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

Santiago-Tirado FH, Peng T, Yang M, Hang HC, Doering TL - PLoS Pathog. (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

Model of Pfa4 function and relationship to morphology, stress tolerance, and virulence.
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ppat.1004908.g008: Model of Pfa4 function and relationship to morphology, stress tolerance, and virulence.

Mentions: The combination of impaired cell wall synthesis and inability to appropriately respond to this condition results in weak and disorganized walls. This may impair other key virulence attributes of C. neoformans, such as the polysaccharide capsule, which associates with the cell wall. A perturbed wall, even in cells where the capsule is only slightly reduced in radius (as with pfa4Δ), may alter the capsule so that it cannot maintain its normal antiphagocytic role and exposes underlying wall components. This, combined with the changed wall arrangement, could explain our observation of abnormally high surface accessibility of specific cell wall components (Fig 3). These included cell wall mannoproteins [54, 55], which can interact with host phagocyte mannose receptors, and chitosan, which also interacts with macrophage receptors and induces a robust inflammatory response [56, 57]. Greater accessibility of these glycans could in turn explain the increased phagocytosis of pfa4Δ cells by macrophages. Once engulfed, these less robust cells, with defects in cell wall, signal transduction, and virulence factor expression, fare poorly (Fig 5A). Potentially reducing the pathogenicity even of cryptococci that remain outside of host phagocytes, we found important membrane transporters that are not correctly palmitoylated in the mutant. These include a putative carbohydrate transporter, a phosphate transporter, and NIC1 and SIT1, which transport nickel and siderophore-iron complexes, respectively [58, 59]. Because these metals are limiting during infection, incorrect processing or targeting of their transporters could influence pathogenesis. Furthermore, melanin, an important virulence factor in this pathogen, is poorly retained at the cell wall (Fig 7C–7E), a phenotype also seen in chs3Δ cells [32] and associated with reduced virulence. Ultimately, all of these factors combine to result in avirulence of the pfa4Δ mutant (Fig 8).


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

Santiago-Tirado FH, Peng T, Yang M, Hang HC, Doering TL - PLoS Pathog. (2015)

Model of Pfa4 function and relationship to morphology, stress tolerance, and virulence.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4430228&req=5

ppat.1004908.g008: Model of Pfa4 function and relationship to morphology, stress tolerance, and virulence.
Mentions: The combination of impaired cell wall synthesis and inability to appropriately respond to this condition results in weak and disorganized walls. This may impair other key virulence attributes of C. neoformans, such as the polysaccharide capsule, which associates with the cell wall. A perturbed wall, even in cells where the capsule is only slightly reduced in radius (as with pfa4Δ), may alter the capsule so that it cannot maintain its normal antiphagocytic role and exposes underlying wall components. This, combined with the changed wall arrangement, could explain our observation of abnormally high surface accessibility of specific cell wall components (Fig 3). These included cell wall mannoproteins [54, 55], which can interact with host phagocyte mannose receptors, and chitosan, which also interacts with macrophage receptors and induces a robust inflammatory response [56, 57]. Greater accessibility of these glycans could in turn explain the increased phagocytosis of pfa4Δ cells by macrophages. Once engulfed, these less robust cells, with defects in cell wall, signal transduction, and virulence factor expression, fare poorly (Fig 5A). Potentially reducing the pathogenicity even of cryptococci that remain outside of host phagocytes, we found important membrane transporters that are not correctly palmitoylated in the mutant. These include a putative carbohydrate transporter, a phosphate transporter, and NIC1 and SIT1, which transport nickel and siderophore-iron complexes, respectively [58, 59]. Because these metals are limiting during infection, incorrect processing or targeting of their transporters could influence pathogenesis. Furthermore, melanin, an important virulence factor in this pathogen, is poorly retained at the cell wall (Fig 7C–7E), a phenotype also seen in chs3Δ cells [32] and associated with reduced virulence. Ultimately, all of these factors combine to result in avirulence of the pfa4Δ mutant (Fig 8).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus