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Stress-dependent expression of a polymorphic, charged antigen in the protozoan parasite Entamoeba histolytica.

ABSTRACT: We have identified a novel stress inducible gene, Ehssp1 in Entamoeba histolytica, the causative agent of amebiasis. Ehssp1 belongs to a polymorphic, multigene family and is present on multiple chromosomes. No homologue of this gene was found in the NCBI database. Sequence alignment of the multiple copies, and genomic PCR data restricted the polymorphism to the central region of the gene. This region contains a polypurine stretch that encodes a domain rich in acidic and basic amino acids. Under normal culture conditions only one copy of this multigene family is expressed, as observed by Northern blot and RT-PCR analysis. The size of this copy of the gene is 1,077 nucleotides, encoding a protein of 359 amino acids. The polymorphic domain in this copy is 64 nucleotides long. However, on exposure of cells to stress conditions such as heat shock or oxidative stress, multiple polymorphic copies of the gene are expressed, suggesting a possible role of this gene in adaptation of cells to stress conditions. The gene copy expressed under normal conditions, and the expression profile of cells under heat stress was identical in two different strains of E. histolytica tested. Interestingly, the extent of polymorphism in this gene was very less in E. dispar, a nonpathogenic sibling species of E. histolytica. Ehssp1 was found to be antigenic in invasive amebiasis patients.

SUBMITTER: Satish S

PROVIDER: S-EPMC166017 | biostudies-literature | 2003 Aug

REPOSITORIES: biostudies-literature

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Stress-dependent expression of a polymorphic, charged antigen in the protozoan parasite Entamoeba histolytica.

Satish S S Bakre Abhijeet A AA Bhattacharya Sudha S Bhattacharya Alok A

Infection and immunity 20030801 8

We have identified a novel stress inducible gene, Ehssp1 in Entamoeba histolytica, the causative agent of amebiasis. Ehssp1 belongs to a polymorphic, multigene family and is present on multiple chromosomes. No homologue of this gene was found in the NCBI database. Sequence alignment of the multiple copies, and genomic PCR data restricted the polymorphism to the central region of the gene. This region contains a polypurine stretch that encodes a domain rich in acidic and basic amino acids. Under ...[more]

PMID: 12874327

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Project description:Histone modification regulates chromatin structure and influences gene expression associated with diverse biological functions including cellular differentiation, cancer, maintenance of genome architecture, and pathogen virulence. In Entamoeba, a deep-branching eukaryote, short chain fatty acids (SCFA) affect histone acetylation and parasite development. Additionally, a number of active histone modifying enzymes have been identified in the parasite genome. However, the overall extent of gene regulation tied to histone acetylation is not known.In order to identify the genome-wide effects of histone acetylation in regulating E. histolytica gene expression, we used whole-genome expression profiling of parasites treated with SCFA and Trichostatin A (TSA). Despite significant changes in histone acetylation patterns, exposure of parasites to SCFA resulted in minimal transcriptional changes (11 out of 9,435 genes transcriptionally regulated). In contrast, exposure to TSA, a more specific inhibitor of histone deacetylases, significantly affected transcription of 163 genes (122 genes upregulated and 41 genes downregulated). Genes modulated by TSA were not regulated by treatment with 5-Azacytidine, an inhibitor of DNA-methyltransferase, indicating that in E. histolytica the crosstalk between DNA methylation and histone modification is not substantial. However, the set of genes regulated by TSA overlapped substantially with genes regulated during parasite development: 73/122 genes upregulated by TSA exposure were upregulated in E. histolytica cysts (p-value = 6 x 10(-53)) and 15/41 genes downregulated by TSA exposure were downregulated in E. histolytica cysts (p-value = 3 x 10(-7)).This work represents the first genome-wide analysis of histone acetylation and its effects on gene expression in E. histolytica. The data indicate that SCFAs, despite their ability to influence histone acetylation, have minimal effects on gene transcription in cultured parasites. In contrast, the effect of TSA on E. histolytica gene expression is more substantial and includes genes involved in the encystation pathway. These observations will allow further dissection of the effects of histone acetylation and the genetic pathways regulating stage conversion in this pathogenic parasite.

