Activity, stability and folding analysis of the chitinase from Entamoeba histolytica

• EhCHT1 is stable over a wide range of pH and temperature, but sensitive to mild concentrations of guanidine hydrochloride.
• EhCHT1 is dependent on in vivo oxidative folding and also reliant on the oxidase activity of EhPDI.
• EhCHT1 has structural features that can be used to identify specific inhibitors with anti-amebic activity.
• Inhibition of EhCHT1 could arrest the parasite's life cycle and, thus, stop the infection.

Human amebiasis, caused by the parasitic protozoan Entamoeba histolytica, remains as a significant public health issue in developing countries. The life cycle of the parasite compromises two main stages, trophozoite and cyst, linked by two major events: encystation and excystation. Interestingly, the cyst stage has a chitin wall that helps the parasite to withstand harsh environmental conditions. Since the amebic chitinase, EhCHT1, has been recognized as a key player in both encystation and excystation, it is plausible to consider that specific inhibition could arrest the life cycle of the parasite and, thus, stop the infection. However, to selectively target EhCHT1 it is important to recognize its unique biochemical features to have the ability to control its cellular function. Hence, to gain further insights into the structure–function relationship, we conducted an experimental approach to examine the effects of pH, temperature, and denaturant concentration on the enzymatic activity and protein stability. Additionally, dependence on in vivo oxidative folding was further studied using a bacterial model. Our results attest the potential of EhCHT1 as a target for the design and development of new or improved anti-amebic therapeutics. Likewise, the potential of the oxidoreductase EhPDI, involved in oxidative folding of amebic proteins, was also confirmed.

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