immune response). UG10 is a strain derived from E. histolytica virulent HM-1:IMSS strain that has lost its virulence in vitro and in vivo as determined by a decrease of hemolytic, cytopathic, and cytotoxic activities, increased susceptibility to human complement, and its inability to form liver abscesses in hamsters. We compared the transcriptome of nonvirulent UG10 and its parental HM-1:IMSS strain. No differences in gene expression of the classical virulence factors were observed. Genes downregulated in the UG10 trophozoites encode for proteins that belong to small GTPases, such as Rab and AIG1. Several protein-coding genes, including iron-sulfur flavoproteins and heat shock protein 70, were also upregulated in UG10. Overexpression of the EhAIG1 gene (EHI_180390) in nonvirulent UG10 trophozoites resulted in augmented virulence in vitro and in vivo. Cocultivation of HM-1:IMSS with E. coli O55 bacteria cells reduced virulence in vitro, and the EhAIG1 gene expression was downregulated. In contrast, virulence was increased in the monoxenic strain UG10, and the EhAIG1 gene expression was upregulated. Therefore, the EhAIG1 gene (EHI_180390) represents a novel virulence determinant in E. histolytica.
... To understand the underlying mechanism, gene expression changes were studied in collagen-activated trophozoites. A shotgun genomic DNA microarray containing 9600 random inserts from E. histolytica DNA was used (Debnath et al., 2004). The fourteen differentially up regulated clones obtained included signaling components, adapter proteins for vesicle formation and cytoskeletal reorganization and locomotion proteins. ...
... Thus this data, obtained from a relatively low-coverage microarray, highlighted the major virulence determinants required for tissue invasion. The same genomic microarray (Debnath et al., 2004) was further used to study other virulence-related factors. Intestinal invasion of E. histolytica trophozoites requires attachment to the colonic mucous layer, and cytolysis of host epithelial and inflammatory cells. ...
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.
... Gene expression has been examined in trophozoites recovered from ALA after 7 days post inoculation, and the results showed that most of the up-regulated genes are associated with stress response, regulation of transcription, and vesicular trafficking, and two hypothetical proteins are mainly downregulated [53]. Trophozoites interacting with collagen up-regulate the expression of components of signaling cascade, adapter proteins for vesicle formation, cytoskeletal proteins, HSP70, cysteine proteinases, and amebaporo [54]. On the other hand, the interaction of trophozoites with Caco-2 cells did not appears to induce major transcriptional changes in the parasite, despite the fact that the parasites lysing the cell monolayer [55]. ...
Entamoeba histolytica is an intestinal protozoan parasite of humans and is endemic in developing countries. E. histolytica has two low molecular weight protein tyrosine phosphatase (LMW-PTP) genes, EhLMW-PTP1 and EhLMW-PTP2, which are expressed in cultured trophozoites, clinical isolates, and cysts. The amino acid sequences of proteins EhLMW-PTP1 and EhLMW-PTP2 showed only one amino acid difference between them at position A85V, respectively. Both genes are expressed in cultured trophozoites, mainly EhLMW-PTP2, and in trophozoites recovered from amoebic liver abscess, the expression of EhLMW-PTP1 is downregulated. We cloned the two genes and purified the corresponding recombinant (rEhLMW-PTPs) proteins. Antibodies anti-rEhLMW-PTP2 showed that during red blood cells uptake by E. histolytica, the EhLMW-PTPs were found in the phagocytic cups based on analysis of fluorescence signals. On the other hand, rEhLMW-PTPs showed an optimum phosphatase activity at pH 6.0 with p-nitrophenyl phosphate as the substrate. They dephosphorylate phosphotyrosine and 3-O-methylfluorescein phosphate, but not phosphoserine or phosphothreonine, and the enzymatic activity is inhibited by orthovanadate. rEhLMW-PTP1 and rEhLMW-PTP2 exhibited optimum temperatures of activities at 60 °C and 58 °C, respectively, with high thermal stability at 50 °C. Also, the rEhLMW-PTPs showed high specific activities and specific km value with pNPP or OMFP as the substrates at the physiological temperature (37 °C).
... E. histolytica trophozoites (HM1:IMSS strain from ATCC) were maintained in TYI-S-33 medium (20 g casein digest, 10 g yeast extract, 10 g glucose, 2 g sodium chloride, 1 g L-cysteine HCl, 0.2 g ascorbic acid, 1 g K 2 HPO 4 , 0.6 g KH 2 PO 4 , 28.75 mg ferric nitrate per liter) that was supplemented with penicillin (100 U/mL), streptomycin (100 µg/mL), and 10% adult bovine serum, under axenic conditions according to the methods of Diamond (Diamond et al., 1978;Debnath et al., 2004Debnath et al., , 2005. All of the experiments were conducted with trophozoites that had been harvested during the logarithmic phase of growth. ...
Hsp90 is an essential chaperone responsible for trafficking a vast array of client proteins, which are substrates that Hsp90 regulates in eukaryotic cells under stress conditions. The ATP-binding N-terminal domain of Hsp90 (also known as a GHKL type ATPase domain) can serve as a specific drug target, because sufficient structural diversity in the ATP-binding pocket of Hsp90 allows for ortholog selectivity of Hsp90 inhibitors. The primary objective of this study is to identify inhibitors specific for the ATP-binding domain of Entamoeba histolytica Hsp90 (EhHsp90). An additional aim, using a combination of site-directed mutagenesis and a protein in vitro assay, is to show that the antiparasitic activity of Hsp90 inhibitors is dependent on specific residues within the ATP-binding domain. Here, we tested the activity of 43 inhibitors of Hsp90 that we previously identified using a high-throughput screen. Of the 43 compounds tested, 19 competed for binding of the EhHsp90 ATP-binding domain. Five out of the 19 EhHsp90 protein hits demonstrated activity against E. histolytica in vitro culture: rifabutin, rutilantin, cetylpyridinium chloride, pararosaniline pamoate and gentian violet. These 5 top E. histolytica Hsp90 inhibitors showed 30-100% inhibition of E. histolytica in culture in the micromolar range. These data suggest that E. histolytica-specific Hsp90 inhibitors are possible to identify and provide important lead compounds for the development of novel antiamebic drugs.
... In addition, clustering methods may be used to identify genomic fingerprints which represent co-regulated genes [ Figure 1]. Microarrays technology has been successfully applied in E. histolytica to obtain transcriptional profiling in several conditions, including virulence, stress and DNA damage, during cyst-trophozoites conversion and to evaluate the role of epigenetic control on gene expression [11][12][13][14][15][16][17][18][19][20][21][22][23]. Next, we discuss some of the genomic studies performed to understand different topics of E. histolytica biology. ...
... Microarray-based analyses can be utilised in conjunction with genome sequencing to assign functional roles to annotated genes and to clarify genomic architecture. A number of groups have utilised DNA microarrays in E. histolytica (made from random genomic DNA fragments or long or short oligonucleotides based on annotated genes) to successfully study transcriptional differences between virulent and avirulent E. histolytica as well transcriptional responses to heat shock, collagen and calcium exposure, tissue invasion and cyst development (Debnath et al., 2004;Gilchrist et al., 2006;MacFarlane and Singh, 2006;Weber et al., 2006). Additionally, using a genomic DNA microarray, comparative genomic hybridisations (CGH) between strains and species of Entamoeba have been performed (Shah et al., 2005). ...
... In fact, EDGs antigens have been identified in the intestinal content of hamsters during the early phase of invasive amebiasis, using two monoclonal antibodies (Arias-Negrete et al., 2001). As has been previously reported, attachment of E. histolytica trophozoites to collagen stimulus destruction mechanisms of the parasite (Debnath et al., 2004). In agreement with these observations, in our in vitro experiments we observed that EDGs were abundant in recovered trophozoites during the initial phase of adhesion to MDCK monolayers, when compared with trophozoites obtained from continuous culture. ...
Electron dense granules (EDGs) were identified by transmission electron microscopy in Entamoeba histolytica trophozoites recovered from hamster liver lesions. Abundant granules were present in trophozoites recovered after 15 min of liver inoculation. Variation in the size and morphology of these EDGs was also observed. Numerous granules were present in the plasma membrane when these parasites were incubated for 5 min with MDCK monolayers. Release of these EDGs was suggested by the presence of granules in contact with the surface of the target cell plasma membrane. Parasite phagocytic invaginations were observed after 10 min of parasite-monolayer interaction. In these structures, scarce granules were seen. Granules secretion was corroborated by obtaining of a pellet of these small structures from the incubation of trophozoites with collagen supernatant. Collagenase and gellatinase activity of this pellet was identified in SDS-PAGE gels. EDGs were also present in amebic hamster liver lesions. Our observations corroborate that these granules are secreted and suggest that may participate in the cytopathic effect of E. histolytica both in vitro and in vivo.
... It is particularly striking that, in contrast to the intricate multicellular associations of Dictyostelium, the Entamoeba life cycle is morphologically rather simple. Underlying this apparently simplicity, however, is probably complex turnover and change to plasma membrane protein sets accompanying life cycle transitions and invasive contact with host tissues293031 . It might be hypothesized that the large complement of myotubularin genes found in this organism is necessary for precise spatial and temporal regulation of these membrane trafficking events, over and above the " constitutive " requirements of any eukaryotic cell. ...
Phosphorylated phosphatidylinositol (PtdIns) lipids, produced and modified by PtdIns kinases and phosphatases, are critical to the regulation of diverse cellular functions. The myotubularin PtdIns-phosphate phosphatases have been well characterized in yeast and especially animals, where multiple isoforms, both catalytically active and inactive, occur. Myotubularin mutations bring about disruption of cellular membrane trafficking, and in humans, disease. Previous studies have suggested that myotubularins are widely distributed amongst eukaryotes, but key evolutionary questions concerning the origin of different myotubularin isoforms remain unanswered, and little is known about the function of these proteins in most organisms.
We have identified 80 myotubularin homologues amidst the completely sequenced genomes of 30 organisms spanning four eukaryotic supergroups. We have mapped domain architecture, and inferred evolutionary histories. We have documented an expansion in the Amoebozoa of a family of inactive myotubularins with a novel domain architecture, which we dub "IMLRK" (inactive myotubularin/LRR/ROCO/kinase). There is an especially large myotubularin gene family in the pathogen Entamoeba histolytica, the majority of them IMLRK proteins. We have analyzed published patterns of gene expression in this organism which indicate that myotubularins may be important to critical life cycle stage transitions and host infection.
