Solid-state NMR molecular snapshots of Aspergillus fumigatus cell wall architecture during a conidial morphotype transition

Abstract

While establishing an invasive infection, the dormant conidia of Aspergillus fumigatus transit through swollen and germinating stages, to form hyphae. During this morphotype transition, the conidial cell wall undergoes dynamic remodeling, which poses challenges to the host immune system and antifungal drugs. However, such cell wall reorganization during conidial germination has not been studied so far. Here, we explored the molecular rearrangement of Aspergillus fumigatus cell wall polysaccharides during different stages of germination. We took advantage of magic-angle spinning NMR to investigate the cell wall polysaccharides, without employing any destructive method for sample preparation. The breaking of dormancy was associated with a significant change in the molar ratio between the major polysaccharides β-1,3-glucan and α-1,3-glucan, while chitin remained equally abundant. The use of various polarization transfers allowed the detection of rigid and mobile polysaccharides; the appearance of mobile galactosaminogalactan was a molecular hallmark of germinating conidia. We also report for the first time highly abundant triglyceride lipids in the mobile matrix of conidial cell walls. Water to polysaccharides polarization transfers revealed an increased surface exposure of glucans during germination, while chitin remained embedded deeper in the cell wall, suggesting a molecular compensation mechanism to keep the cell wall rigidity. We complement the NMR analysis with confocal and atomic force microscopies to explore the role of melanin and RodA hydrophobin on the dormant conidial surface. Exemplified here using Aspergillus fumigatus as a model, our approach provides a powerful tool to decipher the molecular remodeling of fungal cell walls during their morphotype switching.

Structural Adaptation of Fungal Cell Wall in Hypersaline Environment

“…11 and Table 6 ). This molecule has been identified in multiple Aspergillus and Cryptococcus species and was reported to modulate membrane fluidity 38,61 . However, due to the severe overlap of its putative signals with those from other lipids and proteins, and due to the broad distribution of lipid polymers in the cell, more biochemical studies should be undertaken here to explain these changes.…”

“…SsNMR experiments were conducted using experimental schemes applied to fungal cell walls in multiple recent studies 22,35,36,38,61 . Methods include 13 C- 13 C through-space correlations using PDSD, DARR, and CORD sequences 81 , and through-bond correlation experiments such as refocused J -INADEQUATE and refocused INEPT 46,60 .…”

“…11 and Table 6). This molecule has been identified in multiple Aspergillus and Cryptococcus species and was reported to modulate membrane fluidity 38,61 .…”

“…Recently, the use of solid-state NMR (ssNMR) spectroscopy has led to a better understanding of the molecular architecture and dynamics of fungal cell walls [19][20][21][22] . Through integrated ssNMR and biochemical analyses of Aspergillus fumigatus, it has been discovered that a poorly hydrated, mechanically stiff core is formed by physically associated chitin and α-1,3-glucan [23][24][25] , which is conserved in both mycelia and conidia 26 , but with altered molecular composition during morphotype transition 27 . Highly branched β-1,3/1,6-glucans and linear terminal threads of β-1,3/1,4-glucans comprise the mobile and well-hydrated meshes.…”

“…In the case of Aspergillus fumigatus , a prevalent airborne pathogenic fungus of the same Trichocomaceae family, integrated ssNMR and biochemical analyses of the intact mycelia have discovered that a poorly hydrated and mechanically stiff core formed by physically colocalized chitin and α-1,3-glucan 24,35,36 , which is conserved in both mycelia and conidia 37 , but with altered molecular composition during morphotype transition 38 . Highly branched β- 1,3/1,6-glucans and linear terminal threads of β-1,3/1,4-glucans comprise the mobile and well-hydrated meshes.…”