The Aspergillus fumigatus sitA Phosphatase Homologue Is Important for Adhesion, Cell Wall Integrity, Biofilm Formation, and Virulence

Abstract

h Aspergillus fumigatus is an opportunistic pathogenic fungus able to infect immunocompromised patients, eventually causing disseminated infections that are difficult to control and lead to high mortality rates. It is important to understand how the signaling pathways that regulate these factors involved in virulence are orchestrated. Protein phosphatases are central to numerous signal transduction pathways. Here, we characterize the A. fumigatus protein phosphatase 2A SitA, the Saccharomyces cerevisiae Sit4p homologue. The sitA gene is not an essential gene, and we were able to construct an A. fumigatus null mutant. The ⌬sitA strain had decreased MpkA phosphorylation levels, was more sensitive to cell wall-damaging agents, had increased ␤-(1,3)-glucan and chitin, was impaired in biofilm formation, and had decreased protein kinase C activity. The ⌬sitA strain is more sensitive to several metals and ions, such as MnCl 2 , CaCl 2 , and LiCl, but it is more resistant to ZnSO 4 . The ⌬sitA strain was avirulent in a murine model of invasive pulmonary aspergillosis and induces an augmented tumor necrosis factor alpha (TNF-␣) response in mouse macrophages. These results stress the importance of A. fumigatus SitA as a possible modulator of PkcA/MpkA activity and its involvement in the cell wall integrity pathway.

Genome-wide transcriptome analysis ofAspergillus fumigatusexposed to osmotic stress reveals regulators of osmotic and cell wall stresses that are SakAHOG1and MpkC dependent

“…Both Δ atfA Afs35 and Δ atfA CEA17 had decreased growth in 1.2 M sorbitol (Figure a), while Δ atfB‐D CEA17 showed comparable growth with the wild‐type strain (data not shown). Alterations in osmolarity can have an impact on the composition and structure of the cell wall (Valiante et al, ; Altwasser et al, ; Winkelströter et al, a; Bom et al, ; Bruder Nascimento et al, ). In accordance, both Δ atfA Afs35 and Δ atfA CEA17 mutants were sensitive to the cell wall‐damaging agents calcofluor white and Congo red.…”

Functional characterization of clinical isolates of the opportunistic fungal pathogenAspergillus nidulans

“…Phagocytosis and killing assays of A. nidulans conidia by wild-type and gp91phox knockout macrophages were carried out according to Bom et al (2015) (70) with modifications. 24-well plates containing a 15-mm-diameter coverslip in each well (phagocytosis assay) or without any coverslip (killing assay) and 2 x 10 5 macrophages per well were incubated in 1 ml of RPMI-FBS [(RPMI medium (Gibco) supplemented with 10% inactivated fetal bovine serum (FBS) (Gibco), 2 mM glutamine and 100 units/mL of penicillin-streptomycin (Life Technologies)] at 37 °C, 5% CO2 for 24 h. Wells were washed with 1 ml of PBS before the same volume of RPMI-FBS medium supplemented with 1 x 10 6 conidia (1:5 macrophage/conidium ratio) was added in the same conditions.…”

“…Virulence of the A. nidulans strains was determined in immunocompetent, CGD and neutropenic mice. A. nidulans conidial suspensions were prepared and viability experiments carried out as described previously (70). Eight to twelve weeks old wild-type (n=10) and gp91 phox knockout (n=7) C57BL/6 male mice were used as immunocompetent and CGD models, respectively.…”

“…(intratracheal) inoculation, which allowed dispensing of the fungal conidia directly into the lungs. Neutropenic mice were infected by intranasal instillation of 1.0 x 10 4 conidia as described previously (70). PBS (phosphate buffered saline) was administered as a negative control for each murine model.…”

Biofilm Exopolysaccharides of Pathogenic Fungi: Lessons from Bacteria

“…Production of GAG is dependent on the developmental regulatory proteins MedA (8,41,42), StuA (8), and SomA (43). Activation of the cell wall integrity pathway through deletion of the phosphatases SitA (44) or PtcB (45) has been linked to suppression of biofilm formation and extracellular matrix production. Although in bacteria, there is a well established role for cyclic di-GMP as a signaling molecule that stimulates exopolysaccharide production, there is no evidence that fungi, including A. fumigatus, produce this molecule.…”