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The Aspergillus fumigatus Sialidase (Kdnase) Contributes to Cell Wall Integrity and Virulence in Amphotericin B-Treated Mice.

ABSTRACT: Aspergillus fumigatus is a filamentous fungus that can cause a life-threatening invasive pulmonary aspergillosis (IPA) in immunocompromised individuals. We previously characterized an exo-sialidase from A. fumigatus that prefers the sialic acid substrate, 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (Kdn); hence it is a Kdnase. Sialidases are known virulence factors in other pathogens; therefore, the goal of our study was to evaluate the importance of Kdnase in A. fumigatus. A kdnase knockout strain (?kdnase) was unable to grow on medium containing Kdn and displayed reduced growth and abnormal morphology. ?kdnase was more sensitive than wild type to hyperosmotic conditions and the antifungal agent, amphotericin B. In contrast, ?kdnase had increased resistance to nikkomycin, Congo Red and Calcofluor White indicating activation of compensatory cell wall chitin deposition. Increased cell wall thickness and chitin content in ?kdnase were confirmed by electron and immunofluorescence microscopy. In a neutropenic mouse model of invasive aspergillosis, the ?kdnase strain had attenuated virulence and a significantly lower lung fungal burden but only in animals that received liposomal amphotericin B after spore exposure. Macrophage numbers were almost twofold higher in lung sections from mice that received the ?kdnase strain, possibly related to higher survival of macrophages that internalized the ?kdnase conidia. Thus, A. fumigatus Kdnase is important for fungal cell wall integrity and virulence, and because Kdnase is not present in the host, it may represent a potential target for the development of novel antifungal agents.

SUBMITTER: Nesbitt JR

PROVIDER: S-EPMC5778107 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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The Aspergillus fumigatus Sialidase (Kdnase) Contributes to Cell Wall Integrity and Virulence in Amphotericin B-Treated Mice.

Nesbitt Jason R JR Steves Elizabeth Y EY Schonhofer Cole R CR Cait Alissa A Manku Sukhbir S SS Yeung Juliana H F JHF Bennet Andrew J AJ McNagny Kelly M KM Choy Jonathan C JC Hughes Michael R MR Moore Margo M MM

Frontiers in microbiology 20180118

Aspergillus fumigatus is a filamentous fungus that can cause a life-threatening invasive pulmonary aspergillosis (IPA) in immunocompromised individuals. We previously characterized an exo-sialidase from A. fumigatus that prefers the sialic acid substrate, 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (Kdn); hence it is a Kdnase. Sialidases are known virulence factors in other pathogens; therefore, the goal of our study was to evaluate the importance of Kdnase ...[more]

PMID: 29403452

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Project description:Histone acetyltransferase (HAT)-mediated epigenetic modification is essential for diverse cellular processes in eukaryotes. However, the functions of HATs in the human pathogen Aspergillus fumigatus remain poorly understood. In this study, we characterized the functions of MOZ, Ybf2/Sas3, Sas2, and Tip60 (MYST)-family histone acetyltransferase something about silencing (Sas3) in A. fumigatus. Phenotypic analysis revealed that loss of Sas3 results in significant impairments in colony growth, conidiation, and virulence in the Galleria mellonella model. Subcellular localization and Western blot analysis demonstrated that Sas3 localizes to nuclei and is capable of acetylating lysine 9 and 14 of histone H3 in vivo. Importantly, we found that Sas3 is critical for the cell wall integrity (CWI) pathway in A. fumigatus as evidenced by hypersensitivity to cell wall-perturbing agents, altered cell wall thickness, and abnormal phosphorylation levels of CWI protein kinase MpkA. Furthermore, site-directed mutagenesis studies revealed that the conserved glycine residues G641 and G643 and glutamate residue E664 are crucial for the acetylation activity of Sas3. Unexpectedly, only triple mutations of Sas3 (G641A/G643A/E664A) displayed defective phenotypes similar to the Δsas3 mutant, while double or single mutations did not. This result implies that the role of Sas3 may extend beyond histone acetylation. Collectively, our findings demonstrate that MYST-family HAT Sas3 plays an important role in the fungal development, virulence, and cell wall integrity in A. fumigatus.ImportanceEpigenetic modification governed by HATs is indispensable for various cellular processes in eukaryotes. Nonetheless, the precise functions of HATs in the human pathogen Aspergillus fumigatus remain elusive. In this study, we unveil the roles of MYST-family HAT Sas3 in colony growth, conidiation, virulence, and cell wall stress response in A. fumigatus. Particularly, our findings demonstrate that Sas3 can function through mechanisms unrelated to histone acetylation, as evidenced by site-directed mutagenesis experiments. Overall, this study broadens our understanding of the regulatory mechanism of HATs in fungal pathogens.

Project description:Aspergillus fumigatus is a major cause of human disease. The survival of this fungus is dependent on the cell wall organization and function of its components. The cell wall integrity pathway (CWIP) is the primary signaling cascade that controls de novo synthesis of the cell wall in fungi. Abundant conidiation is a hallmark in A. fumigatus, and uptake of conidia by a susceptible host is usually the initial event in infection. The formation of conidia is mediated by the development of fungus-specific specialized structures, conidiophores, which are accompanied by cell wall remodeling. The molecular regulation of these changes in cell wall composition required for the rise of conidiophore from the solid surface and to disperse the conidia into the air is currently unknown. Here, we investigated the role of CWIP in conidiation. We show that CWIP pkcAG579R, ΔmpkA, and ΔrlmA mutants displayed reduced conidiation during synchronized asexual differentiation. The transcription factor RlmA directly regulated the expression of regulators of conidiation, including flbB, flbC, brlA, abaA, and rasB, as well as genes involved in cell wall synthesis and remodeling, and this affected the chitin content in aerial hyphae. Phosphorylation of RlmA and MpkA was increased during asexual differentiation. We also observed that MpkA physically associated with the proteins FlbB, FlbC, BrlA, and RasB during this process, suggesting another level of cross talk between the CWIP and asexual development pathways. In summary, our results support the conclusion that one function of the CWIP is the regulation of asexual development in filamentous fungi.IMPORTANCE A remarkable feature of the human pathogen Aspergillus fumigatus is its ability to produce impressive amounts of infectious propagules known as conidia. These particles reach immunocompromised patients and may initiate a life-threatening mycosis. The conidiation process in Aspergillus is governed by a sequence of proteins that coordinate the development of conidiophores. This process requires the remodeling of the cell wall so that the conidiophores can rise and withstand the chains of conidia. The events regulating cell wall remodeling during conidiation are currently unknown. Here, we show that the cell wall integrity pathway (CWIP) components RlmA and MpkA directly contribute to the activation of the conidiation cascade by enabling transcription or phosphorylation of critical proteins involved in asexual development. This study points to an essential role for the CWIP during conidiation and provides further insights into the complex regulation of asexual development in filamentous fungi.

Dataset Information

The Aspergillus fumigatus Sialidase (Kdnase) Contributes to Cell Wall Integrity and Virulence in Amphotericin B-Treated Mice.

Publications

The <i>Aspergillus fumigatus</i> Sialidase (Kdnase) Contributes to Cell Wall Integrity and Virulence in Amphotericin B-Treated Mice.

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