Cell wall melanin impedes growth of the Cryptococcus neoformans polysaccharide capsule by sequestering calcium

Cryptococcus neoformans has emerged as a frontrunner among deadly fungal pathogens and is particularly life-threatening for many HIV-infected individuals with compromised immunity. Multiple virulence factors contribute to the growth and survival of C. neoformans within the human host, the two most prominent of which are the polysaccharide capsule and melanin. As both of these features are associated with the cell wall, we were interested to explore possible cooperative or competitive interactions between these two virulence factors. Whereas capsule thickness had no effect on the rate at which cells became melanized, build-up of the melanin pigment layer resulted in a concomitant loss of polysaccharide material, leaving melanized cells with significantly thinner capsules than their non-melanized counterparts. When melanin was provided exogenously to cells in a transwell culture system we observed a similar inhibition of capsule growth and maintenance. Our results show that melanin sequesters calcium thereby limiting its availability to form divalent bridges between polysaccharide subunits required for outer capsule assembly. The decreased ability of melanized cells to incorporate exported polysaccharide into the growing capsule correlated with the amount of shed polysaccharide, which could have profound negative impacts on the host immune response. Significance StatementCryptococcus neoformans is an opportunistic fungal pathogen that presents a significant health risk for immunocompromised individuals. We report an interaction between the two major cryptococcal virulence factors, the polysaccharide capsule and melanin. Melanin impacted the growth and maintenance of the polysaccharide capsule, resulting in loss of capsular material during melanization. Our results suggest that melanin can act as a sink for calcium, thereby limiting its availability to form ionic bridges between polysaccharide chains on the growing surface of the outer capsule. As polysaccharide is continuously exported to support capsule growth, failure of melanized cells to incorporate this material results in a higher concentration of shed polysaccharide in the extracellular milieu, which is expected to interfere with host immunity.