A heterogeneously expressed gene family modulates biofilm architecture and hypoxic growth of Aspergillus fumigatus

The 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. Yet, 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. Importance The manipulation of microbial biofilms in industrial and clinical applications remains a difficult task. The problem is particularly acute with regard to filamentous fungal biofilms for which molecular mechanisms of biofilm formation, maintenance, and function are only just being elucidated. Here we describe a family of small genes heterogeneously expressed across Aspergillus fumigatus strains that are capable of modifying colony biofilm morphology and microscopic hyphal architecture. Specifically, these genes are implicated in the formation of a hypoxia-locked colony morphotype that is associated with increased virulence of A. fumigatus. Synthetic introduction of these gene family members, here referred to as biofilm architecture factors, in both A. fumigatus and A. niger additionally modulates low oxygen growth and surface adherence. Thus, these genes are candidates for genetic manipulation of biofilm development in Aspergilli.


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The primary sequence FASTQ sequence reads for the strains are all available in the 361 NCBI SRA database and detailed in Table S2.

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The native 5' sequence to cgnA is required to complement the loss of cgnA in EVOL20

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We have previously characterized a role for the HAC gene cluster in the

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confirmed that similar to hrmA, the number of strains positive for the presence of bafA is 487 low (n=24) (Fig. 2). Similarly, bafB is present within ~27% of the A. fumigatus genomes 488 analyzed (n=24) (Fig. 2). Strains positive for encoding bafC are more abundant (n=35), 489 but this is complicated by the presence of another bafC ortholog in AF293 (Afu1g00770) 490 that is not encoded near a putative hrmC ortholog (Fig. 2)

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Many of the analyzed genomes also encode a predicted pseudogene with high 498 similarity to bafB (Pseudobaf) (Fig.2). The pseudogenes are degenerate ORFs that have To determine if bafB from CEA10, whose protein sequence is 78.35% identical to 518 bafA, could complement the loss of cgnA in EVOL20 (∆cgnA EVOL ), we introduced bafB 519 with the constitutively active gpdA promoter (∆cgnA EVOL ; bafB OE ). The resulting strain 520 reverted the N-MORPH phenotype of ∆cgnA EVOL to the H-MORPH phenotype of EVOL20 521 with significantly increased colony furrows and percent vegetative mycelia (Fig. 3A, B).

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As mentioned above, the majority of HAC genes are not altered in expression as a result

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Even in the absence of hrmA, bafB is sufficient to generate H-MORPH and significantly 530 increase colony furrows and percent vegetative mycelia (Fig. 3A, B). In addition to H-

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The overexpression of bafB significantly increases hypoxic fitness of ∆hrmA EVOL and 534 ∆cgnA EVOL (Fig. 3C); and significantly reduces adherence of these strains to a plastic 535 surface (Fig. 3D). Importantly, bafB is sufficient to complement these phenotypes in 536 EVOL20 without increasing HAC gene mRNA levels (Fig. 3E). In fact, the mRNA levels 537 of hrmA are slightly, but significantly, reduced as a result of constitutive bafB expression 538 (Fig. 3E).

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To test whether bafB expression alters biofilm architecture, a HAC-dependent 540 phenotype of EVOL20, we cultured submerged biofilms for 24 hours and imaged the 541 bottom ~300 µm of the biofilm. As a metric for biofilm architecture, we measured the 542 angle of hyphal deviation from the vertical axis. As has been described for the N-  (Fig. S5C), and concentrated toward the distal hyphal region (Fig.   567   S5D). At the distal region, the GFP signal is present within circular structures, or puncta, 568 as well as localized along the sides of the hyphae (Fig. S5D). Time-lapse imaging 569 23 reveals that these BafB puncta are dynamic and move rapidly within the hyphae (Video 570 S1, Fig. S5E). Co-staining with the membrane dye FM4-64 indicate overlap in the 571 patterns of BafB localization and endosome localization (Fig. S5F). This subcellular 572 pattern and the presence of the N-terminal secretion signal peptide (Fig. S5A) support to 573 the hypothesis that BafB localizes extracellularly at the hyphal tips or is secreted (48).

