Besides the Gal/GalNAc lectin, another important adhesive molecule on the virulent trophozoite surface is a serine-threonine-isoleucine rich protein (EhSTIRP). These surface proteins are encoded by a multigene gene family containing four members: EHI_004340, EHI_012330, EHI_025700 and EHI_073630. Strikingly, most of these protein family members exhibit the unusual feature of expression profiles between life cycle stages and between strains of parasite. EhSTIRPs were reported to be highly expressed in all virulent trophozoites, i.e. HM-1:IMSS, 200:NIH and the invasive trophozoites isolated from infected
colon, and to be not or very lowly expressed in nonvirulent conditions including E. dispar
and E. histolytica Rahman and cystic stage of virulent E. histolytica strains [11,74,77,141]. Based on the HM-1:IMSS genomic reference and annotation in the AmoebaDB
database version 4.2, three of four members of EhSTIRP gene family: EHI_004340,
EHI_012330 and EHI_025700 are similar in a very large size of approximately 8 kb in the
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largest annotated gene in the genome with a length of 15.2 kb. All these family memberscontain a single transmembrane domain and a short portion of 34 amino acid cytoplasmic tail and show very high conservation at their 3’ end with greater than 99% nucleotide identity and less conservation at the 5’ end with 88-94% identity between isotypes [11].
Relevant to their virulent functions, the cytotoxic ability of EhSTIRPs has been proven by comparing the release of lactate dehydrogenase from damaged Caco-2 colonic monolayer cells treated with wild type HM-1:IMSS and EhSTIRP dsRNA-treated HM-1:IMSS
trophozoites and found to be drastically reduced to ~55% in EhSTIRP-downregulated
parasites at all time points [11]. In addition, it was found that EhSTIRP dsRNA-treated HM-
1:IMSS trophozoites showed the decrease in host cell adhesion compared to the wild type parasite after incubating on ice and washing [11]. Altogether, EhSTIRPs have putative functions in virulent parasites for host cell adhesion and cause subsequent host cell damage.
Expectedly, two EhSTIRP gene members in this study: EHI_012330 and EHI_025700
are markedly upregulated with absolute log2FC ≥ 2 in all three virulent strains whereas
another member EHI_004340 shows strong upregulation only in HM-1:IMSS and PVBM08B
as reported in Appendix Table 1.1. For these three EhSTIRP members, HM-1:IMSS and
PVBM08B have much higher transcript levels (log2FC = 5.10-8.43) than less virulent
IULA:1092:1 (log2FC = 1.72-2.63), inferring that differential EhSTIRP gene expression
strongly contributes to virulence variability across virulent strains..
Conversely, it is interesting that the largest EhSTIRP gene (EHI_073630) was
significantly downregulated in all these three virulent strains relative to Rahman with
log2FC = -1.69, -1.52, -0.82 for HM-1:IMSS, PVBM08B and IULA:1092:1, respectively. Based
on the recent microarray, Thibeaux et al., 2013 recently reported that EhSTIRP transcript
EHI_073630 was only significantly upregulated in HM-1:IMSS with fold change = 2.5, FDR-
adjusted P-value = 2.70e-22, compared to Rahman in contact with the colon mucus whereas
other three members showed significant upregulation in HM-1:IMSS both in culture and upon contact with the human colon [82]. Also, the authors demonstrated the co-expression of other functional transcripts involved in DNA-RNA regulation, cell signaling, stress response, proteolysis, translation-protein maturation, subcellular trafficking, cytoskeleton and biomolecular metabolism, shown to be upregulated solely during host mucosal contact [82]. This could be inferred that the expression of this gene set including this EhSTIRP gene EHI_073630 in virulent parasites is not ubiquitous and solely upregulated under the invasive condition, e.g. during contact to the mucus. In other words, it may be stated in the principle of allocation that virulent trophozoites possibly adapt to allocate their limited
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energy for their cell division and growth by reducing the production of such nonessentialtranscripts in the non-enriched axenic culture condition.
Moreover, Weedall et al., 2012 demonstrated that this EhSTIRP gene EHI_073630
which locates on scaffold DS571171 exhibits the most polymorphisms in the genome and is
distantly related to the other three EhSTIRP gene members [70]. Based on SNP data in the
AmoebaDB database, EHI_073630 contains the highest SNPs across all strains: total SNPs = 100, nonsynonymous SNPs = 63, synonymous SNPs = 37 and nonsyn/syn ratio = 1.7 [26]. As demonstrated in Figure 2.12, most of the polymorphisms found in this gene show homozygous sequence pattern in each strain. Strikingly, it was found that HM-1:IMSS and its two derived clones (i.e. HM-1A and HM-1B) have distinctive sequence divergence from the other eight strains that resemble each other, consistent with allelic dimorphism found in Plasmodium genes encoding merozoite surface proteins [70,142,143]. However, the question raising whether the large sequence divergence in HM-1:IMSS is associated with its downregulation in axenic condition still needs to be further investigated.
For three expressed EhSTIRP gene members ubiquitously in virulent strains, it is
intriguing that these three gene members are conversely very low in expression in Rahman, implying that gene silencing exists in the nonvirulent parasite. MacFarlane and Singh, 2007 found that most of EhSTIRP coding sequences as well as their promoters in Rahman are very similar (≥ 98%) to those found in HM-1:IMSS, suggesting that the possible epigenetic mechanisms such as DNA methylation and histone deacetylation might be responsible for EhSTIRP gene silencing in Rahman [11,144-146]. However, no change in EhSTIRP expression was observed after treating Rahman trophozoites with a DNA methyltransferase inhibitor, i.e. 5-azacytidine, and a histone deacetylase inhibitor, i.e. trichostatin A [11]. It is
interesting that EhSTIRP expression could be downregulated in HM-1:IMSS trophozoites
transfected with a plasmid with construct encoding dsRNA specific to the highly conserved 3’ end but this dsRNA-based silencing reverted to the normal wide type after one year of subculture [11].
Recently, an endogenous RNA interference (RNAi) pathway has been identified for its role in gene silencing in several human parasites including G. lamblia, T. vaginalis, T. gondii, T. brucei and E. histolytica [83-86]. The RNAi pathway in E. histolytica is mediated by a population of 27 nt small RNAs and their partners, Argonaute proteins (EhAGOs).
Zhang et al., 2013 demonstrated the presence of abundant 27 nt sRNAs which antisense
mapped to the EhSTIRP genes (EHI_025700 and EHI_012330) only in Rahman but were
absent in HM-1:IMSS [92]. Furthermore, overexpression of a Myc-tagged EhSTIRP1
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Rahman, suggesting that antisense 27 nt sRNAs likely regulate the expression of theseadhesion molecules in the nonvirulent Rahman strain [92]. More details regarding the antisense sRNA-mediated gene silencing in a strain-specific manner will be discussed in Chapter 5.
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Figure 2.12: Sequence polymorphism of EhSTIRP gene EHI_073630 located on scaffold DS571171. The top three rows represent the reference HM-1:IMSS and its derivative strains: HM-1A and HM-1B, respectively. Across the full length of the gene, HM-1:IMSS and its derivatives exhibit sequence divergence compared to the other strains shown in the lower eight rows: Rahman, 2592100, PVBM08B, PVBM08F, IULA:1092:1, HK-9, MS84-1373 and MS27-5030, respectively. Polymorphic positions are indicated by different colours as follows: black for homozygous positions; grey for heterozygous positions; light grey for positions different from the reference; white for base not available. This figure is reproduced with permission from Weedall et al., 2012 [70].