Aspergillus fumigatus biofilms: Toward understanding how growth as a multicellular network increases antifungal resistance and disease progression

Abstract

Aspergillus fumigatus is a saprophytic, filamentous fungus found in soils and compost and the causative agent of several pulmonary diseases in humans, birds, and other mammals. A. fumigatus and other filamentous fungi grow as networks of filamentous hyphae that have characteristics of a classic microbial biofilm. These characteristics include production of an extracellular matrix (ECM), surface adhesion, multicellularity, and increased antimicrobial drug resistance. A. fumigatus biofilm growth occurs in vivo at sites of infection, highlighting the importance of defining mechanisms underlying biofilm development and associated emergent properties. We propose that there are 3 distinct phases in the development of A. fumigatus biofilms: biofilm initiation, immature biofilm, and mature biofilm. These stages are defined both temporally and by unique genetic and structural changes over the course of development. Here, we review known mechanisms within each of these stages that contribute to biofilm structure, ECM production, and increased resistance to contemporary antifungal drugs. We highlight gaps in our understanding of biofilm development and function that when addressed are expected to aid in the development of novel antifungal therapies capable of killing filamentous fungal biofilms.

Fig 1. Overview of Aspergillus fumigatus biofilm development.

(A) Representative fluorescence confocal microscopy images of A. fumigatus CEA10 submerged biofilms grown in liquid minimal media during key stages of development. (B) Representative XY and XZ views of 24-hour A. fumigatus submerged biofilm in minimal liquid media color coded to show height of hyphae within the biofilm. (C) XYZ view of 24-hour A. fumigatus submerged biofilm in liquid minimal media. The 20× magnification scale bar is 100 um. The 100× magnification scale bar is 10 um.

Fig 2. Summary of key stages of biofilm development.

Summary of each stage of biofilm development showing XZ view of a representative fluorescence confocal microscopy image of A. fumigatus CEA10 submerged biofilms. Here, we summarize features of each stage as well as key questions that remain to be answered. All images are 20× magnification. Scale bar is 100 um. ECM, extracellular matrix; eDNA, extracellular DNA; MIC, minimum inhibitory concentration.

Fig 3. Model of mature A. fumigatus biofilms.

Illustration depicting known contributors to A. fumigatus mature biofilm complexity. Gradients of oxygen and metabolic activity develop along a vertical axis within the biofilm which likely creates a shift from respiratory metabolism to fermentative or other alternative metabolisms at the bottom of the biofilm due to oxygen depletion. This change in metabolic activity also plays a key role in azole resistance at the base of the biofilm. ECM coats hyphae and collects at the base of the biofilm. Key questions remain about the spatial distribution of certain molecules, the identities of things such as quorum sensing molecules, the expression or function within biofilms of cellular components such as mediators of polarity, and the signaling mechanisms for processes like quorum sensing. ECM, extracellular matrix; RNS, reactive nitrogen species; ROS, reactive oxygen species.