This study presents an overall framework of eukaryotic myotubularin gene evolution. Inactive myotubularin homologues with distinct domain architectures appear to have arisen on three separate occasions in different eukaryotic lineages. The large and distinctive set of myotubularin genes found in an important pathogen species suggest that in this organism myotubularins might present important new targets for basic research and perhaps novel therapeutic strategies.
... Vorläuferproteine synthetisiert werden und zusammen wirken (Ferlinz et al., 1994Frosches bekannt (Terry et al., 1988;Zasloff, 1987(Kishimoto et al., 1992;Morimoto et al., 1989;Zaltash und Johansson, 1998Granulysin bereits diskutiert wird (Bruhn et al., 2003;Leippe, 1995(Akbar et al., 2004;Bruchhaus et al., 2002;Eichinger, 2001b)(Bracha et al., 1999;Bracha et al., 2003).Abhängigkeit von den 5'– nichtkodierenden Bereichen, wurde für das ribosomale Protein L21 von E. histolytica beschrieben (Moshitch-Moshkovitch et al., 1998)(Akbar et al., 2004;Debnath et al., 2005;Debnath et al., 2004Miteva et al., 1999;Ruysschaert et al., 1998).kationische, antibakterielle Peptide, wie z. ...
Saposin-ähnliche Proteine (SAPLIPs) sind membraninteragierende Proteine, die sich durch die konservierte Position von drei Disulfidbrücken, einer typischen alpha-helikalen Proteinfaltung und der Fähigkeit mit Lipiden zu interagieren, auszeichnen. Ihre zellulären Funktionen sind äußerst vielfältig. Bis zum Beginn des Genomsequenzierungsprojektes waren die Amoebapores die einzigen bekannten und charakterisierten SAPLIPs von Entamoeba histolytica, dem Erreger der humanen Amöbenruhr. Aufgrund ihrer antimikrobiellen Aktivität stellen sie für diesen parasitischen Einzeller, der sich von phagozytierten Bakterien ernährt, wichtige Effektormoleküle dar. Sie können aber auch cytolytisch auf Wirtszellen wirken und werden deshalb als bedeutender Pathogenitätsfaktor angesehen. Die theoretische computergestützte Datenbankanalyse nach Abschluss der Genomsequenzierung ergab, dass es 16 weitere Gene kodierend für SAPLIPs zusätzlich zu den drei Amoebapore-Genen gibt. Die Sequenzen der neuen SAPLIPs sind abgesehen von dem Cysteinmotiv divers und auch die Größe der Proteine ist sehr unterschiedlich (77 - 1009 Aminosäuren). Alle besitzen sie jedoch eine einzige, C-terminal gelegene SAPLIP Domäne. Außer der SAPLIP-Domäne konnten keine weiteren bekannten funktionellen oder strukturellen Domänen in den relevanten Datenbanken identifiziert werden, die auf mögliche Funktionen hätten hinweisen können. Alle SAPLIP-Gene werden gleichzeitig in axenisch kultivierten Trophozoiten transkribiert wie durch reverse Transkriptions-PCR gezeigt wurde. Die vergleichende transkriptionelle Analyse im Mikroarray ergab, dass nach Kontakt mit menschlichen Kolonzellen keine Hochregulierung dieser Gene mit Ausnahme des Amoebapore A Gens stattfindet. Für die parallele Klonierung der verschiedenen SAPLIP-Domänen wurde ein Expressionsscreening in E.coli mit dem grün fluoreszierenden Protein als Reporterprotein etabliert, das die erfolgreiche Klonierung und Expression eines Fragments aufgrund der Fluoreszenz der Bakterienkolonie bereits auf der Ebene der Transformation anzeigt. Die rekombinant exprimierte und bis zur Homogenität gereinigte SAPLIP-Domäne von SAPLIP 12 wies Amoebapore-ähnliche Aktivitäten auf. Unter Verwendung von Liposomen konnte porenbildende Aktivität nachgewiesen werden, wobei diese Aktivität stark an einen sauren pH-Wert gebunden ist. Die SAPLIP-Domäne 12 ist aber auch antibakteriell und dieses sogar mit vergleichbarer Selektivität wie Amoebapore A, nämlich Zelllyse von gram-positiven B. megaterium war nachweisbar, jedoch nicht von gram-negativen E. coli. Strukturell unterscheiden sich die SAPLIP-Domäne 12 und Amoebapore A bezüglich der Exposition positiver Ladungsansammlungen auf der Proteinoberfläche und des Fehlens des für den Mechanismus der Amoebapores essentiellen Histidinrestes an entsprechender Position in der Sequenz. Darüber hinaus übt die SAPLIP-Domäne 12 eine im Vergleich zum Amoebapore A geringere spezifische Aktivität aus. Diese Eigenschaften weisen darauf hin, dass es sich um einen anderen Wirkungsmechanismus handeln könnte. Für die SAPLIP-Domäne 12 wäre eine über die positiven Ladungen der Proteinoberfläche vermittelte Interaktion mit den negativ geladenen Phospholipidköpfen von Membranen denkbar, die bei Erreichen einer bestimmten Konzentration in einer Störung der Lipidordnung und letztendlich in der Auflösung der Membranstruktur resultieren könnte. SAPLIP 3 ähnelt den Amoebapores in der Größe und molekularen Architektur und kann somit als funktionelle Einheit angesehen werden, es unterscheidet sich aber durch eine hohe negative Nettoladung von den Amoebapores. Außerdem ist das rekombinante SAPLIP 3 nicht antibakteriell und die Membraninteraktionen dieses SAPLIPs unterscheiden sich grundlegend von denjenigen, die für die Amoebapores beschrieben sind. SAPLIP 3 zerstört nicht einfach die Liposomenstruktur wie von den porenbildenden Amoebapores bekannt, sondern es vermittelt die Fusion von multilamellaren Liposomen unter Freisetzung des Liposomeninhalts. Diese Aktivität ist abhängig von der Anwesenheit anionischer Lipide und von einem sauren pH-Wert. Die Fähigkeit zur Vesikelfusion sowie die Verteilung der negativen Ladungen von SAPLIP 3 auf der Proteinoberfläche ähneln Merkmalen des humanen Saposin C. Neben der Funktion als Cofaktor von Exohydrolasen, die im Sphingolipid Katabolismus involviert sind, wird angenommen, dass die Fähigkeit von Saposin C, Vesikel zu fusionieren, wichtig für die Reorganisation der humanen lysosomalen Kompartimente ist. Die Saposin C-ähnlichen Charakteristika von SAPLIP 3 geben Grund zu der Annahme, dass es bereits in einem so basalen Organismus wie der Amöbe ein Protein mit Saposin-ähnlichen membranfusionierenden Aktivitäten gibt und dass dieses SAPLIP entsprechende Funktionen während endo- und exozytotischer Transportprozesse in der Amöbe übernehmen könnte. Saposin-like proteins (SAPLIPs) are membrane-interacting proteins that are characterized by the conserved position of three disulphide bonds, a typical alpha-helical fold and the ability to interact with lipids. Their cellular functions are extremely diverse. Until the beginning of the genome-sequencing project, the amoebapores were the only known and characterized SAPLIPs of Entamoeba histolytica, which is the causative agent of human amoebiasis. Due to their antimicrobial activity, these proteins are important effector molecules of this unicellular parasite, which feeds on phagocytozed bacteria. However, they also exert cytolytic activity against host cells and therefore, they are considered to be a major pathogenicity factor of the amoeba. The theoretical computer-based data base analysis after the completion of genome sequencing revealed 16 genes coding for SAPLIPs in addition to the three amoebapore genes. The sequences of the novel SAPLIPs are diverse apart from the cysteine motif and furthermore, the sizes of the proteins are highly different (77 1009 amino acid residues). They all contain a single, C-terminally located SAPLIP domain. Beside the SAPLIP domain, no other functional or structural domains were identified in the relevant databases that would have pointed to possible functions. All SAPLIP-genes are transcribed in axenically cultured trophozoites at the same time as shown by reverse transcription PCR. The comparative transcriptional analysis using microarrays revealed that after six hours of contact with human cells and their phagocytosis none of these genes, with the exception of the amoebapore A gene, was upregulated. In order to clone the different fragments in parallel, an expression screening in E. coli with the green fluorescent protein as reporter protein was established, which indicates the successful cloning and expression of a fragment by the fluorescence of the bacterial colony already at the level of transformation. The SAPLIP domain 12, which has been recombinantly expressed and purified to homogeneity, exerts amoebapore-like activities. Using liposomes, pore-forming activity was detected, which is strongly dependent on acidic pH. Furthermore, the SAPLIP domain 12 is antibacterial even with similar selectivity as amoebapore A in that cell lysis of gram-positive B. megaterium was detectable but not of gram-negative E. coli. Structurally, the SAPLIP domain 12 differs from the amoebapores by exposing patches of positive charges on the protein surface und by lacking a histidine residue at a corresponding position in its sequence, which has been shown to be essential for the mechanism of amoebapore A. Moreover, the SAPLIP domain 12 displays a lower specific activity. These features indicate that this SAPLIP domain may act by means of another mechanism. It may interact with the negatively charged phospholipid head groups of membranes by its positively charged protein surface and after reaching a certain threshold concentration, possibly resulting in a disturbance of the lipid order and finally in the disintegration of the membrane structure. SAPLIP 3 resembles the amoebapores in size and molecular architecture and could therefore be considered as a functional unit, but it differs from the amoebapores by a highly negative net charge. Besides, the recombinant protein is not antibacterial and the membrane interactions of this SAPLIP are completely different from those described for the amoebapores. SAPLIP 3 does not simply destroy the liposome structure as known from the pore-forming amoebapores but induces leaky fusion of multilamellar liposomes. This activity is dependent on the presence of negatively charged lipids and on acidic pH. The capability of vesicle fusion as well as the negative charge distribution of SAPLIP 3 resembles features of the human saposin C. Beside its function as a cofactor of exohydrolases, which are involved in the sphingolipid catabolism, saposin C is considered to be involved in the reorganization of human lysosomal compartments due to its fusogenic activity. The saposin C-like characteristics of SAPLIP 3 suggest that a protein with saposin-like membrane fusogenic activity already exists in such a basal organism like an amoeba and that this SAPLIP fulfils corresponding functions during endo- and exocytotic transport processes in the amoeba.
