Identification of differentially expressed genes in virulent and nonvirulent Entamoeba species: potential implications for amebic pathogenesis

Abstract

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.

FIG. 1.

The hybridization ratios of E. dispar SAW760 and E. histolytica Rahman to E. histolytica HM-1:IMSS are shown for EhActin, CP1, Ariel1, EhSINE1, KERP1, and CP8. Actin has similar expression levels in all Entamoeba species/strains studied. CP1, Ariel1, EhSINE1, and KERP1, all previously shown to be missing at a genomic level in E. dispar SAW760, showed significantly less expression in E. dispar SAW760 than in E. histolytica HM-1:IMSS. CP8, present in E. dispar SAW760 but known to have less expression, shows significantly less hybridization for E. dispar SAW760 than E. histolytica HM-1:IMSS. E. histolytica Rahman had expression levels equivalent to those of E. histolytica HM-1:IMSS for all genes shown. Genes with significantly decreased (P value of <0.05) expression levels compared to those of E. histolytica HM-1:IMSS are denoted with an asterisk. ED, E. dispar; EH, E. histolytica.

FIG. 2.

Northern blot analysis for genes identified as differentially expressed between Entamoeba species. (A) Clone ENTBD72 was used to represent EhSINE2. (B) The genes 5.m00482, 29.m00231, 52.m00161, and 6.m00454 are shown. (C) The genes 6.m00467, 297.m00063, and 9.m00419 are shown. All of the genes in this figure exhibited significantly less expression in E. dispar (ED) SAW760 than in E. histolytica (EH) HM-1:IMSS by microarray analysis. Panels A, B, and C represent different blots. EhActin, which is equally expressed in all species/strains, is shown for each blot as a loading control.

FIG. 3.

Northern blot analysis for genes identified as differentially expressed in both nonvirulent species/strains. (A) The gene 194.m00115 is shown. (B) The genes 238.m00054, 29.m00210, 2.m00567, and EhLINE1, represented by the clone ENTOB31, are shown. All of the genes in this figure exhibited significantly less expression in both E. dispar (ED) SAW760 and E. histolytica (EH) Rahman than in E. histolytica HM-1:IMSS by microarray analysis. Panels A and B represent different blots. EhActin, which is equally expressed in all species/strains, is shown for each blot as a loading control.

FIG. 4.

EhLINEs and EhSINEs have altered expression in the nonvirulent Entamoeba species/strains. (A) Diagrammatic representation of the sequence data for EhLINE1. E. histolytica Rahman data obtained by sequence analysis and E. dispar SAW760 obtained from BLASTN analysis versus genome sequence data. The numbers above the lines represent nucleotide positions, and the reverse transcriptase domain in ORF2 is noted. The shading is indicative of the nucleotide identity (▩, ≥95% identity; ░⃞, >90% nucleotide identity; , >85% nucleotide identity; □, <80% nucleotide identity). No high-homology hit was found for ORF1 in E. dispar SAW760; however, a 1,600-bp region encompassing the reverse transcriptase domain showed 85% identity in E. dispar SAW760. (B) The average expression levels of EhLINE and EhSINE in E. histolytica Rahman and E. dispar SAW760 relative to those in E. histolytica HM-1:IMSS are shown. For each element, 50 clones with the highest similarity to the consensus sequence for each EhLINE or EhSINE were used. The copy number (adapted from Bakre et al. [5]), median BLASTN E-value for the 50 clones with highest homology, and genomic sequence similarity are also displayed. Expression levels that are significantly different from that of E. histolytica HM-1:IMSS are labeled by an asterisk and denote a P value of <0.05.

FIG. 5.

Diagrammatic representation of the sequence data for the genes with differential expression levels in the nonvirulent species/strains. The shading is indicative of the nucleotide identity (▩, ≥95% identity; ░⃞, >90% nucleotide identity; , >85% nucleotide identity; , >80% nucleotide identity; □, <80% nucleotide identity). ×, stop codons; ▵, mutated stop or start codons.