Project description:Entamoeba histolytica is a protozoan parasite that causes colitis and liver abscesses. Several Entamoeba species and strains with differing levels of virulence have been identified. E. histolytica HM-1:IMSS is a virulent strain, E. histolytica Rahman is a nonvirulent strain, and Entamoeba dispar is a nonvirulent species. We used an E. histolytica DNA microarray consisting of 2,110 genes to assess the transcriptional differences between these species/strains with the goal of identifying genes whose expression correlated with a virulence phenotype. We found 415 genes expressed at lower levels in E. dispar and 32 genes with lower expression in E. histolytica Rahman than in E. histolytica HM-1:IMSS. Overall, 29 genes had decreased expression in both the nonvirulent species/strains than the virulent E. histolytica HM-1:IMSS. Interestingly, a number of genes with potential roles in stress response and virulence had decreased expression in either one or both nonvirulent Entamoeba species/strains. These included genes encoding Fe hydrogenase (9.m00419), peroxiredoxin (176.m00112), type A flavoprotein (6.m00467), lysozyme (6.m00454), sphingomyelinase C (29.m00231), and a hypothetical protein with homology to both a Plasmodium sporozoite threonine-asparagine-rich protein (STARP) and a streptococcal hemagglutinin (238.m00054). The function of these genes in Entamoeba and their specific roles in parasite virulence need to be determined. We also found that a number of the non-long-terminal-repeat retrotransposons (EhLINEs and EhSINEs), which have been shown to modulate gene expression and genomic evolution, had lower expression in the nonvirulent species/strains than in E. histolytica HM-1:IMSS. Our results, identifying expression profiles and patterns indicative of a virulence phenotype, may be useful in characterizing the transcriptional framework of virulence.
Project description:The ability of Entamoeba histolytica to phagocytose host cells correlates to observed virulence in vivo. To better understand the mechanism of phagocytosis we used paramagnetic beads coated with host ligand and sorted trophozoites based on phagocytic ability. Gene expression was then measured in both the sorted phagocytic and non-phagocytic populations using a custom Affymetrix chip for E. histolytica. Feed forward regulation of phagocytosis by Entamoeba histolytica. Infection and Immunity. PMID 23045476
Project description:Up until now, the existence of Dnmt2-mediated DNA methylation has mostly been supported by focal analyses in organisms that contain Dnmt2, but no Dnmt1 or Dnmt3 DNA methyltransferase. In these organisms, several independent studies have also provided support for a biologically important function of Dnmt2-dependent DNA methylation. For example, Dnmt2-dependent methylation in Entamoeba histolytica, the causative agent of amebic dysentery, has been connected to the parasite s virulence. However, global DNA methylation levels in Entamoeba have been found to be very low. In addition, no specific features, such as CpG-specificity and specificity for certain genetic subcompartments have been described. This distinguishes Dnmt2-dependent methylation patterns from all other known methylomes and has raised questions about the validity of the underlying results. We have used whole-genome bisulfite sequencing for an unbiased characterization of the Entamoeba histolytica methylome at single-base resolution in a E.histolytica strain HM-1:IMSS devoid of significant level of EhDnmt2 (Ehmeth) expression.
Project description:To know the role of Rab7 isotypes in enteric protozoan parasite Entamoeba histolytica, one out of nine Rab7 isotypes, Rab7D, was specifically silenced and examined the effect on virulence-related phenotypes. To clarify transcriptomic difference caused by rab7d gene silencing, RNA-seq analysis was conducted.
Project description:The ability of Entamoeba histolytica to phagocytose host cells correlates to observed virulence in vivo. To better understand the mechanism of phagocytosis we used paramagnetic beads coated with host ligand and sorted trophozoites based on phagocytic ability. Gene expression was then measured in both the sorted phagocytic and non-phagocytic populations using a custom Affymetrix chip for E. histolytica. Feed forward regulation of phagocytosis by Entamoeba histolytica. Infection and Immunity. PMID 23045476 M280 Streptavidin Dynabeads (Invitrogen) were labeled with 20ug/mL biotinylated Human C1q (Quidel). Entamoeba histolytica (strain HM1) were washed twice with PBS then resuspended with the labeled beads at a 10:1 ratio of beads to trophozoites. Samples were incubated at 37°C for 45 minutes, washed twice with agitation to remove adherent beads, then resuspended in MACS buffer. Samples were loaded into magnetic columns (Miltenyi Biotec) and trophozoites were seperated according to manufacturer's protocols. phagocytic vs. non-phagocytic Entamoeba histolytica populations
Project description:To know the role of lysosomal hydrolase receptors in virulecne of enteric protozoan parasite Entamoeba histolytica, eleven cysteine protease binding protein family (CPBF) proteins were specifically silenced and examined the effect on virulence-related phenotypes. Among them, CPBF2 gene silence caused defect in Matrigel invasion. To clarify transcriptomic difference in CPBF2 gene silenceing strain, RNA-seq analysis was conducted.
Project description:Amoebiasis is a disease caused by the protozoan parasite, Entamoeba histolytica, with or without clinical symptoms . It has been suggested that pathogenic amoebae use three major virulence factors: Gal/GalNAc lectin, cysteine proteinases and amoebapores. However, these molecules cannot exclusively be responsible for amoebic virulence, because most of them are found in pathogenic E. histolytica as well as in non-pathogenic E. dispar, a not close correlation has been found in amoebic strains with different virulence phenotype. Due to the high genetic variability of E. histolytica isolates and strains, differential gene expression might simply reflect inter-isolate genetic variation rather than specific differences linked to virulence. In the current study, we obtained a non-virulent UG10 mutant derived from E. histolytica HM-1:IMSS. Comparing the transcriptome of both strains, no differences in gene expression of the classical virulence factors were observed. RAB family GTPase and AIG1 family members showed higher transcriptional levels in the virulent strain than in the non-virulent mutant. Our study suggests a number of novel gene products in E. histolytica with a role yet to determine in cell physiology and involvement in the pathogenesis process.
Project description:Differential expression was used to access gene differences after Entamoeba histolytica infection. Entamoeba histolytica is an important diarrheal pathogen worldwide, and induces apoptosis of the intestinal epithelium as part of its disease process. Regenerating (REG) 1 protein is anti-apoptotic. We investigated the involvement of REG 1 in E. histolytica colitis. Colonic biopsy samples were obtained from 8 subjects with acute E. histolytica colitis, and again 60 day later during convalescence. Gene expression in the human colon during acute and convalescent E. histolytica disease was evaluated using microarray and confirmed by polymerase chain reaction (PCR). REG 1 protein expression was evaluated with immunohistochemistry. The mechanism of REG 1 involvement in E. histolytica disease was subsequently investigated with a mouse model. REG 1A and REG 1B were the most upregulated genes in the human intestine in acute versus convalescent E. histolytica disease (p=0.003 and p=0.006 respectively). PCR confirmed the microarray results (p=<0.001 and p=0.001 respectively). Increased REG 1A and REG 1B protein expression was similarly observed by immunohistochemistry. REG 1 -/-mice were found to be significantly more susceptible to E. histolytica infection than wild type mice.