neoformans Cryptococcus AMP / Protein Kinase A Pathways in Cross Talk between the Cell Wall Integrity and Cyclic 2014

Cryptococcus neoformans is a fungal pathogen of immunocompromised people that causes fatal meningitis. The fungal cell wall is essential to viability and pathogenesis of C. neoformans, and biosynthesis and repair of the wall is primarily controlled by the cell wall integrity (CWI) signaling pathway. Previous work has shown that deletion of genes encoding the four major kinases in the CWI signaling pathway, namely, PKC1, BCK1, MKK2, and MPK1 results in severe cell wall phenotypes, sensitivity to a variety of cell wall stressors, and for Mpk1, reduced virulence in a mouse model. Here, we examined the global transcriptional responses to gene deletions of BCK1, MKK2, and MPK1 compared to wild-type cells. We found that over 1,000 genes were differentially expressed in one or more of the deletion strains, with 115 genes differentially expressed in all three strains, many of which have been identified as genes regulated by the cyclic AMP (cAMP)/protein kinase A (PKA) pathway. Biochemical measurements of cAMP levels in the kinase deletion strains revealed significantly less cAMP in all of the deletion strains compared to the wild-type strain. The deletion strains also produced significantly smaller capsules than the wild-type KN99 strain did under capsule-inducing conditions, although the levels of capsule they shed were similar to those shed by the wild type. Finally, addition of exogenous cAMP led to reduced sensitivity to cell wall stress and restored surface capsule to levels near those of wild type. Thus, we have direct evidence of cross talk between the CWI and cAMP/PKA pathways that may have important implications for regulation of cell wall and capsule homeostasis. IMPORTANCE Cryptococcus neoformans is a fungal pathogen of immunocompromised people that causes fatal meningitis. The fungal cell wall is essential to viability and pathogenesis of C. neoformans, and biosynthesis and repair of the wall are primarily controlled by the cell wall integrity (CWI) signaling pathway. In this study, we demonstrate that deletion of any of three core kinases in the CWI pathway impacts not only the cell wall but also the amount of surface capsule. Deletion of any of the kinases results in significantly reduced cellular cyclic AMP (cAMP) levels, and addition of exogenous cAMP rescues the capsule defect and some cell wall defects, supporting a direct role for the CWI pathway in regulation of capsule in conjunction with the cAMP/ protein kinase A pathway. Received 30 June 2014 Accepted 3 July 2014 Published 12 August 2014 Citation Donlin MJ, Upadhya R, Gerik KJ, Lam W, Gibbs VanArendonk L, Specht CA, Sharma NK, Lodge JK. 2014. Cross talk between the cell wall integrity and cyclic AMP/protein kinase A pathways in Cryptococcus neoformans. mBio 5(4):e01573-14. doi:10.1128/mBio.01573-14. Editor Joseph Heitman, Duke University Copyright © 2014 Donlin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Address correspondence to Jennifer K. Lodge, lodgejk@wustl.edu. This article is a direct contribution from a Fellow of the American Academy of Microbiology. Cryptococcus neoformans is a fungal pathogen of immunocompromised people that causes an estimated 1 million infections each year among HIV-positive patients globally, resulting in 600,000 deaths annually (1). Much of the disease burden occurs in sub-Saharan Africa, where deaths from cryptococcal infections may exceed those from tuberculosis in some areas (1). Cryptococcal infections can be successfully treated with antifungal agents, but the mortality rate remains 15 to 30%, even in the context of antiviral treatments for HIV (2–4). The closely related species Cryptococcus gattii can infect immunocompetent people and animals. Although C. gattii is more common in Australia, it received global attention in 1999 when it emerged on Vancouver Island in Canada and has since spread into other areas of the Pacific Northwest (5–7). Infection by C. neoformans occurs upon inhalation of spores or desiccated yeast cells, which can then disseminate to the brain and cause fatal meningitis (reviewed in references 8, 9, and 10). C. neoformans rapidly establishes an infection in lung alveoli, where low iron availability and increased carbon dioxide levels induce the formation of a polysaccharide capsule on the surface of the cryptococcal cell. The C. neoformans capsule has been shown to be protective against phagocytosis and oxidative stress as well as reducing the T-cell immune response (11, 12). The capsule is a key virulence factor, and strains with disruptions or deletions of capsule-related genes are usually avirulent or demonstrate attenuated virulence in murine models of infection (reviewed in reference 13). C. neoformans is a soilborne pathogen found ubiquitously in RESEARCH ARTICLE crossmark July/August 2014 Volume 5 Issue 4 e01573-14 ® mbio.asm. org 1 m b.asm . rg on A uust 6, 2014 P ubished by m b.asm . rg D ow nladed fom