Bioassay-Guided Fractionation of Extracts from Codiaeum variegatum against Entamoeba histolytica Discovers Compounds That Modify Expression of Ceramide Biosynthesis Related Genes

Abstract

Leaves of Codiaeum variegatum (“garden croton”) are used against bloody diarrhoea by local populations in Cameroon. This study aims to search for the active components from C. variegatum against Entamoeba histolytica, and thereby initiate the study of their mechanism of action. A bioassay-guided screening of the aqueous extracts from C. variegatum leaves and various fractions was carried out against trophozoites of E. histolytica axenic culture. We found that the anti-amoebic activity of extracts changed with respect to the collection criteria of leaves. Thereby, optimal conditions were defined for leaves' collection to maximise the anti-amoebic activity of the extracts. A fractionation process was performed, and we identified several sub-fractions (or isolated compounds) with significantly higher anti-amoebic activity compared to the unfractionated aqueous extract. Anti-amoebic activity of the most potent fraction was confirmed with the morphological characteristics of induced death in trophozoites, including cell rounding and lysis. Differential gene expression analysis using high-throughput RNA sequencing implies the potential mechanism of its anti-amoebic activity by targeting ceramide, a bioactive lipid involved in disturbance of biochemical processes within the cell membrane including differentiation, proliferation, cell growth arrest and apoptosis. Regulation of ceramide biosynthesis pathway as a target for anti-amoebic compounds is a novel finding which could be an alternative for drug development against E. histolytica.

Lipids in Entamoeba histolytica: Host-Dependence and Virulence Factors

“…First, sphingomyelin (SM) is catabolized by sphingomyelinases (SMA) to produce phosphocholine, which follows the Kennedy pathway (Gulati et al, 2015). In our search, we found a sphingomyelin phosphodiesterase in the E. histolytica genome (EHI_007460) and the previously reported acid sphingomyelinase-like phosphodiesterase (EHI_040600) ( Table 4), detected by transcriptomic assays (Mfotie Njoya et al, 2014). On the other hand, several studies have shown that in eukaryotes SL metabolism is also the main source of ethanolamine, which is incorporated into Kennedy pathway to synthesize PE (Zufferey et al, 2002;Zhang et al, 2007).…”

“…All these data support that SM metabolism has a role in forming the huge amount of ceramides in the amoebae membranes (Mfotie Njoya et al, 2014). Although these enzymes have not been experimentally explored, theses authors postulated that there is a high enzymatic activity of ceramide biosynthesis during the life cycle of trophozoites (Mfotie Njoya et al, 2014), as it happens in Trypansoma, Leishmania and Plasmodium (http://www.llamp.net/). Mfotie Njoya et al (2014) also suggested that alteration in genes expression observed in trophozoites treated with C. variegatum extracts, produces accumulation of ceramides in the plasma membrane.…”

“…These events cause lipid-rafts disorganization responsible of cell death and cell growth inhibition. Reduction of fatty acid metabolism and lipid trafficking is also altered in E. histolytica trophozoites treated with this plant (Mfotie Njoya et al, 2014).…”

“…In the same experiments, authors detected that an acid sphingomyelinaselike phosphodiesterase (EHI_040600) was down regulated. They also found several hypothetical long chain fatty acid synthases (Mfotie Njoya et al, 2014). Furthermore, the E. histolytica genome presents SPT (EHI_069310) and sphingosine kinase (EHI_023410), enzymes involved in sphingolipids metabolism (Table 4).…”

“…There is biochemical evidence of sphingolipids metabolism in amoebae, since they are capable to efficiently degrade and resynthesize CEP (Cerbón and Flores, 1981). Furthermore, several genes encoding for hypothetical enzymes related to ceramides metabolism have been detected by transcriptomic analyses after incubation of trophozoites with extracts of Codiaeum variegatum extracts (Table 4; Mfotie Njoya et al, 2014). This plant, endemic of Cameroon, have been used as an effective treatment against bloody-diarrhea (Moundipa et al, 2005).…”