The parasitic protist Giardia intestinalis lacks most heme proteins yet encodes a flavohemoglobin (gFlHb) that converts nitric oxide to nitrate and likely protects the cell from nitrosative stress. In this work an antibody raised against gFlHb was used to examine both changes in gFlHb expression levels and intracellular localization in Giardia in response to nitrosative stress. Giardia trophozoites exposed to stressors which either directly release nitric oxide (diethyltriamine NONOate, 1 mM) or are sources of other reactive nitrogen intermediates (sodium nitrite 20 mM or S-nitrosoglutathione, 1 or 5 mM) exhibited a 2 to 9-fold increase of gFlHb after 24 hours. Increased expression levels of gFlHb were detectable by 8 hours in S-nitrosoglutathione and diethyltriamine-NONOate-treated trophozoites, and by 12 hours after sodium nitrite exposure; these differences were likely due to differences in the rates of release of RNS from these compounds. In addition to a band of the expected size for gFlHb (52 kDa), western blots detected a second, higher molecular weight band (72 kDa) with comparable or higher intensity upon treatment with these RNS donors, which is consistent with sumoylation of gFlHb. Immunofluorescence microscopy of Giardia trophozoites detected gFlHb diffused throughout the cytoplasm and more punctuated staining along the cell membrane and between the nuclei. The punctuated staining may be due to the association of gFlHb with either peripheral vacuoles or basal bodies.
Author Keywords: Flavohemoglobin, Giardia intestinalis, Nitrosative stress