The Entamoeba histolytica genome: primary structure and expression of proteolytic enzymes

The faecal-oral spread protozoan parasite Entamoeba histolytica is an important human pathogen. Normally, this parasite resides and multiplies in the large bowel and can persist there for months and years causing only an asymptomatic luminal gut infection. However, occasionally E. histolytica penetrates the intestinal mucosa, which leads to ulcerative colitis or it disseminates to other organs, most commonly to the liver, where it induces abscess formation. Cysteine peptidases are considered to play a major role for the pathogenicity of E. histolytica as suggested by a large number of in vitro and in vivo studies [1–9]. Most convincing are results from infections of laboratory animals indicating that E. histolytica trophozoites that have reduced cysteine peptidase activity are greatly impaired in their ability to induce amoebic liver abscesses [8, 9]. In addition, overexpression of cysteine peptidases led to an increase in cytopathic activity, measured by in vitro monolayer disruption, as well as to a significant increase in amoebic liver abscess formation in laboratory animals in comparison to matching controls [10]. Furthermore, the discovery that amoeba cysteine peptidases possess interleukin-1β converting enzyme activity suggests a novel mechanism of these enzymes in amoebic virulence [11].

Homology searches based on the conservation of active site regions revealed that the E. histolytica genome contains a multitude of at least 50 genes coding for cysteine peptidases (reviewed by Clark et al [12]). Of these, the majority is structurally related to the C1 papain superfamily, whereas a few others are more similar to family C2 (calpain-like cysteine proteinases), C19 (ubiquitinyl hydrolase), C48 (Ulp1 peptidase), C54 (autophagin), and C65 (otubain), respectively [12].

Phylogenetic analyses of the 37 C1-family members revealed that they represent 3 distinct clades (A, B, C), each consisting of 13, 11 and 13 members, respectively [12]. EhCP-A and EhCP-B family members are organised as classical pre-pro enzymes with an overall cathepsin L-like structure. They differ in length of the pro regions as well as of the catalytic domains and have specific sequence motifs within the N-terminal regions of the mature enzymes. In addition, most members of the EhCP-B contain hydrophobic stretches near or at the C-terminus [12, 13]. The primary structure prediction of the 13 EhCP-C members indicated a hydrophobic region located 11 to 28 amino acid residues apart from the N-terminus, which is predicted to form a signal anchor. As there is no example of a structural related cysteine peptidase corresponding to the EhCP-C subfamily, any function of this group of molecules remains to be determined.

In addition, two genes encoding for putative cysteine peptidases of the family C2 (calpain-like proteases) were identified within the genome (EhCALP1 and EhCALP2). These molecules are involved in several cellular processes including signal transduction pathways, remodelling of the cytoskeleton and membranes and apoptosis [14].

Another 4 genes were identified coding for enzymes with homology to the peptidase family C54 also termed autophagins (EhAUTO1-4). The process of autophagy has initially been described in other eukaryotic cells as a rescue mechanism that is induced upon starvation or oxidative stress. It is a process by which cells digest parts of their own cytosolic material. This allows the recycling of molecules under conditions of nutritional limitation and remodelling of intracellular structure for cell differentiation [15–17].

Four other genes putatively encoding cysteine peptidases in E. histolytica show homology to members of the C19 and C65 families. These two groups of enzymes are known to be involved in ubiquitin degradation. In addition 3 genes with homology to Ulp1 peptidase (C48 family) were found. Ulp1 is a member of a family of peptidases that control the function of SUMO a small ubiquitin like modifier protein [18].

Only preliminary data are available for other peptidase family members in E. histolytica. So far, a collagenase [19], a high molecular weight proteinase [20], a serine-metallo proteinase [21], a tripeptidyl peptidase I [22] and a serine protease [23] have been reported.

In this study we have analysed the genome of E. histolytica for the presence of additional peptidases belonging to the aspartate, serine and metallo peptidase families. Furthermore, the expression profile of the amoeba genes for the various proteolytic enzymes was assessed in 7 different E. histolytica isolates as well as under heat stress conditions using an oligonucleotide-based microarray and quantitative real time PCR.