... Entamoeba histolytica pathogenicity can be affected by factors in the trophozoite environment, such as calcium and iron levels, temperature, high oxygen pressure, bacteria, collagen binding and inflammation (68,(76)(77)(78)(79)(80)(81)(82). Entamoeba histolytica trophozoites might respond to these environmental stimuli by the release of an intracellular signal that alters the activity of DNA transcription factors, such as AP-1, thus inducing the expression of the genes necessary for the synthesis of the molecules involved in pathogenesis (80,83). ...
The host and parasite factors that influence susceptibility to Entamoeba histolytica infection and disease are not well understood. Entamoeba histolytica pathogenicity has been considered by focusing principally on parasite rather than host factors. Thus, research has concentrated on explaining the molecular differences between pathogenic E. histolytica and non-pathogenic E. dispar. However, the amoeba molecules considered most important for host tissue destruction (amoebapore, galactose/N-acetyl galactosamine inhibitable lectin, and cysteine proteinases) are present in both pathogenic E. histolytica and non-pathogenic E. dispar. In addition, the genetic differences in pathogenicity among E. histolytica isolates are unlikely to completely explain the different outcomes of infection. Considering that the principal difference between pathogenic and non-pathogenic amoebas lies in their surface coats, we propose that pathogenicity of the amoebas is related to the composition and properties of the surface coat components (or pathogen-associated molecular patterns, PAMPs), and the ability of innate immune response to recognize these components and eliminate the parasite. According to this hypothesis, a key feature that may distinguish pathogenic (E. histolytica) from non-pathogenic (E. dispar) strains is whether or not they can overcome innate immune defences. A corollary of this hypothesis is that in susceptible individuals the PAMPs are either not recognized or they are recognized by a set of Toll-like receptors (TLRs) that leads to an inflammatory response. In both cases, the result is tissue damage. On the contrary, in resistant individuals the innate/inflammatory response, induced through the activation of a different set of TLRs, eliminates the parasite.
... arrays. For the reference sample, we utilized a PCR product that labels all the spots on the array relative to the amount of DNA spotted. As all are inferred comparisons , one advantage of this approach is that even samples hybridized at diVerent time points can be compared (Fig. 1). Other groups have also developed microarrays for E. histolytica. Debnath et al. (2004) have used 9600 nonsequenced clones from a randomly sheared genomic library of E. histolytica and used it for identiWcation of transcripts regulated by collagen and calcium exposure. They identiWed cysteine proteinase, amoebapore, and Hsp70 gene as upregulated in ameba after exposure to collagen and calcium. Nancy Guillen's group at the ...
The parasite Entamoeba histolytica is a causative agent of dysentery and liver abscesses. Found predominantly in developing countries, this parasitic infection is responsible for significant morbidity and mortality. We have developed a genomic DNA microarray for E. histolytica. The array composed of 11,328 clones contains >2000 unique genes and was utilized for expression profiling and comparative genomic hybridizations of Entamoeba strains. We present a synopsis of our results to date and potential future applications of microarray technology for the study of Entamoeba biology.
... This interaction triggers the rearrangement of cytoskeleton [7] [8], the recruitment of pp60src and paxillin by pp125 FAK in the signaling cascade [9] and an AP-1 DNA binding activity [10]. During the trophozoites activation , 14 differentially regulated genes have been detected, among them, the SH3 domain-containing protein SH3P18 involved in signal transduction and rearrangement of cytoskeleton [11]. ...
... which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. technology to characterize the amebic transcriptional profile associated with adhesion to collagen, parasite virulence, stage conversion and tissue invasion (8,11,12,13). Although global changes in gene expression were observed, the promoter elements controlling these transcriptional changes were not identified. ...
Few transcriptional regulatory networks have been described in non-model organisms. In Entamoeba histolytica seminal aspects of pathogenesis are transcriptionally controlled, however, little is known about transcriptional regulatory
networks that effect gene expression in this parasite. We used expression data from two microarray experiments, cis-regulatory motif elucidation, and a naïve Bayesian classifier to identify genome-wide transcriptional regulatory patterns
in E. histolytica. Our algorithm identified promoter motifs that accurately predicted the gene expression level of 68% of genes under trophozoite
conditions. We identified a promoter motif (A/TAAACCCT) associated with high gene expression, which is highly enriched in promoters of ribosomal protein genes and tRNA synthetases.
Additionally, we identified three promoter motifs (GAATGATG, AACTATTTAAACATC/TC and TGAACTTATAAACATC) associated with low gene expression. The promoters of a large gene family were highly enriched for
these motifs, and in these genes the presence of ⩾2 motifs predicted low baseline gene expression and transcriptional activation
by heat shock. We demonstrate that amebic nuclear protein(s) bind specifically to four of the motifs identified herein. Our
analysis suggests that transcriptional regulatory networks can be identified using limited expression data. Thus, this approach
is applicable to the multitude of systems for which microarray and genome sequence data are emerging.
... Microarray-based analyses can be utilised in conjunction with genome sequencing to assign functional roles to annotated genes and to clarify genomic architecture. A number of groups have utilised DNA microarrays in E. histolytica (made from random genomic DNA fragments or long or short oligonucleotides based on annotated genes) to successfully study transcriptional differences between virulent and avirulent E. histolytica as well as transcriptional responses to heat shock, collagen and calcium exposure, tissue invasion and cyst development (Debnath et al., 2004;Gilchrist et al., 2006;MacFarlane and Singh, 2006;Weber et al., 2006;Ehrenkaufer et al., 2007). Additionally, using a genomic DNA microarray, comparative genomic hybridisations (CGH) between strains and species of Entamoeba have been performed (Shah et al., 2005). ...
The intestinal parasite Entamoeba histolytica is one of the first protists for which a draft genome sequence has been published. Although the genome is still incomplete, it is unlikely that many genes are missing from the list of those already identified. In this chapter we summarise the features of the genome as they are currently understood and provide previously unpublished analyses of many of the genes.
Protozoan parasitic infections of the gastrointestinal (GI) tract are being increasingly recognized as an important public health problem in the United States. The intestinal protozoan pathogens include both parasites that are extracellular, such as Giardia lamblia, Blastocystis hominis, and Entamoeba histolytica, and the intracellular spore-forming parasites Cryptosporidium parvum, Cyclospora cayetanensis, and Isospora belli. The AIDS epidemic has played an important role in the recognition of intracellular spore-forming parasites as important gastrointestinal protozoan pathogens. Stool microscopic examinations may be performed in clinical laboratories only if specifically requested. In some cases in which infection is suspected on clinical grounds, stool examination may not be revealing, and endoscopy may be necessary to make the diagnosis. The recent rapid development of new molecular assays, will likely facilitate more rapid laboratory diagnoses of not only protozoan, but also viral and bacterial pathogens in the near future.
Protein tyrosine phosphatase of regenerating liver (PRL) is a group of phosphatases that has not been broadly studied in protozoan parasites. In humans, PRLs are involved in metastatic cancer, the promotion of cell migration and invasion. PTPs have been increasingly recognized as important effectors of host-pathogen interactions. We characterized the only putative protein tyrosine phosphatase PRL (PTP EhPRL) in the eukaryotic human intestinal parasite E. histolytica. Here, we reported that the EhPRL protein possessed the classical HCX5R catalytic motif of PTPs and the CAAX box characteristic of the PRL family and exhibited 31-32% homology with the three human PRL isoforms. In amebae, the protein was expressed at low but detectable levels. The recombinant protein (rEhPRL) had enzymatic activity with the 3-o-methyl fluorescein phosphate (OMFP) substrate; this enzymatic activity was inhibited by the PTP inhibitor o-vanadate. Using immunofluorescence we showed that native EhPRL was localized to the cytoplasm and plasma membrane. When the trophozoites interacted with collagen, EhPRL relocalized over time to vesicle-like structures. Interaction with fibronectin increased the presence of the enzyme in the cytoplasm. Using RT-PCR, we demonstrated that EhPRL mRNA expression was upregulated when the trophozoites interacted with collagen but not with fibronectin. Trophozoites recovered from amoebic liver abscesses showed higher EhPRL mRNA expression levels than normal trophozoites. These results strongly suggest that EhPRL may play an important role in the biology and adaptive response of the parasite to the host environment during amoebic liver abscess development, thereby participating in the pathogenic mechanism.
El genoma de Entamoeba histolytica.
La secuencia del borrador del genoma de E. histolytica ha sido publicada (Loftus et al., 2005). Los datos muestran que el genoma de este parásito tiene un tamaño de 23.7 millones de pares de bases, los cuales predicen alrededor de 9,000 genes. En general los genes de E. histolytica son cortos debido principalmente a la perdida de intrones. Las regiones regulatorias 3´ y 5´ no traducidas (3´UTR y 5´UTR) también son cortas, evidenciando una relativa compactación del genoma. La mayoría de los genes predichos contienen solo un exón sencillo, sin embargo cerca del 25% de los genes puede contener intrones y 6% pueden contener 2 o más intrones. En promedio la longitud predicha de una proteína es de 389 amino ácidos (Clark G et al, 2008). El análisis inicial global de la secuencia del genoma reveló que este parásito ha sufrido una serie de adaptaciones metabólicas que incluyen una reducción casi total de los genes que codifican para proteínas involucradas en las rutas metabólicas mitocondriales y el uso de enzimas de estrés oxidativo asociadas generalmente a procariontes anaeróbicos (Loftus et al, 2005) El reporte inicial del análisis de la secuencia del genoma de E. histolytica también muestra evidencia de la existencia de mecanismos de transferencia horizontal de genes que tienen funciones predichas en el metabolismo central del parásito. El genoma también codifica para una larga serie de familias génicas dentro de las cuales destacan un número grande de nuevos receptores-cinasas, así como una expansión de familias asociadas a virulencia tales como proteinasas.
Perfiles de expresión genómica en E. histolytica: aplicación de la tecnología de microarreglos.
El uso de diversas metodologías aplicadas para el análisis genómico han sido aplicadas exitosamente en E. histolytica. La disponibilidad de la secuencia del genoma y la aplicación de diversas herramientas bioinformáticas ha permitido la elaboración de microarreglos de ADN los cuales han permitido descifrar los perfiles de expresión de genes a gran escala en parásitos sometidos a dferentes estímulos y condiciones (Figura 1). En consecuencia se han publicado diversos
AmebiasisBlastocystis hominisGiardiasisDientamoeba fragilisBalantidiasisCoccidia (Cryptosporidium, Isospora, and Cyclospora)
Entamoeba histolytica is the intestinal protozoan parasite responsible for amebic colitis and liver abscesses, which cause mortality in many developing countries. The sequencing of the parasite genome provides new insights into the cellular workings and genome evolution of this major human pathogen. Here, we reviewed recent advances in the efforts to understand virulence and gene expression regulation in E. histolytica by using genomic approaches based on microarray technology and bioinformatic analysis of genome sequence.
Entamoeba histolytica is an intestinal parasite that causes dysentery and liver abscess. Parasite cell surface receptors, such as the Gal/GalNAc lectin, facilitate attachment to host cells and extracellular matrix. The Gal/GalNAc lectin binds to galactose or N-acetylgalactosamine residues on host components and is composed of heavy (Hgl), intermediate (Igl), and light (Lgl) subunits. Although Igl is constitutively localized to lipid rafts (cholesterol-rich membrane domains), Hgl and Lgl transiently associate with this compartment in a cholesterol-dependent fashion. In this study, trophozoites were exposed to biologically relevant ligands to determine if ligand binding influences the submembrane distribution of the subunits. Exposure to human red blood cells (hRBCs) or collagen, which are bona fide Gal/GalNAc lectin ligands, was correlated with enrichment of Hgl and Lgl in rafts. This enrichment was abrogated in the presence of galactose, suggesting that direct lectin-ligand interactions are necessary to influence subunit location. Using a cell line that is able to attach to, but not phagocytose, hRBCs, it was shown that physical attachment to ligands was not sufficient to induce the enrichment of lectin subunits in rafts. Additionally, the mutant had lower levels of phosphatidylinositol (4,5)-bisphosphate (PIP(2)); PIP(2) loading restored the ability of this mutant to respond to ligands with enrichment of subunits in rafts. Finally, intracellular calcium levels increased upon attachment to collagen; this increase was essential for the enrichment of lectin subunits in rafts. Together, these data provide evidence that ligand-induced enrichment of lectin subunits in rafts may be the first step in a signaling pathway that involves both PIP(2) and calcium signaling.
Visceral Leishmaniasis (VL) is the well-recognized infectious disease among the complex of Leishmaniasis (cutaneous, mucocutaneous, visceral) in tropical and subtropical countries. Treatments for VL are unsatisfactory till date and alarming rise of drug resistance is a serious problem. Vaccines to control VL have shown some promise, but none are in current clinical use. Therefore, urgent needs for new and effective anti-leishmanials are prerequisite. To identify the potential factors, DNA microarray an advance, high throughput technology, has open the possibility of discovering new genes that can contribute to vaccine initiatives or serve as potential drug targets. It has been successfully applied to many of the protozoan parasites and identified some new genes as targets. Target discovery is the key step in the biomarker and drug discovery pipeline to diagnose. After the completion of genome sequencing of Leishmania major and L. infantum, advancement in microarray technologies provide new approaches to study the pattern of gene expression profile during differentiation and development of parasite. It has the potential to improve our understanding of pathogenicity, mechanism of drug resistance and virulence factors by identifying up/down regulated gene and characterizing the respective gene expression. Keeping these backgrounds in mind, we reviewed the data obtained from genome-wide wide expression profiling in Leishmania that focuses on applications of microarray in novel vaccine/drug targets discovery for VL and discuss the potential avenues for their future investigation. Ultimately this will be able to translate the findings into the development of novel therapeutic approaches and targets for VL. © arjournals.org, All rights reserved.
Expression profiling with microarray technology has revolutionized exploration of transcriptional regulatory networks on a genome-wide scale. This approach has been successfully applied to the study of Entamoeba histolytica, which causes dysentery and liver abscesses and is a leading parasitic cause of death globally. A variety of microarray platforms have been developed for this system including those generated from genomic DNA, long oligonucleotides, and short oligonucleotides. Using these tools researchers have identified parasite genes whose transcript abundance is differentially regulated during stress, host invasion, and stage conversion. Additionally, novel virulence factors have been identified by identifying genes that are highly expressed in virulent but with low expression in non-virulent Entamoeba strains. All combined, these studies have provided new data on molecular aspects of amebic biology, pathogenic potential and stage conversion and provide investigators with the first insights into potential novel drug targets against amebic disease.
Entamoeba histolytica remains a cause of significant morbidity and mortality in many countries. Although metronidazole has been used for the treatment of amoebiasis for several decades, little is known on how the amoebae react to this challenge on the levels of mRNA and protein expression. In this study, we examined their response using a focused microarray, quantitative RT-PCR, and two-dimensional gel electrophoresis (2DE). The amoebae modestly increased the levels of mRNA coding for superoxide dismutase, peroxiredoxin, ferredoxin, thioredoxin reductase, and the galactose/N-acetylgalactosamine specific lectin light and heavy chains. The mRNAs encoding actin and the 70kDa and 101kDa heat shock proteins were decreased. All the changes occured within 1h of exposition, with very little further changes. In addition, the proteome revealed only very few changes. Taken together, E. histolytica appears to make only modest mRNA and protein expression changes when confronted with an unknown chemical stress.
We have developed an Entamoeba histolytica genomic DNA microarray and used it to develop a transcriptional profile of 1,971 E. histolytica (HM-1:IMSS) genes. The arrays accurately detected message abundance and 31-47% of amebic genes were expressed under standard tissue culture conditions (levels detectable by Northern blot analysis or RT-PCR respectively). Genes expressed at high levels ( approximately 2% of total) included actin (8.m00351), and ribosomal genes (20.m00312). Moderately expressed genes ( approximately 14% of total) included cysteine proteinase (191.m00117), profilin (156.m00098), and an Argonaute family member (11.m00378). Genes with low-level expression ( approximately 15% of total) included Ariel1 (160.m00087). Genes with very low expression ( approximately 16% of total) and those not expressed ( approximately 52% of total) included encystation-specific genes such as Jacob cyst wall glycoprotein (33.m00261), chitin synthase (3.m00544), and chitinase (22.m00311). Transcriptional modulation could be detected using the arrays with 17% of genes upregulated at least two-fold in response to heat shock. These included heat shock proteins (119.m00119 and 279.m00091), cyst wall glycoprotein Jacob (33.m00261), and ubiquitin-associated proteins (16.m00343; 195.m00092). Using Caco-2 cells to model the host-parasite interaction, we verified that host cell killing was dependent on live ameba. However, surprisingly these events did not appear to induce major transcriptional changes in the parasites.
Amoebozoa represent one of the earliest branches from the last common ancestor of all eukaryotes and contain some of the most dangerous human pathogens. Two amoebozoan genomes -- from the model organism Dictyostelium discoideum and the human pathogen Entamoeba histolytica -- have been published this year. Owing to their high A+T content, both genomes were difficult to sequence. In addition to nine amoebozoan expressed sequence tag projects, efforts are underway for comparative sequencing of four additional Entamoeba species. The completed genome sequences of D. discoideum and E. histolytica revealed unusual telomere structures, a high percentage of repetitive elements and a remarkably high gene content that is close to the one of Drosophila melanogaster. Finally, both organisms are brilliant examples of the influence of the lifestyle of an organism on its genome.
Entamoeba histolytica is a protozoan parasite of humans that causes 40,000-100,000 deaths annually. Clinical amoebiasis results from the spread of the normally luminal parasite into the colon wall and beyond; the key development in understanding this complex multistage process has been the publication of the E. histolytica genome, from which has come an explosion in the use of microarrays to examine changes in gene expression that result from changes in growth conditions. The genome has also revealed a unique arrangement of tRNA genes and an extraordinary number of genes for putative virulence factors, many unexpressed under the artificial conditions of growth in culture. The ability to induce apoptosis of mammalian cells and a useful, but as yet little-understood, technique for epigenetic irreversible gene silencing are other exciting developments.
DNA microarrays are high-throughput platforms that take advantage of the vast amount of sequence information and allow scientists to perform gene expression profiling or genotyping studies on a "global" or "genome-wide" scale. The global monitoring of gene expression in hosts and pathogens, either separately or interactively, has given us systemic views of the disease mechanisms. Ongoing improvements in DNA sequencing and microarray technologies continue to open up new opportunities for better understanding and developing more effective approaches in diagnosis, treatments, and preventions of infectious diseases. This review focuses on the latest developments and applications of the DNA microarray technologies designed for studying pathogens in the areas of pathogenesis, host-pathogen interaction, drug response, vaccine development, and disease agent identification. Issues and challenges in the analysis, management and interpretation of microarray data are also addressed.
In this study, the proteome of axenically grown Entamoeba histolytica parasites was explored by two-dimensional gel electrophoresis (2-DE), employing a practical and effective procedure for the solubilization of E. histolytica proteins. Approximately 900 protein species in the pH range between 4 and 7 were detected by Coomassie Blue staining. Ninety-five spots were excised, trypsinated and subjected to mass spectrometry. The resultant data from peptide mass fingerprints were compared with those available in the E. histolytica genome and the (non-redundant) National Center for Biotechnology Information (NCBI) databases for the identification and categorization of proteins. Sixty-three of the proteins identified were predicted to relate to the cytoskeleton, surface, glycolysis, RNA/DNA metabolism, the ubiquitin-proteasome system, vesicular trafficking and signal transduction. The present study demonstrates, for the first time, that corresponding genes are indeed expressed in E. histolytica and provides a foundation for further proteomic studies of this parasite.
