A Single Protein S-acyl Transferase Acts through Diverse Substrates to Determine Cryptococcal Morphology, Stress Tolerance, and Pathogenic Outcome

Abstract

Cryptococcus neoformans is an opportunistic yeast that kills over 625,000 people yearly through lethal meningitis. Host phagocytes serve as the first line of defense against this pathogen, but fungal engulfment and subsequent intracellular proliferation also correlate with poor patient outcome. Defining the interactions of this facultative intracellular pathogen with host phagocytes is key to understanding the latter’s opposing roles in infection and how they contribute to fungal latency, dissemination, and virulence. We used high-content imaging and a human monocytic cell line to screen 1,201 fungal mutants for strains with altered host interactions and identified multiple genes that influence fungal adherence and phagocytosis. One of these genes was PFA4, which encodes a protein S-acyl transferase (PAT), one of a family of DHHC domain-containing proteins that catalyzes lipid modification of proteins. Deletion of PFA4 caused dramatic defects in cryptococcal morphology, stress tolerance, and virulence. Bioorthogonal palmitoylome-profiling identified Pfa4-specific protein substrates involved in cell wall synthesis, signal transduction, and membrane trafficking responsible for these phenotypic alterations. We demonstrate that a single PAT is responsible for the modification of a subset of proteins that are critical in cryptococcal pathogenesis. Since several of these palmitoylated substrates are conserved in other pathogenic fungi, protein palmitoylation represents a potential avenue for new antifungal therapeutics.

A Family of Secretory Proteins Is Associated with Different Morphotypes in Cryptococcus neoformans

“…Cryptococcus WT strain XL280, the dha1Δ mutant, and the reconstituted strain were used for this study. Cryptococcus cells were opsonized with mouse serum at 37°C for 30 min as previously described (39). Freshly grown J774A.1 cells were seeded into a 24-well microtiter plate overnight at 37°C in 5% CO 2 , with each well containing 2.5 ϫ 10 5 Cryptococcus cells per 500 l. The next day, the medium in each well was replaced with fresh medium containing 2.5 ϫ 10 6 Cryptococcus cells in 500 l of medium.…”

All about that fat: Lipid modification of proteins in Cryptococcus neoformans

“…This and several other pfa4 phenotypes are not shared with other PAT mutants and are not complemented by active site mutants of Pfa4, suggesting they are due to dysfunction of one or more Pfa4-specific substrates (Santiago-Tirado et al , 2015). To globally identify Pfa4 substrates and mechanistically explain these defects, a chemical reporter for palmitate was used to label modified proteins in wild-type and pfa4 mutant cells.…”

“…Possible explanations for this widespread apparent redundancy include differing subcellular localization of the enzymes that enables spatial control of protein modification (Ohno et al , 2006; Howie et al , 2014), or distinct substrate specificity (Roth et al , 2006; Ohno et al , 2012). Although the mechanism of specificity remains elusive, recent work in several pathogens, including C. neoformans , has identified specific PATs that regulate critical pathogenic processes through the modification of unique protein substrates (Jones et al , 2012; Frenal et al , 2013; Santiago-Tirado et al , 2015). …”

“…The dominant modifier of C. neoformans Ras1, however, is encoded by PFA4 ; deletion of this gene partly phenocopies a ras1 deletion and alters Ras1 localization. The mutant also displays additional phenotypes unrelated to Ras function, including virulence defects in vitro and in vivo (Nichols et al , 2015; Santiago-Tirado et al , 2015). …”

“…Their occurrence in pathogenic microbes had been relatively unexplored, until recent reports detailing lipid modifications of proteins from bacteria (Hicks and Galan, 2013; Ivanov and Roy, 2013), parasites (Goldston et al , 2014), and fungi. The last group includes medically important organisms such as Candida albicans (Richard et al , 2002; Piispanen et al , 2011), Aspergillus fumigatus (Fontaine et al , 2003; Fortwendel et al , 2012; Fang et al , 2015), and Cryptococcus neoformans (Selvig et al , 2013; Nichols et al , 2015; Santiago-Tirado et al , 2015). …”