Histoplasma capsulatum secreted gamma-glutamyltransferase reduces iron by generating an efficient ferric reductant

Area of Science:

• Medical Mycology
• Molecular Biology
• Biochemistry

Background:

• Histoplasma capsulatum (Hc) is an intracellular fungal pathogen residing in macrophages, causing significant respiratory and systemic diseases.
• Iron acquisition is crucial for microbial pathogenesis, and host organisms employ iron limitation as an antimicrobial defense strategy.
• Hc possesses multiple iron acquisition mechanisms, including secreted glutathione-dependent ferric reductase activity (GSH-FeR).

Purpose of the Study:

• To elucidate the novel extracellular iron reduction strategy employed by Histoplasma capsulatum.
• To characterize the enzyme responsible for gamma-glutamyltransferase (Ggt1) and iron reduction activity.
• To understand the mechanism of iron acquisition involving Ggt1 and glutathione.

Main Methods:

• Purification of the secreted enzyme complex from Hc culture supernatant.
• Biochemical assays to determine enzyme activity (gamma-glutamyl transfer and iron reduction).
• Gene cloning, manipulation (overexpression and RNAi silencing), and enzyme inhibition experiments.

Main Results:

• A 320 kDa enzyme complex containing glycosylated subunits of approximately 50 and 37 kDa was purified.
• The purified enzyme exhibited both gamma-glutamyl transfer and iron reduction activities in the presence of glutathione.
• Overexpression or silencing of the GGT gene concurrently affected both GGT and GSH-FeR activities, indicating a link.
• Enzyme activity involves two steps: Ggt1 cleaves glutathione, releasing cysteinylglycine, which then reduces ferric iron.

Conclusions:

• A novel extracellular iron acquisition strategy in Hc involving gamma-glutamyltransferase (Ggt1) and glutathione was identified.
• Ggt1 plays a dual role, initiating glutathione breakdown and facilitating iron reduction via the released dipeptide.
• This unique Ggt1 function provides efficient iron acquisition over a broad pH range, offering new insights into Hc pathogenesis.