Invasive intestinal amebiasis, caused by Entamoeba histolytica, is initiated with attachment of trophozoites to the colonic mucous layer, mucous disruption and/or depletion, and adherence to and cytolysis of host epithelial and inflammatory cells. A current working model of intestinal amebiasis suggests that the microenvironment of the host intestine, particularly intestinal mucins and the bacterial biofilm, may influence the behavior of pathogenic amebae. The invasive phenotype is dependent on expression of a number of virulence factors of which cysteine proteases provide the most convenient experimental probe because their activity is readily monitored. In the present study, we examined the interaction of E. histolytica with GalNAc, mucin, different epithelial cell lines and bacteria both by biochemical assays of protease release and transcriptional profiling using a previously validated genomic microarray. A significant down-regulation of released cysteine protease activity was observed when amebic trophozoites were grown with GalNAc, specific colonic cell lines and bacteria. Transcriptional profiling during GalNAc interaction revealed enhanced expression of the 170-kDa Gal/GalNAc lectin. Decreased protease activity during GalNAc interaction and enhanced expression of the Gal/GalNAc lectin gene are consistent with a program of commensal infection and mucus coat colonization mediated by the lectin. The down-regulation of cysteine protease activity following interaction with a colonic epithelial cell line parallels the presence of secretory mucin having a complex carbohydrate structure rich in Gal and GalNAc. In contrast, interaction of E. histolytica trophozoites with stomach porcine mucin enhanced cysteine protease (EhCP1 and EhCP2) secretion 3-fold. This suggests the specific composition of mucins may affect the Entamoeba phenotype. Transcriptional profiling revealed interaction of Entamoeba with intestinal bacteria induced protein kinase, ABC transporter, Rab family GTPase and hsp 90 gene expression. The enhanced expression of this gene cluster is consistent with enhanced phagocytosis of E. histolytica during interaction with bacteria.
The genus Entamoeba contains many species, six of which (Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, Entamoeba polecki, Entamoeba coli, and Entamoeba hartmanni) reside in the human intestinal lumen. Entamoeba histolytica is the causative agent of amebiasis and is considered a leading parasitic cause of death worldwide in humans. Although recent studies highlight the recovery of E. dispar and E. moshkovskii from patients with gastrointestinal symptoms, there is still no convincing evidence of a causal link between the presence of these two species and the symptoms of the host. New approaches to the identification of E. histolytica are based on detection of E. histolytica-specific antigen and DNA in stool and other clinical samples. Several molecular diagnostic tests, including conventional and real-time PCR, have been developed for the detection and differentiation of E. histolytica, E. dispar, and E. moshkovskii in clinical samples. The purpose of this review is to discuss different methods that exist for the identification of E. histolytica, E. dispar, and E. moshkovskii which are available to the clinical diagnostic laboratory. To address the need for a specific diagnostic test for amebiasis, a substantial amount of work has been carried out over the last decade in different parts of the world. The molecular diagnostic tests are increasingly being used for both clinical and research purposes. In order to minimize undue treatment of individuals infected with other species of Entamoeba such as E. dispar and E. moshkovskii, efforts have been made for specific diagnosis of E. histolytica infection and not to treat based simply on the microscopic examination of Entamoeba species in the stool. The incorporation of many new technologies into the diagnostic laboratory will lead to a better understanding of the public health problem and measures to control the disease.
We screened a Xenopus laevis oocyte cDNA expression library with a Src homology 3 (SH3) class II peptide ligand and identified a 1270-amino acid-long
protein containing two Eps15 homology (EH) domains, a central coiled-coil region, and five SH3 domains. We named this protein
Intersectin, because it potentially brings together EH and SH3 domain-binding proteins into a macromolecular complex. The
ligand preference of the EH domains were deduced to be asparajine-proline-phenylalanine (NPF) or cyclized NPF (CX
1–2NPFXXC), depending on the type of phage-displayed combinatorial peptide library used. Screens of a mouse embryo cDNA library with
the EH domains of Intersectin yielded clones for the Rev-associated binding/Rev-interacting protein (RAB/Rip) and two novel
proteins, which we named Intersectin-binding proteins (Ibps) 1 and 2. All three proteins contain internal and C-terminal NPF
peptide sequences, and Ibp1 and Ibp2 also contain putative clathrin-binding sites. Deletion of the C-terminal sequence, NPFL-COOH,
from RAB/Rip eliminated EH domain binding, whereas fusion of the same peptide sequence to glutathione S-transferase generated strong binding to the EH domains of Intersectin. Several experiments support the conclusion that the
free carboxylate group contributes to binding of the NPFL motif at the C terminus of RAB/Rip to the EH domains of Intersectin.
Finally, affinity selection experiments with the SH3 domains of Intersectin identified two endocytic proteins, dynamin and
synaptojanin, as potential interacting proteins. We propose that Intersectin is a component of the endocytic machinery.
Proteolytic activities of the protozoan parasite Entamoeba histolytica strain HM1:IMSS and the cytochalasin D-resistant mutant BG-3 were analyzed following stimulation with collagen type I and
Ca2+, which induces electron-dense associated collagenase secretion. The mutant BG-3 had a protease activity of 73 kDa and secretion
of total protease activity was not stimulated by collagen type I and Ca2+, which produced, in contrast, a 2-fold increase in protease secretion by the parental strain. This collagen-stimulated protease
secretion was inhibited by cytochalasin D at a concentration of 1 μg/ml. Cytochalasin D did not have any effect on the protease
activity released by the mutant BG-3. These findings suggest that cytoskeleton integrity is necessary for collagen-induced
protease secretion.
Cells in culture reveal high levels of protein tyrosine phosphorylation in their focal adhesions, the regions where cells adhere to the underlying substratum. We have examined the tyrosine phosphorylation of proteins in response to plating cells on extracellular matrix substrata. Rat embryo fibroblasts, mouse Balb/c 3T3, and NIH 3T3 cells plated on fibronectin-coated surfaces revealed elevated phosphotyrosine levels in a cluster of proteins between 115 and 130 kD. This increase in tyrosine phosphorylation was also seen when rat embryo fibroblasts were plated on laminin or vitronectin, but not on polylysine or on uncoated plastic. Integrin mediation of this effect was suggested by finding the same pattern of elevated tyrosine phosphorylation in cells plated on the cell-binding fragment of fibronectin and in cells plated on a synthetic polymer containing multiple RGD sequences. We have identified one of the proteins of the 115-130-kD cluster as pp125FAK, a tyrosine kinase recently localized in focal adhesions (Schaller, M. D., C. A. Borgman, B. S. Cobb, R. R. Vines, A. B. Reynolds, and J. T. Parsons. 1992. Proc. Natl. Acad. Sci. USA. 89:5192). A second protein that becomes tyrosine phosphorylated in response to extracellular matrix adhesion is identified as paxillin, a 70-kD protein previously localized to focal adhesions. Treatment of cells with the tyrosine kinase inhibitor herbimycin A diminished the adhesion-induced tyrosine phosphorylation of these proteins and inhibited the formation of focal adhesions and stress fibers. These results suggest a role for integrin-mediated tyrosine phosphorylation in the organization of the cytoskeleton as cells adhere to the extracellular matrix.
In order to study gene expression in the human parasite Entamoeba histolytica, a cDNA library of E. histolytica strain 200:NIH was constructed using the phage vector lambda gt10. Three cDNA clones (A, B and C) were selected for further analysis. Each of the three clones hybridized to a distinct mRNA. Two of these mRNAs were translated in vitro after hybrid selection, and yielded distinct translation products. One of these mRNAs, selected by hybridization to clone A, encodes the most abundantly expressed protein in E. histolytica. DNA sequence analysis of this cDNA clone identified the DNA as that encoding actin. The deduced amino acid sequence of E. histolytica actin resembles both cytoplasmic and muscle actins and has an unusual N-terminal glycine residue. We have shown that a family of actin genes is present in E. histolytica. Six different E. histolytica actin clones were obtained from a lambda gt10 genomic library using subcloned cDNA probes. Southern analysis of three different E. histolytica strains (200:NIH, Rhaman, and HM-1:IMSS) revealed at least four different actin genes. Strain HM-1:IMSS, however, differs by the presence of an additional actin gene.
The integrin family of heterodimeric cell surface receptors play critical roles in multiple biological processes by mediating cellular adhesion to the extracellular matrix (ECM). Adhesion triggers intracellular signaling cascades, including tyrosine phosphorylation and elevation of [Ca2+]i. The Focal Adhesion Kinase (FAK or pp125FAK), a protein tyrosine kinase that colocalizes with integrins in cellular focal adhesions, is a prime candidate for a mediator of integrin signaling events. Here we report an analysis of the domain structure of FAK in which we have identified a contiguous stretch of 159 amino acids within the COOH terminus essential for correct subcellular localization. When placed in the context of an unrelated cytosolic protein, this Focal Adhesion Targeting (FAT) sequence functions to efficiently mediate the focal adhesion localization of this fusion protein. Furthermore, this analysis suggests that pp125FAK cannot be activated oncogenically by mutation. This result could be explained if pp125FK either exhibits a narrow substrate specificity or is diametrically opposed by cellular phosphatases or other cellular processes.
We have analyzed three independent genomic loci of the protozoan parasite Entamoeba histolytica that contain coding regions for the iron-containing superoxide dismutase, the pore-forming peptide, and the galactose-inhibitable lectin. All of the three structural genes were found to be closely linked unidirectionally to other coding sequences. The intergenic regions did not exceed 1,350 nucleotides. Nuclear run-on data demonstrated that at least the galactose-inhibitable lectin gene is transcribed in a monocistronic fashion. Comparison of the genomic sequences described here with several others reported previously for E. histolytica revealed a number of invariable peculiarities for the gene organization of this parasite: (i) Coding sequences are not interrupted by introns; (ii) 5' untranslated regions are rather short and transcription starts at the consensus sequences ATTCA or ATCA; (iii) an unusual TATA-motif is located about 30 nucleotides upstream of the start of transcription and comprises the sequence TATTTAAA, which reveals protein binding activity as determined by gel retardation assays; (iv) the conserved pentanucleotide motif TAA/TTT is found within the relatively short 3' untranslated regions and functions putatively as the transcription termination signal; and (v) a stretch of up to 12 pyrmidine residues is located at the end of transcribed sequences.
