Aspergillus fumigatus Trehalose-Regulatory Subunit Homolog Moonlights To Mediate Cell Wall Homeostasis through Modulation of Chitin Synthase Activity

Abstract

Trehalose biosynthesis is found in fungi but not humans. Proteins involved in trehalose biosynthesis are essential for fungal pathogen virulence in humans and plants through multiple mechanisms. Loss of canonical trehalose biosynthesis genes in the human pathogen Aspergillus fumigatus significantly alters cell wall structure and integrity, though the mechanistic link between these virulence-associated pathways remains enigmatic. Here we characterize genes, called tslA and tslB, which encode proteins that contain domains similar to those corresponding to trehalose-6-phosphate phosphatase but lack critical catalytic residues for phosphatase activity. Loss of tslA reduces trehalose content in both conidia and mycelia, impairs cell wall integrity, and significantly alters cell wall structure. To gain mechanistic insights into the role that TslA plays in cell wall homeostasis, immunoprecipitation assays coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to reveal a direct interaction between TslA and CsmA, a type V chitin synthase enzyme. TslA regulates not only chitin synthase activity but also CsmA sub-cellular localization. Loss of TslA impacts the immunopathogenesis of murine invasive pulmonary aspergillosis through altering cytokine production and immune cell recruitment. In conclusion, our data provide a novel model whereby proteins in the trehalose pathway play a direct role in fungal cell wall homeostasis and consequently impact fungus-host interactions.IMPORTANCE Human fungal infections are increasing globally due to HIV infections and increased use of immunosuppressive therapies for many diseases. Therefore, new antifungal drugs with reduced side effects and increased efficacy are needed to improve treatment outcomes. Trehalose biosynthesis exists in pathogenic fungi and is absent in humans. Components of the trehalose biosynthesis pathway are important for the virulence of human-pathogenic fungi, including Aspergillus fumigatus Consequently, it has been proposed that components of this pathway are potential targets for antifungal drug development. However, how trehalose biosynthesis influences the fungus-host interaction remains enigmatic. One phenotype associated with fungal trehalose biosynthesis mutants that remains enigmatic is cell wall perturbation. Here we discovered a novel moonlighting role for a regulatory-like subunit of the trehalose biosynthesis pathway in A. fumigatus that regulates cell wall homeostasis through modulation of chitin synthase localization and activity. As the cell wall is a current and promising therapeutic target for fungal infections, understanding the role of trehalose biosynthesis in cell wall homeostasis and virulence is expected to help define new therapeutic opportunities.

Keywords: Aspergillus fumigatus; cell wall; chitin; filamentous fungi; pathogenesis; trehalose.

FIG 1

Loss of TslA and TslB decreases trehalose production in both conidia (A) and hyphae (B). Quantitation of trehalose production in conidia and mycelia was performed using glucose oxidase (GO) assays (Sigma) after trehalase enzyme incubation. For the conidial stage, 2 × 10⁸ conidia were used to extract trehalose by boiling at 100°C for 20 min and collecting the supernatant to perform GO assays. For the mycelial stage, 1 × 10⁸ conidia were cultured in 10 ml LGMM at 37°C for 16 h, the mycelia were weighed and lyophilized, and trehalose extraction was performed. Data are presented as means ± SE of results from three biological replicates. ***, P < 0.0001 (unpaired two-tailed Student’s t test compared to the wild-type CEA10 results).

FIG 2

Loss of TslA increases fungal susceptibility to cell wall-perturbing agents (A), and growth of the ΔtslA strain is restored on sorbitol minimal media (SMM) and Sabouraud dextrose media (SDA) in the presence of 50 μg/ml calcofluor white (B). Dropout assays were performed at 37°C for 2 days by using 10⁵ to 10² conidia for each strain inoculated on GMM with or without cell wall-perturbing agents, i.e., 1 mg/ml Congo red, 50 μg/ml calcofluor white, and 1 μg/ml caspofungin. Images and data are representative of three independent experiments with similar results.

FIG 3

Loss of TslA decreases fungal cell wall thickness and results in accumulation of electron-dense material at the outer layer of the cell wall. Mycelia from each strain were prepared for TEM as previously described (19, 39). Cell wall thickness was analyzed by ImageJ. Data are presented as means ± SE of 10 measurements from two biological replicates of each strain. **, P value = 0.002 (unpaired two-tailed Student’s t test compared to the wild-type CEA10 results). Bars, 500 nm.

FIG 4

Loss of TslA alters MAMP cell wall exposure. (A and B) The ΔtslA strain has increased chitin levels/exposure as measured by calcofluor white (CFW) staining (A) or wheat germ agglutinin (WGA) (B) compared to the results seen with wild-type CEA10 and the reconstituted ΔtslA+tslA strain. Each strain was cultured into the germling stage under normoxic conditions at 37°C. The germlings were UV irradiated and stained with 25 μg/ml CFW or with 5 μg/ml WGA. The mean intensity was analyzed using ImageJ and the corrected total cell fluorescence (CTCF) was calculated (69, 70). **, P value = 0.0074 for CFW and 0.0017 for WGA compared to CEA10 (unpaired two-tailed Student’s t test compared to the wild-type CEA10 results). DIC, differential interference contrast. (B) The ΔtslA strain has decreased β-glucan exposure as measured by s-dectin-1 staining compared to the wild-type CEA10 and the reconstituted ΔtslA+tslA strain. Each strain was cultured to the germling stage under normoxic conditions at 37°C. The germlings were UV irradiated, blocked, and stained with a conditioned medium containing s-dectin1-hFc followed by Alexa Fluor 488-conjugated, goat anti-human IgG1. The corrected total cell fluorescence (CTCF) was calculated. ***, P value = 0.0005 (unpaired two-tailed Student’s t test compared to the wild-type CEA10 results). Data are presented as means ± SE of 15 images from three biological replicates. Bar, 3 μm.

