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Regulation of antioxidant enzymes in Entamoeba histolytica (Schaudinn, 1903)
Regulation der Antioxidantenzyme in Entamoeba histolytica (Schaudinn, 1903)
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Freie Schlagwörter (Deutsch):
Freie Schlagwörter (Englisch):
Entamoeba, histolytica, antioxidant, genes
Tannich, Egbert (Prof. Dr.)
Tag der mündlichen Prüfung:
Kurzfassung auf Englisch:
Amoebiasis is the infection caused by the human parasitic protozoan E. histolytica. Although the parasite is distributed worldwide, it predominantly affects individuals of lower hygiene conditions especially in developing countries. Usually the infection with E. histolytica remains asymptomatic. In some cases the infection leads to intestinal colitis, and if the parasite succeeds to invade the intestinal mucosal barrier, the parasites migrate to different organs of the body especially the liver where an amoebic liver abscess (ALA) may develop. Whether or not the infection results in the formation of liver abscess may be influenced by the E. histolytica strain. Nowadays it is known that this parasite uses different mechanisms to invade the intestinal wall reaching the liver with the blood stream. One of the mechanisms, that help the microaerophilic parasite E. histolytica to overcome the elevated levels of Oxygen and its derivatives in blood and liver, is the use of antioxidant enzymes. This mechanism is considered as a defensive mechanism that contributes to the degree of virulence of the parasite.
In this work two syngenic amoeba clones (clone A and clone B), which are derived from the E. histolytica isolate HM-1: IMSS were investigated. The two clones differ in their virulence. Clone A is incapable of inducing liver abscess in rodents, while clone B is capable of producing large liver abscesses. Investigating the regulation of antioxidant enzymes in both clones may provide a clue for a better understanding of virulence in this parasite. With the help of different analytical methods difference between the two clones were investigated on the physiological, transcriptional and translational levels.
On the physiological level, there was no significant difference between the trophozoites of clone A and clone B upon the treatment with oxidative reagents that produce intracellular superoxide anions, hydrogen peroxide, and extracellular superoxide radicals. On the other hand, clone A was found to be significantly more sensitive to oxidative reagents that produce nitric oxide radicals. The overexpression of the antioxidant genes antioxidants ehprx, ehtrxr, ehfesod, ehrbr, and ehfehyd in both clones did not reflect always an increased level of protection against oxidative and nitrosative stress
Based on their differential behavior toward nitrosative stress, the differential gene expression of antioxidant genes in both clones A and B was investigated by means of real-time PCR (RT-PCR). Among the 15 investigated genes, 4 genes (ehprx, ehfesod, ehfehyd, and ehtrx4) were differentially higher expressed in the pathogenic clone B.
In order to correlate the transcriptional level of the four differentially expressed antioxidant genes with the translational level, the respective proteins were localized in both clones A and B. In addition, two more proteins that correspond to the genes thioredoxin reductase (ehtrxr) and rubrerythrin (ehrbr) were localized in both clones. These two genes are predicted to play a role in detoxifying hydrogen peroxide radicals. Due to the fact that there was no specific antibody for the gene ehtrx4 availabe, localization studies could not be performed.
The localization analysis reflected that there is no significant difference between both clones in terms of the amount of the enzymes thioredoxin reductase and rubrerythrin. Both were found to be cytosolic localized. Rubrerythrin was found to be associated with the cell membrane. The amount of the enzymes peroxiredoxin, iron-superoxide dismutase, and iron-hydrogenase was found to be more in clone B than in clone A. All three enzymes were cytosolic localized. Peroxiredoxin and iron-superoxide dismutase were found to be associated with the cell membrane. The translational level of antioxidant enzymes correlated with the transcriptional level.
The overexpression of the antioxidant genes ehprx, ehtrxr, ehfesod, ehrbr, and ehfehyd in both clones was targeted to investigate whether their overexpression has an effect on the expression of other antioxidant genes. Surprisingly, the overexpression of the gene ehtrxr lead to the silencing of this gene in trophozoites of the clone A. Moreover, the overexpression of these genes had an influence on the expression of other antioxidant genes.