Cysteine proteinases are hypothesized to be important virulence factors of Entamoeba histolytica, the causative agent of amebic dysentery and liver abscesses. The release of a histolytic cysteine proteinase from E. histolytica correlates with the pathogenicity of both axenic strains and recent clinical isolates as determined by clinical history of invasive disease, zymodeme analysis, and cytopathic effect. We now show that pathogenic isolates have a unique cysteine proteinase gene (ACP1). Two other cysteine proteinase genes (ACP2, ACP3) are 85% identical to each other and are present in both pathogenic and nonpathogenic isolates. ACP1 is only 35 and 45% identical in sequence to the two genes found in all isolates and is present on a distinct chromosome-size DNA fragment. Presence of the ACP1 gene correlates with increased proteinase expression and activity in pathogenic isolates as well as cytopathic effect on a fibroblast monolayer, an in vitro assay of virulence. Analysis of the predicted amino acid sequence of the ACP1 proteinase gene reveals homology with cysteine proteinases released by activated macrophages and invasive cancer cells, suggesting an evolutionarily conserved mechanism of tissue invasion. The observation that a histolytic cysteine proteinase gene is present only in pathogenic isolates of E. histolytica suggests that this aspect of virulence in amebiasis is genetically predetermined.
The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons,
a variety of definitional, algorithmic and statistical refinements described here permits the execution time of the BLAST
programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering
the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program
that runs at approximately three times the speed of the original. In addition, a method is introduced for automatically combining
statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using
this matrix. The resulting Position-Specific Iterated BLAST (PSIBLAST) program runs at approximately the same speed per iteration
as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities. PSI-BLAST
is used to uncover several new and interesting members of the BRCT superfamily.
DNA microarrays containing virtually every gene ofSaccharomyces cerevisiae were used to carry out a comprehensive investigation of the temporal program of gene expression accompanying the metabolic
shift from fermentation to respiration. The expression profiles observed for genes with known metabolic functions pointed
to features of the metabolic reprogramming that occur during the diauxic shift, and the expression patterns of many previously
uncharacterized genes provided clues to their possible functions. The same DNA microarrays were also used to identify genes
whose expression was affected by deletion of the transcriptional co-repressorTUP1 or overexpression of the transcriptional activatorYAP1. These results demonstrate the feasibility and utility of this approach to genomewide exploration of gene expression patterns.
In this study we demonstrate that stimulation with platelet-derived growth factor (PDGF) leads to a marked reorganization of the vimentin filaments in porcine aortic endothelial (PAE) cells ectopically expressing the PDGF beta-receptor. Within 20 minutes after stimulation, the well-spread fine fibrillar vimentin was reorganized as the filaments aggregated into a dense coil around the nucleus. The solubility of vimentin upon Nonidet-P40-extraction of cells decreased considerably after PDGF stimulation, indicating that PDGF caused a redistribution of vimentin to a less soluble compartment. In addition, an increased tyrosine phosphorylation of vimentin was observed. The redistribution of vimentin was not a direct consequence of its tyrosine phosphorylation, since treatment of cells with an inhibitor for the cytoplasmic tyrosine kinase Src, attenuated phosphorylation but not redistribution of vimentin. These changes in the distribution of vimentin occurred in conjunction with reorganization of actin filaments. In PAE cells expressing a Y740/751F mutant receptor that is unable to bind and activate phosphatidylinositol 3'-kinase (PI3-kinase), the distribution of vimentin was virtually unaffected by PDGF stimulation. Thus, PI3-kinase is important for vimentin reorganization, in addition to its previously demonstrated role in actin reorganization. The small GTPase Rac has previously been shown to be involved downstream of PI3-kinase in the reorganization of actin filaments. In PAE cells overexpressing dominant negative Rac1 (N17Rac1), no change in the fine fibrillar vimentin network was seen after PDGF-BB stimulation, whereas in PAE cells overexpressing constitutively active Rac1 (V12Rac1), there was a dramatic change in vimentin filament organization independent of PDGF stimulation. These data indicate that PDGF causes a reorganization of microfilaments as well as intermediate filaments in its target cells and suggest an important role for Rac downstream of PI3-kinase in the PDGF stimulated reorganization of both actin and vimentin filaments.
Thousands of genes are being discovered for the first time by sequencing the genomes of model organisms, an exhilarating reminder that much of the natural world remains to be explored at the molecular level. DNA microarrays provide a natural vehicle for this exploration. The model organisms are the first for which comprehensive genome-wide surveys of gene expression patterns or function are possible. The results can be viewed as maps that reflect the order and logic of the genetic program, rather than the physical order of genes on chromosomes. Exploration of the genome using DNA microarrays and other genome-scale technologies should narrow the gap in our knowledge of gene function and molecular biology between the currently-favoured model organisms and other species.
While comparing gene expression in the pathogenic organism Entamoeba histolytica and the closely related but nonpathogenic species Entamoeba dispar, we discovered that the E. histolytica abundant polyadenylated transcript 2 (ehapt2) and corresponding genomic copies are absent in E. dispar. Although polyadenylated, ehapt2 does not contain any overt open reading frame. Southern blot and sequence analyses revealed
that about 500 copies of ehapt2 genomic elements were present in each cell and that the copies were distributed throughout
the ameba genome. The various ehapt2 elements are regularly located in the vicinity of protein-encoding genes, downstream
of pyrimidine-rich sequence stretches (40 to 125 bp; CT content, 79.2 to 85.5%), and are flanked by duplicated target sites
of variable length. Target site duplications were obviously generated during integration of ehapt2 into the E. histolytica genome as one copy of the flanking repeat and the complete ehapt2 element are specifically absent in orthologous E. dispar genomic sequences. ehapt2 shares 3′ sequences with EhRLE, a recently identified non-long-terminal-repeat (non-LTR) retrotransposon-like
element of E. histolytica, which contains a conceptual open reading frame for reverse transcriptase. Thus, ehapt2 has all of the properties of nonautonomous
non-LTR retrotransposons. A comparison of various E. histolytica isolates suggested that transposition of ehapt2 takes place at a very low frequency as the genomic localization of ehapt2
elements was found to be well conserved. A mobile element such as ehapt2 could be a suitable mechanism to explain the infrequent
and late transition of E. histolytica from a harmless gut commensal to an invasive pathogen.
The integrin family of heterodimeric cell surface receptors play critical roles in multiple biological processes by mediating cellular adhesion to the extracellular matrix (ECM). Adhesion triggers intracellular signaling cascades, including tyrosine phosphorylation and elevation of [Ca2+]i. The Focal Adhesion Kinase (FAK or pp125FAK), a protein tyrosine kinase that colocalizes with integrins in cellular focal adhesions, is a prime candidate for a mediator of integrin signaling events. Here we report an analysis of the domain structure of FAK in which we have identified a contiguous stretch of 159 amino acids within the COOH terminus essential for correct subcellular localization. When placed in the context of an unrelated cytosolic protein, this Focal Adhesion Targeting (FAT) sequence functions to efficiently mediate the focal adhesion localization of this fusion protein. Furthermore, this analysis suggests that pp125FAK cannot be activated oncogenically by mutation. This result could be explained if pp125FK either exhibits a narrow substrate specificity or is diametrically opposed by cellular phosphatases or other cellular processes.
HM-1:IMSS, a pathogenic strain of Entamoeba histolytica, and its mutant BG-3, Identified by resistance to cytochalasin D, were tested for their capacity to: (i) secrete electron-dense granules; (ii) adhere and digest native type I collagen gels; and (iii) produce liver abscesses in new-born hamsters. The results demonstrate that the mutant has low adherence to collagen, low electron-dense granule secretion and collagenolytic activity, and low capacity to produce liver lesions in vivo, compared with the parental strain HM1:IMSS.
Molecules expressed by the pathogenic ameobaEntamoeba histolytica but weakly expressed or absent from the non-pathogenic ameobaEntamoeba dispar could be used by intestinal epithelial cells to discriminate between the two species and to initiate an appropriate inflammatory
response. Among the possible molecules involved in this identification are the Gal/GalNac lectin and the lipophosphoglycan.
Once the inflammatory response is initiated,E. histolytica trophozoites have to protect themselves against reactive nitrogen intermediates produced by intestinal epithelial cells,
oxygen intermediates, and cytotoxic molecules released by activated neutrophils. By screening theE. histolytica genome, we have identified proteins that may play a role in the defence strategy of the parasite. One of these proteins,
a serine proteinase inhibitor, inhibits human neutrophil cathepsin G, a key component of the host defence.
The interaction ofEntamoeba histolyticatrophozoites with collagen involves cell adherence, formation, and release of electron dense granules (EDGs) containing collagenase activity leading to the degradation of the bound protein. The binding is thought to be mediated by an “integrin-like” collagen receptor. Since the signal transduction mechanisms triggered by the collagen–trophozoite interaction are unknown, but clearly involve cytoskeletal organization, we decided to explore the role of protein tyrosine phosphorylation in this process. Collagen induces a time-dependent increase in the phosphorylation of several polypetides migrating around 67 and 110 kDa. One polypeptide of the high-molecular-weight component was identified as a 125-kDa protein with very similar epitopes to the focal adhesion kinase, pp125FAK. Another protein that became tyrosine phosphorylated upon collagen treatment was a 42-kDa polypeptide related to the mitogen activated protein kinase (MAPK) family. Our results suggest that tyrosine phosphorylation is involved in collagen signaling in amoebas and that pp125FAKand p42MAPKhomologs may play an active role in turning on the genetic program that enables the parasite to invade its host.
Amoebapore, a 77-residue peptide with pore-forming activity from the human pathogen Entamoeba histolytica, is implicated in the killing of phagocytosed bacteria and in the cytolytic reaction of the amoeba against host cells. Previously, we structurally and functionally characterized three amoebapore isoforms in E. histolytica but recognized only one homolog in the closely related but non-pathogenic species Entamoeba dispar. Here, we identified two novel amoebapore homologs from E. dispar by molecular cloning. Despite strong resemblance of the primary structures of the homologs, molecular modeling predicts a species-specific variance between the peptide structures. Parallel isolation from trophozoite extracts of the two species revealed a lower amount of pore-forming peptides in E. dispar and substantially higher activity of the major isoform from E. histolytica towards natural membranes than that from E. dispar. Differences in abundance and activity of the lytic polypeptides may have an impact on the pathogenicity of amoebae.
