Solving the puzzle of beta-1,6-glucan in the cryptococcal cell wall

Solving the puzzle of beta-1,6-glucan in the cryptococcal cell wall ABSTRACT Cryptococcus neoformans is a global pathogen responsible for roughly 150,000 deaths each year, mainly in HIV+ individuals but with increasing morbidity in non-AIDS patient populations. Like other fungi, C. neoformans is bounded by a complex and resilient cell wall, which maintains cell integrity while allowing morphologic flexibility. Wall synthesis is a compelling research area, because it is a validated target of antifungal drugs. Unfortunately, these do not work against C. neoformans infection, revealing a pressing need to elucidate the biosynthesis - and potential vulnerabilities - of the cryptococcal wall. Our long-term goal is to define cell wall biosynthesis in C. neoformans, to deepen our understanding of this critical process and potentially identify targets for antifungal therapy. Much of the wall is composed of various polymers of glucose, or glucans, which differ in their linkage and branching patterns. Our focus here is on β-1,6- glucan, which is a key component of cell walls because it helps interconnect other wall components but whose synthesis is poorly understood. This material is particularly abundant in C. neoformans, even though the crypto- coccal genome does not encode homologs of several proteins that have been implicated in its synthesis in other yeast. We have discovered that cryptococcal cells lacking a putative glycosyltransferase called Ggt2 are dra- matically impaired in wall β-1,6-glucan, cell integrity, and virulence. Based on our preliminary results and struc- tural features of this protein, we hypothesize that Ggt2 acts directly in the formation of β-1,6-glucan. To pursue our promising initial findings, we propose to determine how Ggt2 affects cryptococcal wall structure, its cellular role, and its biochemical activity. We further propose to identify the C. neoformans machinery required for β-1,6-glucan synthesis. We plan to achieve these goals by pursuing three aims in parallel: Aim 1 is to combine biochemical and biophysical methods to define the cell wall of wild-type and ggt2∆ cells. Aim 2 is to establish the biochemical function of Ggt2 and its role in cell wall synthesis. Aim 3 is to apply genetic strategies to discover proteins that either participate in β-1,6-glucan synthesis or compensate for its loss, and to initiate mechanistic studies of these candidates. We propose highly feasible studies in pursuit of aims that are designed to be independent yet complementary. Our results will increase fundamental understanding of the cryptococcal wall and processes that are crucial for its integrity, and possibly open the way to new therapeutic strategies. Achieving our aims will also leave us poised for exciting future studies, including further structural studies of fungal walls, potential screens for inhibitors of Ggt2, and mechanistic dissection of β-1,6-glucan synthesis with potential application to other fungi. Project Number: 1R01AI192892-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Tamara Doering (+1 co-PI) | Institution: WASHINGTON UNIVERSITY, SAINT LOUIS, MO | Award Amount: $773,053 | Activity Code: R01 | Study Section: Pathogenic Eukaryotes Study Section[PTHE] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R01AI19289201