In an attempt to annotate all E. histolytica peptidase genes, a total of 86 putative or known proteolytic enzymes were identified within the E. histolytica genome. Such a great number of peptidase genes is not unusual for protozoans. So far, 110 annotated peptidases were found for Plasmodium falciparum and 70 for Giardia lamblia. Entamoeba, Plasmodium and Giardia contain aspartic peptidases of the A22 family. In addition, P. falciparum contains genes belonging to the A1 family known as plasmepsins. Of the various cysteine peptidases, the autophagin-like as well as the OTU-like enzymes are only present in E. histolytica. On the other hand, several cysteine peptidase families found in P. falciparum, such as C2, C12, C13, C14, C44 and C56 have no counterpart in Entamoeba. Regarding the serine and metallo peptidases no striking differences between the families of Entamoeba, Plasmodium and Giardia became obvious, except 10 additional peptidase families, that are peculiar for P. falciparum. The leishmanolysin-like peptidases of the M8 family are specific for E. histolytica and absent in P. falciparum and G. lamblia, respectively.

Since only a fraction of the 86 putative amoeba peptidases have been biochemically and functionally characterized so far, the function and localization of most of the molecules can only be predicted from the deduced primary structure.

All four aspartic peptidases identified within the Entamoeba histolytica genome may belong to intramembrane-cleaving proteases, which usually perform downstream functions such as cell signalling, regulation and intercellular communications [32]. So far, three families of peptidases are known to promote intramembrane cleavage. These are metallo peptidases represented by the human site-2 protease [33], serine peptidases represented by Drosophila melanogaster rhomboid-1 [34], and aspartic proteases including human presenilins [35], as well as signal peptide peptidases [24]. Within the Entamoeba genome, homologous to only the presenilins and signal peptide peptidases have been found.

One of the amoebic aspartic peptidases (EhAsp22-4) shows highest identity to presenilins. So far, the physiological function of presenilins is not fully understood. Presenilin is one of the subunits that form a multiprotein complex called gamma-secretase [36]. Homologues are found in various organisms of different origin such as Caenorhabditis elegans, Drosophila melanogaster, and even plants [37, 38]. It has been shown that mutations within this protein are associated with Alzheimer's disease [36]. However, homologues to other subunits of this complex, such as nicastrin have not been identified within the Entamoeba genome. EhAsp22-4 contains seven putative transmembrane domains. Interestingly, the active site residues were found within one predicted outside loop of the protein. This is different to the other known presenilins [39].

The 11 identified serine peptidases can be grouped into 3 families. The two amoeba serine peptidases characterized so far, belong to family S28, previously designated EhSP1 and EhSP2 and now renamed EhSP28-1 and EhSP28-2 [23]. Biochemical analysis revealed that these peptidases prefer the substrate Suc-AAF-AMC. This enzymatic feature is identical to that of the E. histolytica tripeptidyl serine peptidase purified by Flockenhaus and colleagues [22]. Unfortunately, no sequence of the purified tripeptidyl peptidase is available. In addition to these two described serine peptidases, one more serine peptidase gene belonging to family S28 has been identified. Microarray analysis indicated that EhSP28-1, EhSP28-2 and EhSP28-3 are not expressed or expressed at a very low level, which is in contrast to the results of Barrios-Ceballos and colleagues [23]. They postulated that the identified peptidase activity corresponds to EhSP28-2 and that this protein is associated with the trophozoite membrane. Using bioinformatic tools, no hydrophobic stretches or transmembrane domains could be deduced within EhSP28-2. Due to these controversial results, the amoeba serine peptidases require further investigation.

The function of the two S26 family serine peptidases identified within E. histolytica is unknown. Peptidases of this family are usually membrane proteins and their function is the processing of newly synthesised secretory proteins. They remove the hydrophobic, N-terminal signal peptides as the proteins are translocated across membranes [40].

Four genes belong to the S9 family (homologous to dipeptidyl-peptidases). One peptidase (SP9-2) shows the highest expression of all serine peptidases analysed in this study. However, the function of these enzymes in E. histolytica remains to be determined.