In order to identify molecules that might be responsible for the difference in pathogenicity between the two closely related protozoan parasites Entamoeba histolytica and Entamoeba dispar, we focussed on cysteine proteinases because this class of enzymes has been considered important for pathogenic tissue destruction. By screening a genomic library derived from an E. histolytica isolate, a total of six distinct genes (ehcp1–ehcp6) encoding typical prepro-forms of cysteine proteinases were identified which differed from each other by 40% to 85% of their nucleotide sequences. Three of these genes, ehcp1, ehcp2, and ehcp5, which exhibited high levels of expression, were found to be responsible for approximately 90% of cysteine proteinase transcripts, whereas the remaining three were either not or only marginally expressed. Expression of the different genes directly correlated with the level of activity of the respective enzymes in trophozoite lysates. Purification of the enzymes and N-terminal sequencing revealed that virtually all cysteine proteinase activity of E. histolytica can be attributed to three enzymes namely EhCP1, EhCP2 and EhCP5. Southern blot analysis indicated that just two of these abundantly expressed genes are missing in E. dispar. On the other hand, genes analogous to four of the six genes identified in E.histolytica were found to be present in E. dispar, but only two of these are expressed within the trophozoite stage.
A peculiar characteristic of Entamoeba histolytica trophozoites is their capacity to invade human tissues. One of the cellular determinants of invasion may include adhesion to extracellular matrix components such as collagen, induction, and secretion of electron-dense granules (EDG) and tissue digestion. The mechanism and receptors involved in this process are not well understood. Previous results suggested that cytoskeleton plays a very important role during EDG secretion. We present evidence suggesting that adhesion to collagen and activation of EDG secretion are integrin-dependent events, since β1 subunits detected by antibodies are concentrated at membrane sites where collagen and actin were colocalized. Furthermore, the involvement of actin, vimentin, and tubulin in restructuring cytoskeleton during EDG secretion was evident, since cytoskeleton isolation was possible exclusively in activated cells. Studies of immunolocalization of tubulin, actin, and vimentin by immunofluorescence and transmission electron microscopy suggest a role for cytoskeleton in EDG secretion. Cell Motil. Cytoskeleton 50:45–54, 2001. © 2001 Wiley-Liss, Inc.
Entamoeba histolytica cells secrete electron-dense granules (EDGs) that have collagenase activity. To study the possible involvement of calmodulin (CaM) on EDG secretion, the effect of several CaM antagonists (TFP, R24571, W-7, W-5, dibucaine and DL-propranolol) was tested on this cellular function. Except for W-5 and dibucaine, the rest of these compounds inhibited EDG secretion. Transmission electron microscopy of collagen-activated trophozoites showed numerous EDGs located in or near the surface membrane. In contrast, trophozoites incubated with TFP showed no EDGs. Protein kinase C inhibitors (H-7, ML-9) had no effect on EDG secretion, suggesting that CaM antagonists acted by selectively inhibiting CaM. These results suggest that a CaM-dependent process is involved in EDG secretion.
A polypeptide that causes pore formation in target-cell membranes is implicated in the potent cytolytic activity of pathogenic Entamoeba histolytica. Pore-forming material was purified to apparent homogeneity by a multistep procedure, and its analysis by NaDodSO4/PAGE revealed one peptide of 4-5 kDa under nonreducing or under reducing conditions. Pore-forming activity was measured by depolarization of liposome membrane potential and was found to be optimally expressed at low pH. Active material preferentially inserted into negatively charged lipid vesicles. Treatment of purified amoeba peptide in solution or bound to liposomes with glutaraldehyde revealed oligomers upon NaDodSO4/PAGE, suggesting functionally relevant peptide-peptide interactions. The NH2-terminal amino acid sequence of the amoeba peptide was determined by protein sequencing and revealed a structural similarity to melittin, the membranolytic peptide of bee venom.
A human ribosomal protein S17 cDNA [Chen et al., Proc. Natl. Acad. Sci. USA 83 (1986) 6907-6911] was used as heterologous probe to isolate S17 clones from Drosophila genomic and cDNA recombinant libraries. Five S17 genomic clones were recognized; all contained overlapping regions of a single chromosomal site. Subsequently the Drosophila RPS17 gene was mapped by in situ hybridization to chromosome 3L, band 67B1-5. The locus spans approximately 1000 bp of DNA and includes four exons. It is preceded by conventional CAAT and TATA RNA polymerase II promoter motifs. The 131 amino acid protein encoded within Drosophila RPS17 is similar to ribosomal proteins from several other eukaryotes. Comparison of eukaryotic S17 proteins' primary structures as well as the number and location of their genes' intervening sequences suggest that S17 is a relatively recent addition to the ribosomal protein family, probably post-dating divergence of eukaryotes and prokaryotes.
Communication between cells assumes particular importance in multicellular organisms. The growth, migration and differentiation of cells in the embryo, and their organization into specific tissues, depend on signals transmitted from one cell to another. In the adult, cell signalling orchestrates normal cellular behaviour and responses to wounding and infection. The consequences of breakdowns in this signalling underlie cancer, diabetes and disorders of the immune and cardiovascular systems. Conserved protein domains that act as key regulatory participants in many of these different signalling pathways are highlighted.
The NADPH oxidase responsible for generation of superoxide anion and related microbicidal oxidants by phagocytes is assembled from at least five distinct proteins. Two are cytosolic components (p47-phox and p67-phox) that contain Src homology 3 (SH3) domains and associate with a transmembrane cytochrome b558 upon activation. We show here that the SH3 domains of p47-phox bind to proline-rich sequences in p47-phox itself and the p22-phox subunit of cytochrome b558. Binding of the p47-phox SH3 domains to p22-phox was abolished by a mutation in one proline-rich sequence (Pro156-->Gln) noted in a distinct form of chronic granulomatous disease and was inhibited by a short proline-rich synthetic peptide corresponding to residues 149-162 of p22-phox. Expression of mutated p22-phox did not restore oxidase activity to p22-phox-deficient B cells and did not enable p22-phox-dependent translocation of p47-phox to membranes in phorbol ester-stimulated cells. We also show that the cytosolic oxidase components associate with one another through the C-terminal SH3 domain of p67-phox and a proline-rich C-terminal sequence in p47-phox. These SH3 target sites conform to consensus features deduced from SH3 binding sites in other systems. We propose a model in which the oxidase complex assembles through a mechanism involving SH3 domains of both cytosolic proteins and cognate proline-rich targets in other oxidase components.
Engagement of antigen receptor complexes induces rapid activation of Src-family kinases and association with phosphatidylinositol-3' kinase (PI-3 kinase). Here it was found that the Src homology 3 (SH3) domain of Lyn and Fyn bound to a proline-rich region (residues 84 to 99) within the 85-kilodalton subunit (p85) of PI-3 kinase. The binding of SH3 to the purified kinase led to a five- to sevenfold increase in the specific activity of PI-3 kinase. Ligand-induced receptor stimulation activated PI-3 kinase, and this activation was blocked by a peptide containing residues 84 to 99 of p85. These data demonstrate a mechanism for PI-3 kinase activation and show that binding of SH3 domains to proline-rich target sequences can regulate enzymatic activity.
Src homology 3 (SH3) domains have been implicated in mediating protein-protein interactions in receptor signaling processes; however, the precise role of this domain remains unclear. In this report, affinity purification techniques were used to identify the GTPase dynamin as an SH3 domain-binding protein. Selective binding to a subset of 15 different recombinant SH3 domains occurs through proline-rich sequence motifs similar to those that mediate the interaction of the SH3 domains of Grb2 and Abl proteins to the guanine nucleotide exchange protein, Sos, and to the 3BP1 protein, respectively. Dynamin GTPase activity is stimulated by several of the bound SH3 domains, suggesting that the function of the SH3 module is not restricted to protein-protein interactions but may also include the interactive regulation of GTP-binding proteins.
Clustering of integrin receptors during cell adhesion stimulates signal transduction across the cell membrane. Second messengers are generated, activating cytosolic proteins and causing cytoskeletal assembly and rearrangement. HeLa cell adhesion to a collagen substrate has been shown to initiate an arachidonic acid-mediated signaling pathway, leading to the activation of protein kinase C (PKC) and cell spreading. To determine the role of integrin receptors in triggering this signaling pathway, monoclonal antibodies to beta 1 integrins were used to either cluster integrins on the cell surface or to provide an integrin-dependent substrate for cell adhesion. Using this approach, we have defined a pathway required for cell spreading that can be initiated by the ligation of integrins and leads to the activation of PKC. Specifically, our results indicate that clustering beta 1 integrins results in the activation of phospholipase A2 leading to the production of arachidonic acid and the activation of PKC.
The interaction of Entamoeba histolytica with collagen induces the intracellular formation and release of electron-dense granules (EDGs) containing collagenase activity which are important in the pathogenicity of this parasite. Purified EDGs contain at least 25 polypeptides with acidic pIs, nine gelatinase activities, small molecules, including inorganic phosphate (Pi), pyrophosphate (PP) and other elements, including Na, Mg, S, Cl, K, Ca and Fe as measured by scanning transmission electron microscopy. Six of these polypeptides with apparent molecular weights of 108, 106, 104, 97, 68 and 59 kDa and two protease activities with apparent molecular weights of 40 and 85 kDa were detected exclusively in the EDGs and were not observed in total trophozoite extracts. Actin was also detected in the EDGs. Therefore, EDGs are a complex of mainly cationic proteins, which contains numerous proteolytic activities, actin and small molecules such as P(i), PP and cations.
The interaction of Entamoeba histolytica trophozoites with collagen induces the synthesis and release of electron-dense granules containing a collagenase activity that is an important factor in the pathogenicity of the parasite. The binding is thought to be mediated by an 'integrin-like' collagen receptor. In the signal transduction mechanisms activated by collagen, pp125FAK and p42MAPK are involved. Using immunoprecipitation assays coupled to Western blot analysis, we demonstrate here the collagen-dependent association of paxillin and Src with pp125FAK. Furthermore, collagen induces a time-dependent increase in the DNA binding activity of the activator protein 1, which is well correlated with an increase in Fos expression. Our results suggest that a stimulus-transcription coupling triggered by collagen in E. histolytica trophozoites might activate or repress genes involved in tissue invasiveness.
Models of the bacterial ribosome based on recent structural analyses are beginning to provide new insights into the protein synthetic machinery. Central to evolving models are the high-resolution structures of individual ribosomal proteins, which represent detailed probes of their local RNA and protein environments. Ribosomal proteins are extremely ancient molecules; the structures therefore also provide a unique window into early protein evolution. Many of the proteins contain domains that are present in more recently evolved families of RNA- and DNA-binding proteins. Such structural homology can be used to predict mechanisms by which proteins interact with RNA in the ribosome.
