Molecular Biology

E. histolytica is a eukaryotic organism with unusual cellular characteristics. It lacks organelles that morphologically resemble rough endoplasmic reticulum, Golgi or mitochondria (Figure 9.3; Table 9.2) (Rosenbaum and Wittner, 1970; McLaughlin and Aley, 1985; Hasegawa and Hashimoto, 1993; Clark and Roger, 1995; Mann et al., 1991); however, the presence of nuclear-encoded mitochondrial genes, such as pyridine nucleotide transhydrogenase and hsp60, is consistent with E. histolytica having contained mitochondria at one time. Cell surface and secreted proteins contain signal sequences, and tunicamycin inhibits protein glycosylation, implicating functional rough endoplasmic reticulum or Golgi apparatus (Mann et al., 1991). Ribo-somes form aggregated crystalline arrays in the cytoplasm of the trophozoite (Rosenbaum and Wittner, 1970). Unique biochemical pathways from metazoans include the lack of glutathione and enzymes required for glutathione metabolism, the use of pyrophosphate instead of

Fig. 9.3 Electron micrograph of an E. histolytica trophozoite. Note the nucleus with peripheral and central chromatin, the lack of structures resembling mitochondria or rough endoplasmic reticulum, and the prominent intracytoplasmic vacuoles

Table 9.2 Some unusual features of the cell biology and biochemistry of E. histolytica

• Lack of mitochondria, rough endoplasmic reticulum or Golgi

• Presence of crystalline arrays of aggregated ribosomes

• Ribosomal RNA genes on multicopy circular DNA molecules

• Lack of glutathione and enzymes of glutathione metabolism

• Use of pyrophosphate instead of ATP at several steps in glycolysis

• Inability to synthesize purine nucleotides de novo

From Petri (1996), with permission.

ATP at several steps in glycolysis, and the inability to synthesize purine nucleotides de novo. Glucose is actively transported into the cytoplasm, where the end-products of carbohydrate metabolism are ethanol, C02 and, under aerobic conditions, acetate (McLaughlin and

Aley, 1985). E. histolytica genomic organization and transcriptional control appear to be distinct from both metazoan and better-characterized protozoan organisms. The genome is relatively small for a eukaryote (3.2x107bp; Gelderman et al., 1971) and extremely AT-rich (67% within coding regions and 78% overall; Gelderman et al., 1971; Tannich and Horstmann, 1992). Transcription of protein-encoding genes is by an RNA polymerase that is resistant to 1 mg/ml a-amanitin (Lioutas and Tannich, 1995). Introns are rarely identified (Lohia and Samuelson, 1993; Plaimauer et al., 1994), suggesting that cis-splicing is a rare event, and there is no evidence of trans-splicing or polycistronic transcription (Bruchhaus et al., 1993). However, recent work suggests that coding regions are tightly packed, with all four intergenic regions characterized to date smaller than 1.35 kb (Bruchhaus et al., 1993). The structure of the mRNA is remarkable as well, with the 5'-untranslated region having an average length of 11 bases compared to a metazoan average of 60-80 bases (Kozak, 1984). The 3'-untranslated region is also short, with an average size of only 33 bases (Bruchhaus et al., 1993). Ribosomal RNA is not contained within the genome but is encoded on a circular, 24 kb DNA episome (Bhattacharya et al., 1989).

Both transient and stable DNA-mediated transfection of E. histolytica with heterologous gene expression have been recently accomplished (Buss et al., 1995; Nickel and Tannich, 1994; Purdy et al., 1994; Vines et al., 1995; Hamann et al., 1995). Gene expression in E. histolytica appears to involve species-specific transcription factors. An inducible promoter based on the tetracycline repressor system has also been developed (Hamann et al., 1997; Ramakrishnan et al., 1997). Analysis of the hgl5 gene has revealed four positive upstream regulatory elements and one negative upstream regulatory element in the 200 bases upstream of the start of transcription (Figure 9.4). The architecture of the core promoter is unique and differs even from more closely related protozoa, consisting of three conserved elements: a TATA box, an initiator and a third conserved region, the GAAC element, which are all able to direct the site of transcription initiation (Singh et al., 1997; Singh and Rogers, 1998). Recent work has implicated inside-out signaling from the cytoplasmic tail of

Fig. 9.4 Structure of the promoter of the hgl5 gene of E. histolytica. Four positive and one negative upstream regulatory regions have been identified by linker scanner mutagenesis and transient transfection system using the reporter gene luciferase. Three regions have also been identified in the core promoter, which appears to control gene expression

Fig. 9.4 Structure of the promoter of the hgl5 gene of E. histolytica. Four positive and one negative upstream regulatory regions have been identified by linker scanner mutagenesis and transient transfection system using the reporter gene luciferase. Three regions have also been identified in the core promoter, which appears to control gene expression the surface lectin, involved in adherence and cytotoxicity of the ameba (Vines et al., 1998). The carbohydrate recognition domain of the lectin has been identified and interestingly has sequence conservation with hepatocyte growth factor, implicating a possible molecular mechanism of tropism of the ameba for the liver (Dodson et al., 1999).

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