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.
Highlights
Entamoeba histolytica is the etiological agent of human amoebiasis, which presents clinical manifestations including colitis, dysentery, and hepatic liver abscess (Stanley, 2003; Santi-Rocca et al, 2009)
We further demonstrated that EhArfX2 and the EhArfX2-mediated traffic pathway are involved in the defense against nitrosative stress, which is known to occur during liver abscess formation
Gene expression profiles were compared between two isogenic strains with the identical genetic background but maintained in different ways: one that had been passaged through the hamster liver and one that had been continuously cultured in vitro and was no longer capable of producing abscesses in hamsters (a-HM1)
Summary
Entamoeba histolytica is the etiological agent of human amoebiasis, which presents clinical manifestations including colitis, dysentery, and hepatic liver abscess (Stanley, 2003; Santi-Rocca et al, 2009). The survival of trophozoites in the tissues and subsequent formation of niche for parasitism require mechanisms for inactivation of or evasion from the hostile host environments including the immune response. Multiple cellular mechanisms such as motility, adherence, and cytolysis are coordinately involved in the pathogenesis of E. histolytica (Faust and Guillen, 2012). KERP1 is implicated in the virulence because KERP1 is involved in the binding to epithelial cells, and its gene expression is upregulated during liver abscess development (Santi-Rocca et al, 2008) and shown to be necessary for the progression of liver abscess (Seigneur et al, 2005). Anti-sense repression of CP-A5 (Ankri et al, 1999) or AP-A (Zhang et al, 2004) caused decreased abscess size, suggesting that both factors are essential for abscess formation
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