We screened a Xenopus laevis oocyte cDNA expression library with a Src homology 3 (SH3) class II peptide ligand and identified a 1270-amino acid-long protein containing two Eps15 homology (EH) domains, a central coiled-coil region, and five SH3 domains. We named this protein Intersectin, because it potentially brings together EH and SH3 domain-binding proteins into a macromolecular complex. The ligand preference of the EH domains were deduced to be asparajine-proline-phenylalanine (NPF) or cyclized NPF (CX1-2NPFXXC), depending on the type of phage-displayed combinatorial peptide library used. Screens of a mouse embryo cDNA library with the EH domains of Intersectin yielded clones for the Rev-associated binding/Rev-interacting protein (RAB/Rip) and two novel proteins, which we named Intersectin-binding proteins (Ibps) 1 and 2. All three proteins contain internal and C-terminal NPF peptide sequences, and Ibp1 and Ibp2 also contain putative clathrin-binding sites. Deletion of the C-terminal sequence, NPFL-COOH, from RAB/Rip eliminated EH domain binding, whereas fusion of the same peptide sequence to glutathione S-transferase generated strong binding to the EH domains of Intersectin. Several experiments support the conclusion that the free carboxylate group contributes to binding of the NPFL motif at the C terminus of RAB/Rip to the EH domains of Intersectin. Finally, affinity selection experiments with the SH3 domains of Intersectin identified two endocytic proteins, dynamin and synaptojanin, as potential interacting proteins. We propose that Intersectin is a component of the endocytic machinery.
A system of cluster analysis for genome-wide expression data from DNA microarray hybridization is described that uses standard statistical algorithms to arrange genes according to similarity in pattern of gene expression. The output is displayed graphically, conveying the clustering and the underlying expression data simultaneously in a form intuitive for biologists. We have found in the budding yeast Saccharomyces cerevisiae that clustering gene expression data groups together efficiently genes of known similar function, and we find a similar tendency in human data. Thus patterns seen in genome-wide expression experiments can be interpreted as indications of the status of cellular processes. Also, coexpression of genes of known function with poorly characterized or novel genes may provide a simple means of gaining leads to the functions of many genes for which information is not available currently.
Genome projects are providing vast amounts of sequence data. This raw material makes possible a completely new era of experimental approaches. Among these, DNA array technology, which allows one to assay thousands of unique nucleic acid samples simultaneously, will be important in genomic research, and the results of this research are likely to affect virtually every field of biology. DNA array technology is still in its infancy, but many have demonstrated its power by using it for such diverse applications as global monitoring of gene expression, mutation detection, and genetic mapping.
For the identification of quantitative genetic differences between pathogenic Entamoeba histolytica and the closely related but nonpathogenic species E. dispar, a set of 68 independent probes that had previously been isolated from an E. histolytica cDNA library were hybridized to total genomic DNA of both amoeba species. Besides ehcp5, the sequence that codes for cysteine proteinase 5 and has recently been shown to be missing in E. dispar, only one of the probes exclusively reacted with E. histolytica DNA, whereas the remainder hybridized to DNA of both species. Sequence analysis revealed that the specific probe represents a copy of the multicopy ariel gene family, which has 80% sequence identity to srehp, the gene encoding a serine-rich E. histolytica membrane protein. In contrast to ariel, srehp is present in both amoeba species, suggesting that the ariel gene product might have a particular function in E. histolytica.
Amoebapore, a 77-residue peptide with pore-forming activity from the human pathogen Entamoeba histolytica, is implicated in the killing of phagocytosed bacteria and in the cytolytic reaction of the amoeba against host cells. Previously, we structurally and functionally characterized three amoebapore isoforms in E. histolytica but recognized only one homolog in the closely related but non-pathogenic species Entamoeba dispar. Here, we identified two novel amoebapore homologs from E. dispar by molecular cloning. Despite strong resemblance of the primary structures of the homologs, molecular modeling predicts a species-specific variance between the peptide structures. Parallel isolation from trophozoite extracts of the two species revealed a lower amount of pore-forming peptides in E. dispar and substantially higher activity of the major isoform from E. histolytica towards natural membranes than that from E. dispar. Differences in abundance and activity of the lytic polypeptides may have an impact on the pathogenicity of amoebae.
Upon analysis of 304 expressed sequence tags derived from the protozoan parasite Entamoeba histolytica, a number of novel protein encoding amoeba sequences were isolated. In addition, two unrelated, abundantly expressed transcripts were identified, and designated, ehapt1 and ehapt2. Although these transcripts do not contain any overt open reading frame, both are polyadenylated and together represent about 19% of total polyA+-RNA(11.6% for ehapt1 and 7.5% for ehapt2), thus being the most highly expressed polyA-containing transcripts so far identified in E. histolytica trophozoites. Northern blot and primer extension analyses revealed single-sized transcripts of 0.5 and 0.6 kb for ehapt1 and ehapt2, respectively, and Southern blot analysis suggests that both are encoded by multiple genes, which are distributed throughout the amoeba genome. Comparison between various ehapt1- and ehapt2-derived cDNAs indicated that both transcripts are highly polymorphic. Whereas nucleotide substitutions in ehapt2 are distributed throughout the sequence, variations in ehapt1 are mainly restricted to two regions, one of which comprises a deletion of variable length within an 8 nt tandem repeat unit. At present there is no convincing explanation for the possible role of ehapt1 and ehapt2 in E. histolytica, and analogous sequences have not been described so far for any other organism. Most likely they might represent regulatory RNAs or transcribed transposable elements.
To study the role of cysteine proteinases in the pathogenicity of Entamoeba histolytica, we have attempted to overexpress the three main cysteine proteinases (EhCP1, EhCP2, EhCP5) of this parasite in trophozoites of E. histolytica as well as in non-pathogenic Entamoeba dispar by episomal transfection. Although each of the corresponding coding sequences were cloned in identical expression plasmids, we were unable to overexpress EhCP1 and EhCP5, respectively, but could substantially induce expression of EhCP2 in both amoeba species by sevenfold, leading to a threefold increase in total cysteine proteinase activity. Overexpression of EhCP2 did not influence expression of other cysteine proteinases and could be attributed to an increase of a single 35 kDa activity band in substrate gel electrophoresis. In contrast to previous findings, which indicated that amoeba cysteine proteinases are involved in erythrophagocytosis and liver abscess formation, cells overexpressing EhCP2 showed no difference in erythrophagocytosis or liver abscess formation compared with respective controls. However, overexpression of EhCP2 in both amoeba species resulted in a marked increase of in vitro monolayer destruction.
Entamoeba histolytica, a human parasite, crosses the natural barriers of the intestine and, in turn, spreads into the deeper organs, resulting in amoebiasis. The motility of the parasite and its ability to lyse or phagocytose human cells facilitates passage of the amoeba through the intestinal epithelium. Little is known about the uptake of material by this parasite; nevertheless, the cytoskeleton is believed to play a role in phagocytosis. Myosin IB, an actin-binding protein, localizes to the phagocytic cup and, with time, surrounds the internalized phagosome itself. The role of unconventional myosins in phagocytosis has also been demonstrated in other cell types, suggesting that this molecular mechanism is a common denominator in phagocytic events. Here, we summarize the emerging view of the role of unconventional myosins as well as other cytoskeleton-associated proteins in pseudopod formation at early stages of phagocytosis and during the late step of this process in E. histolytica.
The majority of human infections with the intestinal protozoan parasite Entamoeba histolytica remain asymptomatic. In a small proportion of infections, however, E. histolytica trophozoites penetrate the intestinal mucosa and disseminate to other organs, most commonly to the liver, where they induce abscess formation. It is believed that the ability of E. histolytica trophozoites to destroy host tissues and to survive within the liver is accomplished by a strong adaptive response, which requires the specific regulation of a number of amoeba proteins. Using differential display polymerase chain reaction (DD-PCR), we compared RNA expression between E. histolytica trophozoites isolated from liver abscesses of infected gerbils and those grown under normal culture conditions. A total of 3000 cDNA-derived amplicons were compared between the two groups of amoebae, which were calculated to represent about one-third of all E. histolytica mRNA species (transcriptome). Among these, 55 were found to be specifically present or absent in abscess-derived amoebae, of which 42 were successfully cloned and sequenced. Database searches and Northern blot analyses revealed that the 42 amplicons correspond to 29 independent E. histolytica genes, of which at least seven are specifically upregulated and five are downregulated in abscess-derived amoebae. Specific expression of most of these genes was not simply the result of a heat shock response, which might be expected during abscess formation, as only five of the genes revealed an expression profile similar to that found in amoebae cultured under elevated temperatures. The two genes specifically downregulated in abscess-derived amoebae encode members of a family of so far unknown proteins, which contain repetitive stretches of sequences that are rich in lysine and glutamic acid residues. In contrast, a diverse set of genes is specifically upregulated, encoding ribosomal proteins (S30, L37A), cyclophilin, ferredoxin 2 and GTP-binding protein RAB7D, supporting the notion that liver abscess formation requires the regulation and concerted action of a variety of amoeba proteins. These proteins are associated with stress response, signal transduction, regulation of transcription and vesicular trafficking. However, transcriptome analysis will not be sufficient to identify all proteins specifically upregulated during abscess formation, as at least an increase in the expression of actin was found to be regulated at the post-transcriptional level.
A microarray comprising 21,024 different PCR products spotted on glass slides was constructed for gene expression studies on Trypanosoma brucei. The arrayed fragments were generated from a T. brucei shotgun clone library, which had been prepared from randomly sheared and size-fractionated genomic DNA. For the identification of stage-specific gene activity, total RNA from in vitro cultures of the human, long slender form and the insect, procyclic form of the parasite was labelled and hybridised to the microarray. Approximately 75% of the genomic fragments produced a signal and about 2% exhibited significant differences between the transcript levels in the bloodstream and procyclic forms. A few results were confirmed by Northern blot analysis or reverse-transcription and PCR. Three hundred differentially regulated clones have been selected for sequencing. So far, of 33 clones that showed about 2-fold or more over-expression in bloodstream forms, 15 contained sequences similar to those of VSG expression sites and at least six others appeared non-protein-coding. Of 29 procyclic-specific clones, at least eight appeared not to be protein-coding. A surprisingly large proportion of known regulated genes was already identified in this small sample, and some new ones were found, illustrating the utility of genomic arrays.