In order to identify transcripts that are involved in or even responsible for pathogenicity, we compared two highly related E. histolytica isolates that differ substantially in their pathogenicity. The E. histolytica HM-1:IMSS cell line B is highly pathogenic and produces large liver abscesses in an animal model, whereas HM-1:IMSS cell line A appears to have lost its ability to induce abscess formation .
The microarray applied in this study had already been used to identify the differences between the non-pathogenic isolate Rahman and the pathogenic isolate HM-1:IMSS . Using this array, which covers 75% of E. histolytica genes, 1.4% of the analyzed genes showed a two-fold or greater difference in expression between cell line A and cell line B. Only 0.3% of the genes showed a five-fold and higher differential transcription.
Astonishingly, there is only a small overlap between our transcriptomic and proteomic studies that compare the two cell lines. Only two genes were fond to be differentially expressed both on a transcript and protein level; Fe-hydrogenase 2 [GenBank:XM_647747] at higher abundance in cell line B and one C2 domain protein [GenBank:XM_650207] at a higher abundance in cell line A . A similar phenomenon was also observed when comparing the transcriptomes and proteomes of Rahman and HM-1:IMSS with each other [2, 3]. Davis and colleagues identified only one molecule (alcohol dehydrogenase 3, [GenBank:XM_650038]), where the expression profile was comparable on protein and RNA levels. This discrepancy between regulation at the transcript- and proteome level appears therefore to be a general characteristic of Entamoeba, indicating that this primordial eukaryote has a more complex way of expression regulation.
The strategy of identifying pathogenicity genes in E. histolytica by comparing pathogenic and non-pathogenic strains has already been used applied by other groups. They compared the pathogenic isolate HM-1:IMSS and the non-pathogenic isolate Rahman using microarray techniques [3–5]. A direct comparison of our results with all three microarray studies exhibits only poor overlaps. Of the 152 transcripts that were found in higher levels in the pathogenic isolate HM-1:IMSS in comparison to Rahman in microarray study performed by Davis and colleagues, only five are expressed in higher levels in the pathogenic cell line B (EhCP-A4 [GenBank:XM_651510], AIG family proteins [GenBank:XM_648115, XM_643009, homolog to HSP70 [GenBank:XM_648787], hypothetical protein [GenBank:XM_648447]). We identified only one gene expressed at higher rates in the non-pathogenic cell line A (hypothetical protein [GenBank:XM_644469]) among the 201 genes that are expressed in higher levels in Rahman compared to HM-1:IMSS .
Only 22 of the genes found to be differentially expressed between cell line A and cell line B were also found to be regulated in the study of Ehrenkaufer and colleagues and only eleven of them are regulated in both studies in the same direction . These molecules include one of the two identified C2 domain proteins [GenBank:XM_650951], cell surface gp63 [GenBank:XM_647540], AIG family protein [GenBank:XM_648725] and eight hypothetical proteins [GenBank:XM_645291, XM_649962, XM_644469, XM_646066, XM_647129, XM_651187, XM_645139, XM_649961].
The low level of consensus between our study comparing two cell lines with the same genetic background and the remaining studies comparing two isolates with a different genetic backgrounds, leads to the assumption that the mechanism that determine the loss of virulence in Rahman differs from that observed for HM-1:IMSS cell line A.
Interestingly, only 19 of the investigated genes (0.3% of the predicted transcriptome) show a differential expression higher than five-fold between non-pathogenic cell line A and pathogenic cell line B. It can be assumed that genes where the level of respective transcripts differs to such an extent are involved in virulence. One of these molecules is a cell surface protease gp63 (EhMP8-2), which is transcribed at a more than 20-fold higher level in cell line A than in cell line B. In contrast to ehmp8-2, ehmp8-1 transcripts are found in similar abundance in both cell lines. In E. histolytica, neither of the gp63 proteases (EhMP8-1 and EhMP8-2) have been characterized. In Leishmania, the homolog, named leishmanolysin, occurs predominantly as a heavily-glycosylated protein that is attached to the outer membrane of Leishmaniapromastigates by a glycosylphosphatidylinositol anchor. It has been demonstrated that leishmanolysin plays a role in resistance of promastigotes to complement-mediated lysis and in receptor-mediated uptake of the parasite by phagocytic host cells . It appears that most eukaryotes have homologs of this protein [11, 12].
