טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentShahi Preeti
SubjectAdaptive Response of the Parasite Entamoeba Histolytica
to Nitrosative and Oxidative Stresses
DepartmentDepartment of Medicine
Supervisor Professor Serge Ankri
Full Thesis textFull thesis text - English Version


Abstract

Entamoeba histolytica is a causative protozoan parasite of amebiasis, a disease which is characterized by acute inflammation of the colon. Adaptation of the parasite to toxic levels of nitric oxide (NO) that are produced by phagocytes 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, trophozoites were progressively adapted to increasing concentrations of the NO donor drug, S-nitroso glutathione up to a concentration of 110 µM. These NO-adapted trophozoites (NAT) were more resistant to an acute exposure of 350μM GSNO and activated macrophages and were better at invading porcine colon explants than wild-type trophozoites.  The transcriptome of NATs was investigated by RNA sequencing and the results of the analysis revealed the existence of a weak overlap with the transcriptome of acute nitrosatively-stressed trophozoites (TEANS). N-acetyl ornithine deacetylase (NAOD) was among the 208 genes that were upregulated in NATs. NAOD catalyzes the deacylation of N-acetyl-L-ornithine to yield ornithine and acetate. Overexpression of NAOD resulted in significant overproduction of putrescine and in a better adaptation to NS. Surprisingly, overexpression of a catalytically inactive NAOD (mNAOD) resulted in a better adaptation to NS but did not result in putrescine overexpression. These results suggest that the formation of putrescine is not essential for the adaptation of the parasite to NS. We also demonstrated that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is detrimental to E.histolytica exposed to NS and that this detrimental effect is neutralized by NAOD or by mNAOD. These results establish NAOD as a moonlighting protein, and highlight the unexpected role of this metabolic enzyme in the adaptation of the parasite to NS.

Moreover, In the large intestine, the invading trophozoites is confronted with reactive oxygen species (ROS) that are produced and released by cells of the innate immune system at the site of infection. The ability of the parasite to survive oxidative stress (OS) is essential for a successful invasion. Although the effects of OS on the regulation of gene expression in this parasite that play an important role in the parasite defense against reactive species have been previously investigated, our knowledge on oxidized proteins in E.histolytica is lacking and these amoebic proteins which are modified by OS may be new components of the parasite's antioxidant machinery. In order to fill this knowledge gap, we performed a large-scale identification and quantification of the parasite's proteins that are oxidized following an oxidative challenge to the parasite by resin-assisted capture (RAC) mass spectrometry (MS). We found that 154 proteins involved in antioxidant activity, cytoskeleton, translation, and transport are among the oxidized proteins that were enriched by OX-RAC. We provide evidences that arginase, an enzyme involves in the conversion of L-arginine into L-ornithine and urea, is involved in the protection of the parasite against OS. Collectively, these results emphasize the importance of OS as a regulator of key physiological functions in E.histolytica and indicate a new role for arginase in the resistance of E.histolytica to OS.