Interactions between copper homeostasis and the fungal cell wall affect copper stress resistance

Abstract

Copper homeostasis mechanisms are essential for microbial adaption to changing copper levels within the host during infection. In the opportunistic fungal pathogen Cryptococcus neoformans (Cn), the Cn Cbi1/Bim1 protein is a newly identified copper binding and release protein that is highly induced during copper limitation. Recent studies demonstrated that Cbi1 functions in copper uptake through the Ctr1 copper transporter during copper limitation. However, the mechanism of Cbi1 action is unknown. The fungal cell wall is a dynamic structure primarily composed of carbohydrate polymers, such as chitin and chitosan, polymers known to strongly bind copper ions. We demonstrated that Cbi1 depletion affects cell wall integrity and architecture, connecting copper homeostasis with adaptive changes within the fungal cell wall. The cbi1Δ mutant strain possesses an aberrant cell wall gene transcriptional signature as well as defects in chitin / chitosan deposition and exposure. Furthermore, using Cn strains defective in chitosan biosynthesis, we demonstrated that cell wall chitosan modulates the ability of the fungal cell to withstand copper stress. Given the previously described role for Cbi1 in copper uptake, we propose that this copper-binding protein could be involved in shuttling copper from the cell wall to the copper transporter Ctr1 for regulated microbial copper uptake. Microorganisms must be equipped to readily acquire essential micro-nutrients like copper from nutritionally poor environments while simultaneously shielding themselves from conditions of metal excess. We explored mechanisms of microbial copper homeostasis in the human opportunistic fungal pathogen Cryptococcus neoformans (Cn) by defining physiological roles of the newly described copper-binding and release protein Cn Cbi1/Bim1. Highly induced during copper limitation, Cbi1 has been shown to interact with the high-affinity copper transporter Ctr1. We defined Cbi1-regulated changes in the fungal cell wall, including controlling levels and surface exposure of the structural carbohydrates chitin and chitosan during copper and host-derived stress. These polysaccharides are embedded deeply in the cell wall and are known to avidly bind copper. Our data suggest a model in which the fungal cell wall, especially the chito-oligomer layer, serves as a copper-binding structure. Also, alterations in the cell wall polysaccharide content affect the ability to withstand excess copper. Given its ability to bind and release copper, the Cbi1 protein likely shuttles copper from the cell wall to copper transporters for regulated copper acquisition.