ArfX2 GTPase Regulates Trafficking From the Trans-Golgi to Lysosomes and Is Necessary for Liver Abscess Formation in the Protozoan Parasite Entamoeba histolytica

Abstract

Entamoeba histolytica is the causative agent of amoebic dysentery and liver abscess in humans. The parasitic lifestyle and the virulence of the protist require elaborate biological processes, including vesicular traffic and stress management against a variety of reactive oxygen and nitrogen species produced by the host immune response. Although the mechanisms for intracellular traffic of representative virulence factors have been investigated at molecular levels, it remains poorly understood whether and how intracellular traffic is involved in the defense against reactive oxygen and nitrogen species. Here, we demonstrate that EhArfX2, one of the Arf family of GTPases known to be involved in the regulation of vesicular traffic, was identified by comparative transcriptomic analysis of two isogenic strains: an animal-passaged highly virulent HM-1:IMSS Cl6 and in vitro maintained attenuated avirulent strain. EhArfX2 was identified as one of the most highly upregulated genes in the highly virulent strain. EhArfX2 was localized to small vesicle-like structures and largely colocalized with the marker for the trans-Golgi network SNARE, EhYkt6, but neither with the endoplasmic reticulum (ER)-resident chaperon, EhBip, nor the cis-Golgi SNARE, EhSed5, and Golgi-luminal galactosyl transferase, EhGalT. Expression of the dominant-active mutant form of EhArfX2 caused an increase in the number of lysosomes, while expression of the dominant-negative mutant led to a defect in lysosome formation and cysteine protease transport to lysosomes. Expression of the dominant-negative mutant in the virulent E. histolytica strain caused a reduction of the size of liver abscesses in a hamster model. This defect in liver abscess formation was likely at least partially attributed to reduced resistance to nitrosative, but not oxidative stress in vitro. These results showed that the EhArfX2-mediated traffic is necessary for the nitrosative stress response and virulence in the host.

Proteomic analysis of Atg8-dependent recruitment of phagosomal proteins in the enteric protozoan parasite Entamoeba histolytica

“…Syntaxin belongs to the soluble N-ethylmaleimide-sensitive fusion factor attachment protein (SNAP)-receptors (SNAREs) and is involved in membrane fusion ( Kloepper et al., 2007 ; Koike and Jahn, 2022 ). The E. histolytica genome has 25 SNAREs including at least three plasma membrane, two TGN/lysosome, and one cis-Golgi localized SNAREs, as predicted by phylogenetic analyses, although the localization of other SNAREs remains undetermined ( Saito-Nakano et al., 2021a ). STX found in this study is grouped into a clade that is unique to E. histolytica among the representative SNAREs from Homo sapiens , Arabidopsis thaliana , and Saccharomyces cerevisiae ; thus its function is unpredictable ( Saito-Nakano et al., 2021a ).…”

“…The E. histolytica genome has 25 SNAREs including at least three plasma membrane, two TGN/lysosome, and one cis-Golgi localized SNAREs, as predicted by phylogenetic analyses, although the localization of other SNAREs remains undetermined ( Saito-Nakano et al., 2021a ). STX found in this study is grouped into a clade that is unique to E. histolytica among the representative SNAREs from Homo sapiens , Arabidopsis thaliana , and Saccharomyces cerevisiae ; thus its function is unpredictable ( Saito-Nakano et al., 2021a ). Importantly, only one SNARE, out of 25, is included in a repertoire of 338 phagosomal proteins, strongly suggesting that STX is the predominant SNARE involved in phagosome biogenesis.…”

Diversity and Plasticity of Virulent Characteristics of Entamoeba histolytica

“…To investigate the virulence of E. histolytica in animal models, the liver abscess model in golden hamsters is used [ 59 , 60 , 61 ]. The experimental inoculation and induced liver abscess were analyzed for the various virulent factors of vaccine development [ 62 , 63 , 64 , 65 ]. However, after isolating the clinical E. histolytica strains from the patients and maintaining subcultures, most E. histolytica strains can lose their potential to induce liver abscess in hamsters at a relatively early stage.…”

Attenuation of In Vitro and In Vivo Virulence Is Associated with Repression of Gene Expression of AIG1 Gene in Entamoeba histolytica

“…The amoebae genes with differential expression during stress [ 28 ], host invasion [ 29 ], formation of amoebic liver abscesses [ 30 , 31 ], stage conversion [ 18 ], and interaction with bacteria [ 32 , 33 , 34 ] have been identified, and novel factors or determinants of virulence have been proposed. Indeed, besides some known virulence factors, several genes are upregulated during the expression of amoebic virulence, which are implicated in cellular processes, including stress response, cell signaling, vesicular trafficking [ 35 ], and antibacterial activity.…”