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While the GFP signal corresponding to BafB is largely absent from the hyphal edges

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The strain CEA10 contains HAC, H B AC, and H C AC, but like AF293, bafA 600 expression is below the level of detection by qRT-PCR in biofilm cultures but can be 601 detected following introduction of a second overexpressed bafA allele (Fig. S6B).

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H-MORPH in EVOL20, and other clinical isolates, coincides with reduced 614 adherence and increased hypoxic fitness (hypoxic growth relative to normoxia growth,

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inability for bafA expression to impact CEA10 colony morphology, and its apparent 620 reduced impact on adherence and hypoxic growth relative to AF293 may be explained 25 by the presence of the other baf genes encoded in the CEA10 genome. While bafA 622 mRNA levels are undetectable in CEA10 during normal oxygen growth, mRNA for both 623 bafB and bafC is detected (Fig. 4H). As the amino acid identity between these three 624 proteins ranges from 45-78% we hypothesize that bafB and bafC are also sufficient to 625 impact colony and biofilm morphology. it does significantly increase colony furrows in normoxia relative to CEA10 (Fig. 5B, D).

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However, the percent vegetative mycelia is not significantly increased (Fig. 5D).

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Despite variation in how the baf genes impact colony morphology in the two 642 strain backgrounds, in both AF293 and CEA10 overexpression of bafB or bafC results in 643 significantly reduced adherence to plastic (Fig. 5F). CEA10 adheres less well to plastic 644 compared to AF293, and the difference in adherence is smaller as a result of bafB or 645 bafC overexpression. As these two genes are already present and expressed in CEA10 ( Fig. 4H), it is possible that this native baf expression contributes to this difference 647 between CEA10 and AF293.

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As putative biofilm architecture factors, we sought to confirm an impact of bafB 649 and bafC on biofilm architecture, similar to that observed with elevated expression of 650 bafA (Fig. 3D, E). In AF293, overexpression of bafB visibly impacts biofilm architecture 651 and formation in the XZ (Fig. 5G) and XY dimensions (Fig. S6C). The XY dimension 652 reveals dense hyphal growth and abundant hyphal branching (Fig. S6C). The XZ 653 dimension shows a stunted 24 hour biofilm that reaches heights of only 200-250 µm 654 (Fig. 5G). Similarly, regions of the 24 hour biofilms generated by the overexpression of 655 bafC in AF293 (AF293 bafC OE ) are also stunted with evidence of hyphae that are hyper 656 branching (Fig. 5G, Fig. S6C). In regards to biofilm architecture as defined by hyphal 657 orientation to the vertical axis, overexpression of bafC but not bafB in AF293 results in 658 increased deviation from the vertical axis above 50 µm (Fig. 5 G. H). Notably,

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In CEA10 biofilms, overexpression of bafB and bafC results in increased 662 deviation from the vertical axis above 50 µm in 24 hour biofilms (Fig. 5I, J). There is also 663 qualitative evidence for hyper branching as a result of elevated bafB or bafC expression 664 in CEA10 (Fig. S6E). These data support a role for all three proposed baf genes in 665 biofilm architecture, through multiple metrics, in two independent strain backgrounds of 666  AfbafA is capable of impacting biofilm architecture across fungal species (Fig. 6D).

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Not only does AfbafA impact the colony morphology to generate H-MORPH and 698 modulate the biofilm architecture, but it also generates other H-MORPH and EVOL20 699 associated phenotypes including increased hypoxia fitness and reduced adherence. In 700 AF293 and CEA10 expression of bafA results in increased hypoxia fitness (hypoxic 701 growth normalized to normoxic growth); similarly, the hypoxia fitness of A1144 702 significantly increases with constitutive expression of AfbafA (Fig. 6E). Adherence of A.

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fumigatus is quantified in minimal media, however, the adherence of the reference A.  formation of furrows that potentially increase the colony surface area exposed to 788 ambient oxygen.

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The formation of aerial hyphae and the generation of a 'fluffy' colony morphology 790 would also increase the hyphal surface area exposed to ambient oxygen. In surface