Project description:The protozoan parasite, Entamoeba histolytica, invades the host colon causing significant tissue destruction and inflammation. Upon host infection, the parasite is confronted with reactive oxygen and nitrogen species (ROS/RNS) that cause large-scale changes in gene expression profiles, which likely support the parasite's adaptation to the host environment. We have previously identified oxidative and nitrosative stress responsive genes using whole-genome expression profiling. Functional studies on two such genes are now reported and demonstrate that they have roles in parasite virulence. EHI_056680 encodes a small hypothetical protein named E. histolytica stress-induced adhesion factor (EhSIAF); EHI_188210 encodes a putative phospholipid transporting P-type ATPase/flippase (EhPTPA). Over-expression of each protein in E. histolytica trophozoites enhanced parasite survival in response to oxidative stress. Exposure to oxidative and nitrosative stress did not affect the localization of EhSIAF or EhPTPA but markedly increased EhPTPA protein levels. Interestingly, over-expression of each gene resulted in parasites with increased adherence to healthy mammalian cells, but increased adherence to apoptotic cells was noted only in EhSIAF over-expressing parasites. However, despite having increased adherence to both healthy and apoptotic host cells, EhSIAF-over-expressing parasites were reduced in their ability to destroy mammalian cell monolayers, raising the intriguing possibility that EhSIAF over-expression caused signaling defects or resulted in a dominant negative phenotype. Over-expression of EhSIAF and EhPTPA also resulted in decreased motility in a transwell motility assay. Thus, we have confirmed that two genes that are upregulated by ROS confer increased resistance to oxidative stress and have identified an unexpected role of EhSIAF and EhPTPA in host cell adherence and a role of EhSIAF in parasite virulence. Our data imply that stress response genes may play multi-factorial roles in amoebic pathogenesis.

Project description:BACKGROUND: Entamoeba histolytica, an enteric protozoan parasite, causes amebic colitis and extra intestinal abscesses in millions of inhabitants of endemic areas. E. histolytica completely lacks glutathione metabolism but possesses L-cysteine as the principle low molecular weight thiol. L-Cysteine is essential for the structure, stability, and various protein functions, including catalysis, electron transfer, redox regulation, nitrogen fixation, and sensing for regulatory processes. Recently, we demonstrated that in E. histolytica, L-cysteine regulates various metabolic pathways including energy, amino acid, and phospholipid metabolism. RESULTS: In this study, employing custom-made Affymetrix microarrays, we performed time course (3, 6, 12, 24, and 48 h) gene expression analysis upon L-cysteine deprivation. We identified that out of 9,327 genes represented on the array, 290 genes encoding proteins with functions in metabolism, signalling, DNA/RNA regulation, electron transport, stress response, membrane transport, vesicular trafficking/secretion, and cytoskeleton were differentially expressed (?3 fold) at one or more time points upon L-cysteine deprivation. Approximately 60% of these modulated genes encoded proteins of no known function and annotated as hypothetical proteins. We also attempted further functional analysis of some of the most highly modulated genes by L-cysteine depletion. CONCLUSIONS: To our surprise, L-cysteine depletion caused only limited changes in the expression of genes involved in sulfur-containing amino acid metabolism and oxidative stress defense. In contrast, we observed significant changes in the expression of several genes encoding iron sulfur flavoproteins, a major facilitator super-family transporter, regulator of nonsense transcripts, NADPH-dependent oxido-reductase, short chain dehydrogenase, acetyltransferases, and various other genes involved in diverse cellular functions. This study represents the first genome-wide analysis of transcriptional changes induced by L-cysteine deprivation in protozoan parasites, and in eukaryotic organisms where L-cysteine represents the major intracellular thiol.

Dataset Information

Stress-dependent expression of a polymorphic, charged antigen in the protozoan parasite Entamoeba histolytica.

Publications

Stress-dependent expression of a polymorphic, charged antigen in the protozoan parasite Entamoeba histolytica.

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