Transcripts of three genes coding for members of the Rab7 GTPase family (EhRab7D, 7E, 7G) were detected at much higher levels in the non-pathogenic cell line A. Rab proteins are essential for the regulation of vesicular trafficking in the endocytic and exocytic/secretory pathways of eukaryotic cells . E. histolytica possesses more than 90 rab genes and therefore seems to be an organism with extremely diverse and complex Rab functions . Rab7 in particular, has been described as one of the most important molecules involved in lysosomal biogenesis . In different organisms it plays divergent roles in several distinct steps of endosomal or lysosomal trafficking . E. histolytica encodes nine EhRab7 isotypes (EhRab7A-I), which show 40-65% identity to each other. It was shown that EhRab7A is associated with the post-Golgi compartment and is involved in the fusion of late endosomes. EhRab7B is localized to late endosomes/lysosomes and associated with the formation of lysosomes or the fusion to lysosomes. There is further evidence that all the EhRab7 isotypes are sequentially and coordinately involved in phagosome biogenesis . In addition, we found that Vps35, is also transcribed at higher levels in cell line A (2.6-fold). Together with Vps26 and Vps29, it forms the amoebic retromer-like complex and functions as a EhRab7A-binding protein. This retromer-like complex is linked to the retrograde transport of putative hydrolase receptors from preparatory vacuoles and phagosomes to the Golgi apparatus. Nakada-Tsukui and colleagues showed that overexpression of EhRab7A caused enlargement of lysosomes and a decrease in cellular cysteine peptidase activity. The reduced cysteine peptidase activity was restored by co-expression of EhVps26. Thus, the EhRab7A-mediated transport of cysteine peptidases to phagosomes seems to be regulated by the retromer-like complex . As mentioned above, phenotypic characterization of cell line A and cell line B showed a reduced cysteine peptidase activity in cell line A due to a reduced amount of mature EhCP-A1 and -A2 . Although, it has been described that the interaction of EhRab7A with the retromer-like complex is specific as no association was observed with other isotypes such as EhRab7B or EhRab7D, an altered expression of molecules involved in vesicular trafficking may be responsible for the observed differences in cysteine peptidase activity .
We found two different transcripts of C2 domain-containing proteins in higher levels in the non-pathogenic cell line A. The C2 domain is a Ca2+-dependent membrane-targeting module found in proteins involved in membrane trafficking. Both molecules contain one N-terminal C2 domain and the C-terminal domains show high similarity to the P30 adhesin protein of Mycoplasma pneumoniae and to a hypothetical protein of Paramecium tetraurelia [GenBank:XP_001426443], respectively. P30 is a membrane-bound protein that is oriented with its N-terminus in the cytoplasm and its C-terminus on the cell surface, and is required for cytoadherence. The protein has three types of proline-containing repeats at its carboxy end [19–21]. Similar repeats were also found in the C2 domain-containing protein of E. histolytica [GenBank:XP_655299], giving rise to an overall 56% sequence identity. Nevertheless, the amoebic molecule seems not to be membrane-anchored, since it does not contain a signal sequence or a transmembrane domain. Additionally, the C-terminal region of the second C2-domain containing protein [GenBank:XP_656043] has a repetitive structure consisting of two main stretches.
In contrast to the transcripts described above, most members of the so far uncharacterized family of putative small GTPases, the AIG1 proteins, were found in higher levels in the pathogenic cell line B. At least 47 genes coding for AIG1 molecules are present in the E. histolytica genome. Quantitative real-time PCR analysis indicates that 18 of the 34 investigated aig1 genes are expressed at higher levels in cell line B. So far, the physiological relevance of these molecules is completely unknown in E. histolytica. They share partial homology to the GIMAP/IAN family molecules of vertebrates. Additionally, they have relatives in higher plants but not in most other well studied organisms, including bacteria, nematodes, and the amoeba Dictyostelium discoideum . Nevertheless, aig1 genes are also present in the genome of E. invadens, E. dispar and E. moshkovskii, indicating that this family is conserved within the genus Entamoeba (data not shown). Structurally, all members of the family contain a GTP-binding cassette and several coiled-coil motifs . In the E. histolytica homologs, only a part of the GTP-binding cassette is conserved and coiled-coil motifs are only predicted for some family members. GIMAP proteins are thought to be regulators of cell death in lymphomyeloid cells. It is suggested that the plant homolog AIG1 is involved in cell death regulation following self-defence responses to bacterial infection. Therefore, GIMAP proteins might be involved in self-defence machineries common to vertebrates and plants . There are two further studies that discuss a link between pathogenicity and expression of aig1 genes. Davis and colleagues found aig1 genes expressed at much higher levels in HM-1:IMSS than in Rahman . Additionally, the expression of aig1 genes was highly regulated in HM-1:IMSS trophozoites, obtained from a murine model of amoebic colitis. Trophozoites isolated from the cecal lumen of mice early in infection (day 1) showed either an unregulated or an increased expression of aig1 genes. Late in the infection (day 29), a decrease in the expression was observed . Although this body of evidence points to an important role of aig1 genes for amoebic pathogenicity, their physiological role has to be determined.