Hypothetical proteins play a role in stage conversion, virulence, and the stress response in the Entamoeba species

Entamoeba histolytica is an enteric parasite that causes amoebic dysentery and amoebic liver abscess in humans and nonhuman primates. This parasite has a two-stage life cycle, consisting of the infective, environmentally-stable cyst form and the pathogenic trophozoite form. E. histolytica cysts are transmitted via fecally-contaminated food and water, making this disease prevalent where sanitation is substandard, such as in sub-Saharan Africa and southern Asia, (Shirley et al., 2018). As of 2020, 489 million people worldwide utilize unprotected drinking water sources, including wells, springs, and surface water. Additionally, 494 million people continue to practice open defecation (WHO & UNICEF, 2021). Thus, there is considerable risk for the spread of this disease. Globally, more than 50 million people become infected annually with the parasite, with over 100,000 deaths annually (Shirley et al., 2018).

E. histolytica is ingested as a latent cyst and travels through the digestive system until unknown cues trigger the excystation of trophozoites in the small intestine. Trophozoites colonize the large intestine where they feed on the natural gut flora and mucosal cells that compose the epithelial lining. In some cases, trophozoites will degrade the mucosal/epithelial layer and enter the blood stream where they cause extra-intestinal infections in the liver, and more rarely the lungs, or the brain (König et al., 2021). In the large intestine, unknown signals induce aggregation and encystation of trophozoites, which results in environmentally-stable cysts that are shed into the environment to facilitate host-to-host spread (Bercu et al., 2007). Until recently (Wesel et al., 2021), there was no method for inducing efficient stage conversion of E. histolytica in vitro. However, in vitro stage conversion is easily achievable for Entamoeba invadens; therefore, this reptilian parasite is routinely used as a model organism (Avron et al., 1986; Coppi and Eichinger, 1999).

The molecular mechanisms governing stage conversion and virulence in the Entamoeba species remains unclear. Furthermore, metronidazole, the current treatment for amoebiasis, is associated with high toxicity and severe side effects (Cherian et al., 2015; Ralston and Petri, 2011). Thus, there is a need to further characterize the cellular processes underlying the lifecycle of E. histolytica so that novel therapeutic targets can be identified. Hypothetical proteins are proteins that are postulated to be expressed from an open reading frame. Typically, their function cannot be deduced by bioinformatics analyses and there may or may not be experimental evidence for their translation in vivo (Ijaq et al., 2015). Hypothetical proteins have been explored as drug targets for several communicable diseases including, chlamydia (Turab Naqvi et al., 2017), tuberculosis (Yang et al., 2019), and shigellosis (Sen and Verma, 2020). Therefore, unique hypothetical proteins may also represent a promising source of potential new targets for the treatment of E. histolytica infection.

According to AmoebaDB (amoebadba.org) the genomes of E. invadens and E. histolytica are predominately comprised of hypothetical proteins, (i.e., proteins with unknown functions). In a study of the proteomic profiles of E. histolytica trophozoites, cysts, and cyst-like structures, Luna-Nácar et al. (2016) found that the cyst proteome was different from that of trophozoites, where almost 40% of the cyst proteome was annotated as hypothetical proteins. Similarly, a quantitative proteomics analysis of membrane proteins in avirulent and virulent strains of E. histolytica found 19 hypothetical proteins that were upregulated, and 18 hypothetical proteins that were downregulated in the virulent strain (Ng et al., 2018). In an attempt to elucidate virulence mechanisms, König and colleagues compared the genomes of the pathogenic amoebae, E. histolytica and E. nuttali (the macaque pathogen), to that of a nonpathogenic amoeba, E. dispar (König et al., 2021). One hundred seventy-five proteins were found to be unique to E. histolytica, most of which were annotated as hypothetical proteins. E. histolytica trophozoites possessed 67 unique genes that had putative homologs in E. nuttalli but not in E. dispar, many of which were also hypothetical proteins (König et al., 2021). Furthermore, Matthiesen et al. (2019) silenced hypothetical protein, EHI_127670, in E. histolytica trophozoites and found that parasites with reduced expression of EHI_127670 were less able to form amoebic liver abscesses (ALAs) in mice. Alternatively, overexpression of EHI_127670 in nonpathogenic amoebae lead to restoration of ALA formation ability.

Luna-Nácar et al. (2016) identified 4 hypothetical cyst-specific proteins that could represent promising drug or vaccine targets as they were highly antigenic. Furthermore, a third of the E. histolytica genome is not found within the human host, indicating that many of these hypothetical proteins may be worthy drug targets (Marchat et al., 2020). Ultimately, the genomes of E. histolytica and E. invadens remain highly enigmatic and this represents a vast gap in knowledge. Therefore, the goal of this study was to characterize two hypothetical proteins in the Entamoeba species.

Using an RNAi silencing approach, we reduced expression of two such proteins, EIN_059080 in E. invadens, and EHI_056700 in E. histolytica. E. invadens parasites with reduced expression of EIN_059080 (EIN_059080-KD) possessed a significantly decreased rate of encystation, and an increased rate of phagocytosis, an important virulence function. These mutants were also significantly less viable than control parasites when exposed to oxidative stress. E. histolytica parasites with reduced expression of EHI_056700 (EHI_056700-KD) exhibited significantly lower rates of adhesion to host cells and phagocytosis and were significantly less viable when exposed to oxidative stress and glucose deprivation. This study suggests that hypothetical proteins play important roles in stage conversion, virulence, and the stress response in the Entamoeba species and supports their potential as targets for therapy.

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