FIG 5

Loss of TslA does not affect the cell wall integrity and HOG-MAPK pathways (A) and does not change the expression of fksA and csmA (B). A total of 10⁶ conidia of the wild-type strain and the ΔtslA strain were incubated overnight in liquid GMM, and CFW was added for 0, 15, and 60 min as indicated. Samples were collected, and RNA extraction was performed for measuring rlmA, atfA, fksA, and csmA mRNA abundance using qRT-PCR analysis as previously described (15). Data are presented as means ± SD of results from three biological replicates of each strain.

FIG 6

TslA physically interacts with CsmA. Affinity purification assays from Flag-tagged CsmA strains in the background of S-tagged TslA were performed with S-protein beads (A) and anti-Flag beads (B) to verify interactions. Data are images representative of results from three independent experiments, all with similar results.

FIG 7

Loss of TslA increases chitin synthase activity. Ten micrograms of membrane proteins were used to perform a nonradioactive chitin synthase activity assay. Each strain was cultured at 30°C for 6 h and switched to 37°C for 24 h. Ten micrograms of the wild type’s membrane proteins was used to compare with no substrate, UDP-N-acetyl glucosamine (UDP-GlcNAc) and no trypsin as negative-control assays. *, P = 0.0117; **, P = 0.0013; ***, P < 0.0001 (unpaired two-tailed Student’s t test compared to the wild-type CEA10 results). Data are presented as means ± SE of results from three biological replicates.

FIG 8

TslA promotes CsmA hyphal tip localization. (A) Western blot analysis of C-terminal GFP-tagged CsmA in the wild-type strain, ΔtslA strain, and ΔtslA+tslA strain backgrounds. (B) C-terminal GFP-tagged CsmA was generated in the wild-type strain, ΔtslA, and ΔtslA+tslA mutant backgrounds. Each strain was cultured at 37°C for 12 h, and live-cell imaging was performed using a Quorum Technologies WaveFX spinning disk confocal microscope (magnification, ×1,000). The images were analyzed using Imaris 8.1.4 software. (C) Loss of TslA changes the number of CsmA puncta at the hyphal tip. To quantify the puncta at the subapex region (within 20 μm of the tip), the puncta in the images were counted and analyzed using Imaris 8.1.4 software. ***, P < 0.0001 (unpaired two-tailed t test compared to the wild-type CEA10 results). (D) Localization of GFP-tagged TslA. C-terminal GFP-tagged TslA was generated and cultured at 37°C for 12 h. Live-cell imaging was performed using a Quorum Technologies WaveFX spinning disk confocal microscope (magnification, ×1,000). The three-dimensional (3D) structure of TslA puncta was created by the use of Imaris 8.1.4 software. Data are presented as means ± SE of results corresponding to 15 images from three biological replicates. Bar, 3 μm.

FIG 9

Survival analysis (A) and fungal burden (B) data from the ΔtslA strain are similar to the data from the wild-type and ΔtslA+tslA strains, while the loss of TslA increased inflammation (panels C and D and panels E and F). (A) A total of 10⁶ conidia of each strain were inoculated via the intranasal route in a chemotherapeutic IPA murine model. Ten CD1 mice were used in each group. Survival analysis was performed for 2 weeks. p.i., postinfection. (B) No significant differences in fungal burden were observed in the strains tested. Analysis of the fungal burden of these mice was performed as previously described (36). (C) ΔtslA-infected lungs show more inflammatory cell infiltrations. The fungal histology was performed on day 2 and day 4 to observe the inflammatory cell infiltrations. Arrowheads show the abscess-like structure. Images are representative of results from three mice. Magnification, ×50. (D and E) ΔtslA-infected bronchoalveolar lavage fluid samples (BALs) had increased cell infiltrations, especially macrophages. To observe changes in the inflammatory response in vivo, cell counts and differential counts were performed. (D) P value = 0.0051 (unpaired two-tailed t test compared to the wild-type CEA10 results). (E) For neutrophils, *, P < 0.05 (two-tailed ANOVA for comparisons among the CEA10, ΔtslA, and ΔtslA+tslA strains); for macrophages, **, P < 0.01 (two-tailed ANOVA for comparisons among the CEA10, ΔtslA, and ΔtslA+tslA strains). (F) Luminex assay results from ΔtslA mutant-infected BALs show an increased inflammatory cytokine profile. Data are presented as means ± SE of results from BALF from three mice of each strain. *, P = 0.0286 (unpaired two-tailed Mann-Whitney test).