Purification and characterization of proteins involved in metronidazole resistance in Giardia lamblia
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Giardia lamblia is a common intestinal protozoan parasite, causing the gut infection giardiasis. In humans, the infection may vary from asymptomatic carrier stage to a more severe malabsorption syndrome or chronic sequelae. The first line treatment in Norway is metronidazole (MTZ). Recently, MTZ-refractory cases of giardiasis have increased. The mechanism of MTZ-resistance in Giardia is not well understood. This study examined the flavohemoglobin (FlHb) from the two human-infective Giardia assemblages (A and B). FlHb is an enzyme thought to play a major role in nitric oxide (NO) detoxification in Giardia. In addition to FlHb, the enzymes nitroreductase 1 (NR1) and nitroreductase 2 (NR2) from assemblage B, which are thought to activate and inactivate MTZ respectively, and also be involved in MTZ resistance, were examined. FlHb from assemblage A was expressed using E.coli as host cells, and purified using the Ni- NTA affinity purification system, followed by size exclusion chromatography (SEC). Structural characterization and stability tests including circular dichroism (CD), thermofluor, light scattering (LS), small angle X-ray scattering (SAXS) were performed for FlHb from assemblage A. In addition, screening for crystallization conditions was done to get crystals for crystallographic studies. NR1 and FlHb from assemblage B were cloned and expressed using E.coli as host cells, and initial purification trials were done, but time did not allow further research of these. NR2 was not straightforward to clone, and the time limit put an end for further attempts. From our results, it was observed that FlHb from assemblage A separated into two different variants during SEC. One of the variants containing heme, and one variant without heme. Comparison from structural characterization suggested that the heme-less variant has a more open structural shape and behaved like a dimer. The heme-containing variant, which is the active variant of interest, is monomeric and has a closed, more globular shape. In conclusion, further research needs to be performed and crystals need to be obtained for NR1, NR2 and FlHb in order to obtain more information about the protein functions and to better understand their role in MTZ resistance in Giardia.
PublisherThe University of Bergen
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