title: Histoplasma capsulatum secreted gamma-glutamyltransferase reduces iron by generating an efficient ferric reductant.
abstract: The intracellular fungal pathogen Histoplasma capsulatum (Hc) resides in mammalian macrophages and causes respiratory and systemic disease. Iron limitation is an important host antimicrobial defence, and iron acquisition is critical for microbial pathogenesis. Hc displays several iron acquisition mechanisms, including secreted glutathione-dependent ferric reductase activity (GSH-FeR). We purified this enzyme from culture supernatant and identified a novel extracellular iron reduction strategy involving gamma-glutamyltransferase (Ggt1) activity. The 320 kDa complex was composed of glycosylated protein subunits of about 50 and 37 kDa. The purified enzyme exhibited gamma-glutamyl transfer activity as well as iron reduction activity in the presence of glutathione. We cloned and manipulated expression of the encoding gene. Overexpression or RNAi silencing affected both GGT and GSH-FeR activities concurrently. Enzyme inhibition experiments showed that the activity is complex and involves two reactions. First, Ggt1 initiates enzymatic breakdown of GSH by cleavage of the gamma-glutamyl bond and release of cysteinylglycine. Second, the thiol group of the released dipeptide reduces ferric to ferrous iron. A combination of kinetic properties of both reactions resulted in efficient iron reduction over a broad pH range. Our findings provide novel insight into Hc iron acquisition strategies and reveal a unique aspect of Ggt1 function in this dimorphic mycopathogen.
authors.affiliation: Department of Medical Microbiology and Immunology, University of Wisconsin, Madison, WI, USA. rzarnowski@wisc.edu
authors.forename: Robert
authors.initials: R
authors.lastname: Zarnowski
authors.forename: Kendal G
authors.initials: KG
authors.lastname: Cooper
authors.forename: Laura Schmitt
authors.initials: LS
authors.lastname: Brunold
authors.forename: Jimmy
authors.initials: J
authors.lastname: Calaycay
authors.forename: Jon P
authors.initials: JP
authors.lastname: Woods