It is likely that some of the E. histolytica serine peptidases might play a role during the encystation process is it was shown for E. invadens, the in vitro model organism for en- and excystation [41]. Unfortunately, the serine peptidases involved in the E. invadens encystations processes have not been identified so far.

A total of 22 genes were identified encoding metallo peptidases, which are predicted to belong to 11 different families. The members of two of the identified metallo peptidase families contain transmembrane domains. These are the leishmanolysin-like peptidases and the CAAX prenyl peptidases. EhMP8-1 and EhMP8-2 are homologous to leishmanolysin found in kinetoplastids. Leishmanolysin occurs mainly as a heavily-glycosylated protein that is attached to the outer membrane of Leishmania promastigates 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 [42]. There are other eukaryotes, including Caenorhabditis elegans, Drosophila melanogaster and Homo sapiens that have homologues of this protein. Nevertheless, highest degree of sequence similarity to the classical leishmanolysin is found for the enzymes of E. histolytica and Dictyostelium discoideum. However, the proteins of these two organisms have not been characterised so far.

Interestingly, under standard axenic culture conditions only a relatively small number of peptidase genes is significantly expressed. The results are in agreement with a recent study by Ehrenkaufer et al., in which the expression pattern of 38 of the 50 different cysteine peptidase genes were analysed in the standard laboratory E. histolytica isolate HM-1:IMSS [43]. However, in contrast to the results presented here, Ehrenkaufer et al., found differences in the expression of a considerable number of peptidase genes when recent clinical isolates were compared with strain HM-1:IMSS. The discrepancy between the two studies is most likely due to differences in the culture media used. In the study presented here, all E. histolytica isolates were grown under axenic conditions in a monophasic medium, whereas Ehrenkaufer et al. cultured their recent clinical isolates xenically using a diphasic medium and compared the results with HM-1:IMSS grown under axenic condition in a monophasic medium. As proteolytic enzymes are considered to be involved in nutrition uptake and digestion, differences in the composition of the culture medium and in particular the presence of microorganisms should considerably influence expression of petidase genes in Entamoeba.

However, questions remain about possible functions of all the different peptidases present in E. histolytica. At least some of them may be involved in encystation- or exystation processes, as described for E. invadens or for a cathepsin C like peptidase of G. lamblia, which is involved in processing of cyst-wall specific proteins [44]. Aggressive and invading Entamoeba trophozoites should be endowed with adequate mechanisms that ensure their protection against host defence strategies. In this study, the trophozoites were exposed to a temporary heat stress, which partly mimics the situation during tissue invasion. Heat stressed amoebae revealed downregulation of the genes for EhCP-A1 and EhCP-A2 and elevated expression of the genes for EhCP-A5, EhCP-A6 and EhMP8-2, respectively, which is in accordance with a recent report by Weber and colleagues [31]. As EhCP-A6 and EhMP8-2 are expressed at very low levels during in vitro cultivation, these enzymes are obviously not essential for parasite growth at least at standard culture conditions. It has been postulated that the upregulation of the gene for EhCP-A6 during heat stress is due to its potential role in the degradation of damaged proteins [31]. Recently, in few other studies, regulation of peptidase expression in response to various conditions has been described. In HM-1:IMSS clone L6, which is deficient in virulence, phagocytosis as well as cysteine peptidase activity, expression of the genes for EhCP-A1, EhCP-A2 and EhCP-A5 was significantly decreased [45]. In contrast, during intestinal colonisation expression of the genes for EhCP-A1, EhCP-A4 and EhCP-A6 was found to be increased [46]. This further highlights the importance of peptidases for E. histolytica pathogenicity.

Under standard culture conditions only a relatively small number of at least 86 identified peptidase genes is expressed and only very few variations become apparent between various clinical E. histolytica isolates. Nevertheless, here and in few other studies, it was shown that the peptidase expression can be regulated in response to various conditions. Therefore, further studies are necessary to understand the role of all or at least most of the peptidases in the biochemistry and especially for the virulence of E. histolytica.