Intracellular siderophore but not extracellular siderophore is required for full virulence in Metarhizium robertsii

Authors: DONZELLI, BRUNO G. - Cornell University; Gibson, Donna; Krasnoff, Stuart

Submitted to: Fungal Genetics and Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/27/2015
Publication Date: 6/29/2015
Citation: Donzelli, B.G., Gibson, D.M., Krasnoff, S. 2015. Intracellular siderophore but not extracellular siderophore is required for full virulence in Metarhizium robertsii. Fungal Genetics and Biology. 82:56-68.

Interpretive Summary: The low solubility of iron in nature presents an iron bioavailability challenge that many plants, fungi, and bacteria meet by producing siderophores, small molecules that are specialized for strong binding of iron. Producing organisms secrete external siderophores into the environment to scavenge iron. The siderophore-iron complex can then be recovered by the organism, which then internalizes the iron payload by specialized biochemical processes and transports it to cellular target sites using a second type of internal siderophore. Iron is a key nutrient for virtually all organisms and iron restriction, if successful, can stop pathogens from producing disease. Metarhizium robertsii is an insect-pathogenic fungus that is widespread geographically and infects a wide range of injurious insect species. Thus, this fungus is important because of its potential as a natural biocontrol agent of insects and several related species of Metarhizium have been commercialized as safe pesticides and are now important components of integrated pest management programs. In light of the key role played by microbial siderophores in pathogenic processes we explored the genes that control their biosynthesis. By inactivating them we found that siderophores used to bind iron outside the cell are not required for successful infection. This implies other iron acquisition systems not involving siderophores are deployed by the pathogen during infection. We also inactivated a gene that acts as a brake on the iron acquisition system, but found that this did not increase virulence. However, loss of the internal siderophore reduced M. robertsii virulence of 2-3 fold. These studies advance our knowledge of how this biocontrol fungus acquires and manages iron and how these capabilities contribute to the ability of this fungus to successfully infect and kill insects.

Technical Abstract: Efficient iron acquisition mechanisms are fundamental for microbial survival in the environment and for pathogen virulence within their hosts. M. robertsii produces two known iron-binding natural products: metachelins, which are used to scavenge extracellular iron, and ferricrocin, which is strictly intracellular. To study the contribution of siderophore-mediated iron uptake and storage to M. robertsii fitness, we generated null mutants for each siderophore synthase genes (mrsidD and mrsidC, respectively), as well as uptake-deregulated mutants by inactivating the transcriptional repressor mrsreA. All of these mutants showed impaired germination speed, differential sensitivity to hydrogen peroxide, and differential ability to overcome iron chelation on growth-limiting iron concentrations. RT-qPCR data supported regulation of mrsreA, mrsidC, and mrsidD by supplied iron in vitro and during growth within the insect host, Spodoptera exigua. We also observed strong upregulation of the insect iron-binding proteins, transferrins, during infection. Insect bioassays revealed that ferricrocin is required for full virulence against S. exigua; neither the loss of metachelin production nor the deletion of the transcription factor mrsreA significantly affected M. robertsii virulence.