Abstract

ABSTRACTThe genus Aspergillus encompasses human pathogens such as Aspergillus fumigatus and industrial powerhouses such as Aspergillus niger. In both cases, Aspergillus biofilms have consequences for infection outcomes and yields of economically important products. However, the molecular components influencing filamentous fungal biofilm development, structure, and function remain ill defined. Macroscopic colony morphology is an indicator of underlying biofilm architecture and fungal physiology. A hypoxia-locked colony morphotype of A. fumigatus has abundant colony furrows that coincide with a reduction in vertically oriented hyphae within biofilms and increased low oxygen growth and virulence. Investigation of this morphotype has led to the identification of the causative gene, biofilm architecture factor A (bafA), a small cryptic open reading frame within a subtelomeric gene cluster. BafA is sufficient to induce the hypoxia-locked colony morphology and biofilm architecture in A. fumigatus. Analysis across a large population of A. fumigatus isolates identified a larger family of baf genes, all of which have the capacity to modulate hyphal architecture, biofilm development, and hypoxic growth. Furthermore, introduction of A. fumigatus bafA into A. niger is sufficient to generate the hypoxia-locked colony morphology, biofilm architecture, and increased hypoxic growth. Together, these data indicate the potential broad impacts of this previously uncharacterized family of small genes to modulate biofilm architecture and function in clinical and industrial settings.

Highlights

  • Introduction of the cryptic gene orthologbafB is sufficient to complement the loss of cgnA and hrmA in EVOL20

  • These data suggest that colony furrowing of A. fumigatus occurs in hypoxia in part to increase oxygen diffusion into the colonies and that the furrows of the hypoxia-evolved H-MORPH strain EVOL20 develop even under normoxia to increase oxygen deep within the colonies

  • We identify and characterize here a family of putative orthologous protein-coding genes that are heterogeneously expressed across A. fumigatus strains and impact biofilm architecture and hypoxic growth

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Summary

Introduction

Introduction of the cryptic gene orthologbafB is sufficient to complement the loss of cgnA and hrmA in EVOL20. To determine whether bafB from CEA10, whose protein sequence is 78.35% identical to bafA, could complement the loss of cgnA in EVOL20 (DcgnAEVOL), we introduced bafB with the constitutively active gpdA promoter (DcgnAEVOL; bafBOE). Aspergillus niger strain CBS 513.88 encodes a gene, An08g12010, with 69% nucleotide identity to A. fumigatus bafA and 41.03% amino acid identity to the predicted protein sequence of BafA (see Fig. S7A). This suggests that the role of baf or baf-like genes may be conserved in other Aspergillus species. We sought to determine whether A. fumigatus bafA (AfbafA) could influence colony morphology, biofilm architecture, hypoxic growth, and adherence in the A. niger reference strain A1144. This strain was selected for its robust growth at 37°C and the ease at which it is genetically manipulated

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