Flavohemoglobin and nitric oxide detoxification in the human protozoan parasite Giardia intestinalis

Flavohemoglobins (flavoHbs), commonly found in bacteria and fungi, afford protection from nitrosative stress by degrading nitric oxide (NO) to nitrate. Giardia intestinalis, a microaerophilic parasite causing one of the most common intestinal human infectious diseases worldwide, is the only pathogenic protozoon as yet identified coding for a flavoHb. By NO amperometry we show that, in the presence of NADH, the recombinant Giardia flavoHb metabolizes NO with high efficacy under aerobic conditions (TN = 116 ± 10 s−1 at 1 μM NO, T = 37 °C). The activity is [O2]-dependent and characterized by an apparent KM,O2 = 22 ± 7 μM. Immunoblotting analysis shows that the protein is expressed at low levels in the vegetative trophozoites of Giardia; accordingly, these cells aerobically metabolize NO with low efficacy. Interestingly, in response to nitrosative stress (24-h incubation with ⩾5 mM nitrite) flavoHb expression is enhanced and the trophozoites thereby become able to metabolize NO efficiently, the activity being sensitive to both cyanide and carbon monoxide. The NO-donors S-nitrosoglutathione (GSNO) and DETA-NONOate mimicked the effect of nitrite on flavoHb expression. We propose that physiologically flavoHb contributes to NO detoxification in G. intestinalis.

Research highlights► NO degradation by Flavohemoglobin (FlavoHb) was measured by NO amperometry. ► FlavoHb was detected in Giardia intestinalis cells by immunoblotting. ► G. intestinalis cells were exposed to nitrosative stress using nitrite and NO-donors. ► G. intestinalis overexpresses FlavoHb under nitrosative stress conditions. ► The FlavoHb-overexpressing cells degrade NO more efficiently (∼×10) than controls.