Clinical studies in Bangladesh have showed that certain parasite genotypes are more likely to cause disease [2
]. The large number of genotypes identified demonstrates a high level of genetic diversity, and some genotypes are associated with increased virulence in humans, as well as a propensity to invade the liver [10
]. Studies comparing the transcriptomes of different genotypes are limited at present. The Rahman strain (isolated from an asymptomatic carrier) exhibits a reduced virulence phenotype in multiple in vitro
assays of cytotoxicity, as well as a decreased ability to form lesions in the human colonic xenograft model of amebiasis, compared with HM1:IMSS (isolated from a patient with active amebic dysentery) [11
]. A potential drawback to this comparison is the early derivation of the HMI:IMSS and Rahman axenized strains (1979 and 1980, respectively) and the different geographical location of the infected hosts (Mexico or England, respectively, with unknown etiological origin); therefore, differences might not only be related to disease outcome but also reflect parasite speciation. Nevertheless, the transcriptome profiles of the HM1:IMSS and Rahman strain [6
] revealed some interesting differences between the two isolates, most notably changes in genes involved in oxygen defense and protein degradation (summarized in ).
Figure 2 Comparison of mRNA-expression data from two Entamoeba histolytica isolates. The Rahman strain was isolated from an individual who was colonized and HM1:IMSS was cultured from the rectal biopsy of a patient with amebic colitis. Differences in transcripts (more ...)
Peroxiredoxin was one of the enzymes involved in oxygen metabolism that was expressed at higher levels in HM1:IMSS [6
]. When this transcript was artificially increased in the Rahman strain, the transfected trophozoites had both greater resistance to killing by H2
and an increased pro-inflammatory phenotype in the human colonic xenograft model of amebiasis [11
]. This protein is recruited to the host–parasite interface by the galactose- and N-acetyl-galactosamine-inhibitable (Gal/GalNAc) lectin. Gal/GalNAc lectin mediates amebic adherence to and contact-dependent killing of host cells. Therefore, the recruitment of peroxiredoxin could well protect the trophozoites against the reactive oxygen intermediates (ROS) generated by the host [14
The later spatial regulation could be important in the normal role of peroxiredoxin in response to oxidative stress in particular strains or conditions. In contrast to the earlier work [15
], Vicente et al.
] report that exposure of either HM1:IMSS or Rahman to H2
did not cause a change in peroxiredoxin mRNA levels or upregulate transcripts encoding previously characterized enzymes involved in oxygen detoxification.
This work highlights the need to minimize confounding variables when comparing the results from different laboratories. Preparation of culture media in different laboratories might be important when studying oxidative stress because the cysteine included in the E. histolytica culture media acts as a reducing agent and has an essential role in its oxygen tolerance during in vitro culture.
Vicente et al.
] hypothesize that in their in vitro
growth conditions, the conventional pathways involved in oxygen defense might be expressed at the maximum rate and that the upregulation of a new pathway or pathways occurs during the E. histolytica
response to stress. Although there is little overlap of mRNAs modulated by heat stress, as described by Weber et al.
], this hypothesis is supported by the substantial overlap between the oxygen- and nitric-oxide-stress-responsive transcripts and mRNAs reported by Hackney et al.
] to be modulated by heat stress.
The annotated transcripts identified by Vicente et al.
] were the minority of the modulated transcripts but indicate an important role in cell signaling in response to stress (e.g. Rabl1 and Ram M1 were upregulated and RhoGEF and ArfGAP were downregulated) and the repair or metabolic pathways (e.g. deoxyuridine 5′-triphosphate nucleotidohydrolase was upregulated). The majority of the stress-modulated transcripts, however, encoded proteins of unknown function [18
Comparison of transcripts regulated in response to ROS in Rahman and HM1:IMSS indicated that there was a decrease in both number and amplitude of change occurring in Rahman. Although this correlated with the increased sensitivity of Rahman to H2O2, this difference could also reflect the variation in growth and culture conditions, as discussed above.
Another transcript highly expressed in the HM1:IMSS microarray data, EhSTRIP1 (one of a family of E. histolytica
serine-, threonine- and isoleucine-rich proteins), was apparently absent in Rahman [7
]. Analyzing the functional role of the EhSTIRP gene family and its potential role in virulence was accomplished by decreasing the expression of the EhSTIRP family, including EhSTRIP1, in HM1:IMSS by using RNA interference. As expected, this led to a decrease in two in vitro
assays of ameba virulence, adherence and cytolysis of Chinese hamster ovary cells, supporting the hypothesis that EhSTRIP1 has a role in amebic virulence [19
Surprisingly, one confounding aspect is the variation in the results of two independent microarray comparisons of Rahman and HM1:IMSS, performed by two different laboratories. Although both groups used a two-color spotted microarray platform, the first group arrayed 2110 genomic amplicons and the second group used 70-base oligonucleotides to generate an array representing 6242 genes, based on the then-current genome assembly [20
]. Different techniques of normalization (median adjustment to give a net change between arrays of zero or Loess the local estimation of weighted moving averages) [21
] and differences in programs used to identify significantly modulated transcripts (Student’s t
test p-value of <0.01 versus the Significance Analysis of Microarrays) [22
] might explain some of the apparent differences. A cross-platform comparison using a standardized battery of bioinformatic and statistical analyses would be useful to assess the impact of array design on array sensitivity and specificity [23
Verification of some of the observed changes was performed by northern blot [7
] and reverse transcription quantitative PCR [6
]. Therefore, at least some of the differences observed between the two studies could be due to biological differences in the laboratory cultures of Rahman and HM1:IMSS strain. More work is required to resolve these differences, as well as to compare different genotypes of varying virulence from the same geographic location.