טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentManor Shiran
SubjectAdaptation of Entamoeba histolytica to nitrosative stress:
insights into the role of arginase in the
adaptation process
DepartmentDepartment of Medicine
Supervisor Professor Serge Ankri
Full Thesis textFull thesis text - English Version


Abstract

Entamoeba histolytica trophozoites usually reside in the colon of infected individuals and 90% of these infected individuals are asymptomatic. In the host, the parasite is exposed to environmental challenges. One of these challenges is nitrosative stress (NS) generated by macrophages. NO can regulate cell signaling through post-translational modifications (PTMs). One such well-characterized PTM is S-nitrosylation, which refers to covalent bond formation between a NO moiety and the reduced thiol of a cysteine (Cys) residue present in the target protein. Adaptation of E. histolytica to toxic levels of nitric oxide (NO) may be essential for the establishment of chronic amebiasis and the parasite's survival in its host. In order to obtain insight into the mechanism of E. histolytica's adaptation to NO, E. histolytica trophozoites were progressively adapted to increasing concentrations of the NO donor drug, S-nitrosoglutathione (GSNO) up to a concentration of 110µM. SNO-RAC is a method which is used to identify S-nitrosylated (SNO) proteins. We identified 141 of SNO proteins in trophozoites adapted to NO (NAT). We noticed that arginase was among the SNO proteins. Arginase is a manganese metalloenzyme that catalyzes the conversion of L-arginine to L-ornithine and urea. Arginase and NO synthases (NOSs) both use L-arginine as a common substrate. A previous work showed that arginase released by lysed E.histolytica trophozoites can convert L-arginine to L-ornithine in the media and limit NO production by macrophages. Based on that previous work about arginase, our hypothesis is that arginase is involved in the adaptation of NAT to NS. Our goal was to show that arginase can protect the amoeba from the damage of the NS. In order to reach this goal, we performed variety of experiments which included over expressing of the arginase and checking it's resistance to NS and checking the arginase activity in NAT.   

We demonstrated that arginase activity is upregulated in NAT and we showed that this effect is not caused by an upregulation of arginase expression or by a boosting of arginase activity following its S-nitrosylation. We also showed that arginase is not involved in the adaptation of trophozoites to NS because trophozoites overexpressing arginase or a catalytically inactive form of arginase adapt to a same extend to NS than trophozoites carrying a mock plasmid.

We can conclude from this work that intracellular arginase does not play a role in the adaptation of the parasite to NS. In contrast, arginase contributes to the resistance of the parasite against NS when it is released by lysed parasite in the media. Future work will focus on other nitrosylated proteins identified in NAT like cytoskeletal proteins which are enriched among SNO proteins and are essential for the mobility and virulence of the parasite.