Erratum: AIG1 affects in vitro and in vivo virulence in clinical isolates of Entamoeba histolytica.

[This corrects the article DOI: 10.1371/journal.ppat.1006882.].

AIG1 affects in vitro and in vivo virulence in clinical isolates of Entamoeba histolytica.

The disease state of amebiasis, caused by Entamoeba histolytica, varies from asymptomatic to severe manifestations that include dysentery and extraintestinal abscesses. The virulence factors of the pathogen, and host defense mechanisms, contribute to the outcomes of infection; however, the underlying genetic factors, which affect clinical outcomes, remain to be fully elucidated. To identify these genetic factors in E. histolytica, we used Illumina next-generation sequencing to conduct a comparative genomic analysis of two clinical isolates obtained from diarrheal and asymptomatic patients (strains KU50 and KU27, respectively). By mapping KU50 and KU27 reads to the genome of a reference HM-1:IMSS strain, we identified two genes (EHI_089440 and EHI_176590) that were absent in strain KU27. In KU27, a single AIG1 (avrRpt2-induced gene 1) family gene (EHI_176590) was found to be deleted, from a tandem array of three AIG1 genes, by homologous recombination between the two flanking genes. Overexpression of the EHI_176590 gene, in strain HM-1:IMSS cl6, resulted in increased formation of cell-surface protrusions and enhanced adhesion to human erythrocytes. The EHI_176590 gene was detected by PCR in 56% of stool samples from symptomatic patients infected with E. histolytica, but only in 15% of stool samples from asymptomatic individuals. This suggests that the presence of the EHI_176590 gene is correlated with the outcomes of infection. Taken together, these data strongly indicate that the AIG1 family protein plays a pivotal role in E. histolytica virulence via regulation of host cell adhesion. Our in-vivo experiments, using a hamster liver abscess model, showed that overexpression or gene silencing of EHI_176590 reduced and increased liver abscess formation, respectively. This suggests that the AIG1 genes may have contrasting roles in virulence depending on the genetic background of the parasite and host environment.

Metabolic profiling of the protozoan parasite Entamoeba invadens revealed activation of unpredicted pathway during encystation.

Encystation, which is cellular differentiation from the motile, proliferative, labile trophozoite form to the dormant, resistant cyst form, is a crucial process found in parasitic and free-living protozoa such as Entamoeba, Giardia, Acanthamoeba, and Balamuthia. Since encystation is an essential process to deal with the adverse external environmental changes during the life cycle, and often integral to the transmission of the diseases, biochemical understanding of the process potentially provides useful measures against the infections caused by this group of protozoa. In this study, we investigated metabolic and transcriptomic changes that occur during encystation in Entamoeba invadens, the reptilian sibling of mammal-infecting E. histolytica, using capillary electrophoresis-tandem mass spectrometry-based metabolite profiling and DNA microarray-based expression profiling. As the encystation progressed, the levels of majority of metabolites involved in glycolysis and nucleotides drastically decreased, indicating energy generation is ceased. Furthermore, the flux of glycolysis was redirected toward chitin wall biosynthesis. We found remarkable temporal increases in biogenic amines such as isoamylamine, isobutylamine, and cadaverine, during the early period of encystation, when the trophozoites form large multicellular aggregates (precyst). We also found remarkable induction of γ-aminobutyric acid (GABA) during encystation. This study has unveiled for the first time the dynamics of the transcriptional and metabolic regulatory networks during encystation, and should help in better understanding of the process in pathogenic eukaryotes, and further development of measures controlling infections they cause.

Multisite performance evaluation of an enzyme-linked immunosorbent assay for detection of Giardia, Cryptosporidium, and Entamoeba histolytica antigens in human stool.

A novel fecal antigen detection assay for fresh and frozen human samples that detects but does not differentiate Giardia spp, Cryptosporidium spp, and Entamoeba histolytica, the Tri-Combo parasite screen, was compared to three established enzyme-linked immunosorbent assays (ELISAs) at three international sites. It exhibited 97.9% sensitivity and 97.0% specificity, with positive and negative predictive values of 93.4% and 99.1%, respectively. The Tri-Combo test proved a reliable means to limit the use of individual parasite ELISAs to positive samples.

Amebiasis in HIV-1-infected Japanese men: clinical features and response to therapy.

Invasive amebic diseases caused by Entamoeba histolytica are increasing among men who have sex with men and co-infection of ameba and HIV-1 is an emerging problem in developed East Asian countries. To characterize the clinical and epidemiological features of invasive amebiasis in HIV-1 patients, the medical records of 170 co-infected cases were analyzed retrospectively, and E. histolytica genotype was assayed in 14 cases. In this series of HIV-1-infected patients, clinical presentation of invasive amebiasis was similar to that described in the normal host. High fever, leukocytosis and high CRP were associated with extraluminal amebic diseases. Two cases died from amebic colitis (resulting in intestinal perforation in one and gastrointestinal bleeding in one), and three cases died from causes unrelated to amebiasis. Treatment with metronidazole or tinidazole was successful in the other 165 cases. Luminal treatment was provided to 83 patients following metronidazole or tinidazole treatment. However, amebiasis recurred in 6 of these, a frequency similar to that seen in patients who did not receive luminal treatment. Recurrence was more frequent in HCV-antibody positive individuals and those who acquired syphilis during the follow-up period. Various genotypes of E. histolytica were identified in 14 patients but there was no correlation between genotype and clinical features. The outcome of metronidazole and tinidazole treatment of uncomplicated amebiasis was excellent even in HIV-1-infected individuals. Luminal treatment following metronidazole or tinidazole treatment does not reduce recurrence of amebiasis in high risk populations probably due to amebic re-infection.

Identification of an avirulent Entamoeba histolytica strain with unique tRNA-linked short tandem repeat markers.

Highly polymorphic, non-coding short tandem repeats (STR) are scattered between the tRNA genes in Entamoeba histolytica in a unique tandemly arrayed organization. STR markers that correlate with the virulence of individual E. histolytica strains have recently been reported. Here we evaluated the usefulness of tRNA-linked STR loci as genetic markers in identifying virulent and avirulent strains of E. histolytica from 37 Japanese E. histolytica samples (12 diarrheic/dysenteric, 20 amebic liver abscess (ALA), and 5 asymptomatic cases). Twenty three genotypes, assigned by combining the STR sequence types from all 6 STR loci, were identified. One to 8 new STR sequence types per locus were also discovered. Genotypes found in asymptomatic isolates were highly polymorphic (4 out of 5 genotypes were unique to this group), while in symptomatic isolates, almost half of the genotypes were shared between diarrhea/dysentery and ALA. One asymptomatic isolate (KU27) showed unique STR patterns in 4 loci. This strain, though associated with the typical pathogenic zymodeme II, failed to induce amebic liver abscess by animal challenge, which suggests that inherently avirulent E. histolytica strains exist, that are associated with unique genotypes. Furthermore, STR genotyping and in vivo challenge of 2 other asymptomatic isolates (KU14 and KU26) verified the covert virulence of these strains.