Grant: Discovery Grants Program - Individual

Giardia intestinalis is a parasitic protist found in fresh waters worldwide that can infect humans, livestock, pets, and wildlife. Giardia has many unusual characteristics, and one of these is its inability to make the iron-containing cofactor heme. This versatile molecule has many uses and its most well-known role is as the oxygen-binding group in hemoglobin. Although it cannot make heme, Giardia does have heme proteins, although far fewer types than occur in other organisms. One of these heme proteins is the enzyme flavohemoglobin which protects Giardia from the toxic effects of the free radical nitric oxide that is produced by the host; flavohemoglobin oxidizes this nitric oxide to harmless nitrate. The structure of flavohemoglobin is a fusion of two domains linked on a single protein chain. One domain of flavohemoglobin resembles hemoglobin, and binds the heme cofactor, where oxygen, nitric oxide and an electron combine to make nitrate. The other domain binds a flavin cofactor that feeds the electrons into the globin domain. Flavohemoglobins are also found in other single-celled organisms such as bacteria and yeast, but the Giardia version is unusual in being slightly larger, owing to extra amino acid sequences inserted into each domain of the enzyme that may strengthen the interactions between domains. We are interested in how these inserts affect the functional properties of the Giardia flavohemoglobin compared to flavohemoglobins of other species. We also make precise changes in the amino acid sequences within these inserts and then measure the outcomes of these changes on the properties of these variant versions compared to the original unchanged Giardia flavohemoglobin. We are also interested in the relation between Giardia flavohemoglobin and resistance to two drugs, albendazole and metronidazole, that are commonly used to treat Giardia infections in humans and pets. Interestingly, some of the flavohemoglobins in the metronidazole-resistant strains have altered amino acid sequences within these insert regions. The other four heme proteins of Giardia are all members of the same protein family: the small electron transfer protein cytochrome b5, which participates in biochemical oxidation-reduction reactions. This protein is also found in humans and other species and has been well-studied. However, the presence of four of these proteins in Giardia is still a mystery - it is as if one is at a theatre and knows who the actors are, and where they are on the stage, but not their roles. In a similar way, we know where these different cytochromes are located within the Giardia cell, and know some of their properties, but we do not know what proteins they receive electrons from or which proteins they transfer electrons to. Our research on the cytochromes aims to identify their electron transfer partners, and to identify that factors that direct them to their appropriate location within the Giardia cell.