Microbial populations form intricate macroscopic colonies with diverse morphologies whose functions remain to be fully understood. Despite fungal colonies isolated from environmental and clinical samples revealing abundant intraspecies morphological diversity, it is unclear how this diversity affects fungal fitness and disease progression. Here we observe a notable effect of oxygen tension on the macroscopic and biofilm morphotypes of the human fungal pathogen Aspergillus fumigatus. A hypoxia-typic morphotype is generated through the expression of a subtelomeric gene cluster containing genes that alter the hyphal surface and perturb interhyphal interactions to disrupt in vivo biofilm and infection site morphologies. Consequently, this morphotype leads to increased host inflammation, rapid disease progression and mortality in a murine model of invasive aspergillosis. Taken together, these data suggest that filamentous fungal biofilm morphology affects fungal–host interactions and should be taken into consideration when assessing virulence and host disease progression of an isolated strain.
Subscribe to Journal
Get full journal access for 1 year
only $4.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
RNA sequencing data that support the findings of this study have been deposited under NCBI Gene Expression Omnibus with the identifier GSE133440 under the BioProject PRJNA551460. The genomic sequencing data for EVOL20 and reference AF293 have been deposited under BioProject identifier PRJNA417720. All strains and any other data used to support these findings will be made available upon reasonable request to the corresponding author.
The custom scripts that were used in this study are available at https://github.com/stajichlab/Afum_RNASeq_hrmA, https://github.com/stajichlab/Afum_hrmA_cluster_evolution, https://github.com/stajichlab/Afum_popgenome and https://github.com/stajichlab/Afum_EVOL20. The BiofilmQ software is available for public download at https://drescherlab.org/data/biofilmQ/. Other data that support the findings presented here are available upon request from the corresponding author.
Slutsky, B., Buffo, J. & Soll, D. R. High-frequency switching of colony morphology in Candida albicans. Science 230, 666–669 (1985).
Simpson, L. M., White, V. K., Zane, S. F. & Oliver, J. D. Correlation between virulence and colony morphology in Vibrio vulnificus. Infect. Immun. 55, 269–272 (1987).
Kuthan, M. et al. Domestication of wild Saccharomyces cerevisiae is accompanied by changes in gene expression and colony morphology. Mol. Microbiol. 47, 745–754 (2003).
Workentine, M. L. et al. Phenotypic heterogeneity of Pseudomonas aeruginosa populations in a cystic fibrosis patient. PLoS ONE 8, e60225 (2013).
Haussler, S. et al. Highly adherent small-colony variants of Pseudomonas aeruginosa in cystic fibrosis lung infection. J. Med. Microbiol. 52, 295–301 (2003).
Hagiwara, D. et al. Whole-genome comparison of Aspergillus fumigatus strains serially isolated from patients with aspergillosis. J. Clin. Microbiol. 52, 4202–4209 (2014).
Fong, J. C. & Yildiz, F. H. The rbmBCDEF gene cluster modulates development of rugose colony morphology and biofilm formation in Vibrio cholerae. J. Bacteriol. 189, 2319–2330 (2007).
Drenkard, E. & Ausubel, F. M. Pseudomonas biofilm formation and antibiotic resistance are linked to phenotypic variation. Nature 416, 740–743 (2002).
Miller, M. G. & Johnson, A. D. White-opaque switching in Candida albicans is controlled by mating-type locus homeodomain proteins and allows efficient mating. Cell 110, 293–302 (2002).
Workentine, M. L. et al. Phenotypic and metabolic profiling of colony morphology variants evolved from Pseudomonas fluorescens biofilms. Environ. Microbiol. 12, 1565–1577 (2010).
Jain, N., Guerrero, A. & Fries, B. C. Phenotypic switching and its implications for the pathogenesis of Cryptococcus neoformans. FEMS Yeast Res. 6, 480–488 (2006).
Jain, N., Hasan, F. & Fries, B. C. Phenotypic switching in fungi. Curr. Fungal Infect. Rep. 2, 180–188 (2008).
Kowalski, C. H. et al. Heterogeneity among isolates reveals that fitness in low oxygen correlates with Aspergillus fumigatus virulence. MBio 7, e01515-16 (2016).
Barker, B. M. et al. Transcriptomic and proteomic analyses of the Aspergillus fumigatus hypoxia response using an oxygen-controlled fermenter. BMC Genom. 13, 62 (2012).
Kale, S. D. et al. Modulation of immune signaling and metabolism highlights host and fungal transcriptional responses in mouse models of invasive pulmonary aspergillosis. Sci. Rep. 7, 17096 (2017).
McDonagh, A. et al. Sub-telomere directed gene expression during initiation of invasive aspergillosis. PLoS Pathog. 4, e1000154 (2008).
Fedorova, N. D. et al. Genomic islands in the pathogenic filamentous fungus Aspergillus fumigatus. PLoS Genet 4, e1000046 (2008).
Law, M. J., Chambers, E. J., Katsamba, P. S., Haworth, I. S. & Laird-Offringa, I. A. Kinetic analysis of the role of the tyrosine 13, phenylalanine 56 and glutamine 54 network in the U1A/U1 hairpin II interaction. Nucleic Acids Res. 33, 2917–2928 (2005).
Abdel-Nour, M. et al. The Legionella pneumophila collagen-like protein mediates sedimentation, autoaggregation, and pathogen-phagocyte interactions. Appl. Environ. Microbiol. 80, 1441–1454 (2014).
Chen, S. M. et al. Streptococcal collagen-like surface protein 1 promotes adhesion to the respiratory epithelial cell. BMC Microbiol. 10, 320 (2010).
Wang, C. & St Leger, R. J. A collagenous protective coat enables Metarhizium anisopliae to evade insect immune responses. Proc. Natl Acad. Sci. USA 103, 6647–6652 (2006).
Gravelat, F. N. et al. Aspergillus galactosaminogalactan mediates adherence to host constituents and conceals hyphal beta-glucan from the immune system. PLoS Pathog. 9, e1003575 (2013).
Lee, M. J. et al. Deacetylation of fungal exopolysaccharide mediates adhesion and biofilm formation. mBio 7, e00252–00216 (2016).
Brown, G. D. & Gordon, S. Immune recognition: a new receptor for beta-glucans. Nature 413, 36–37 (2001).
DePas, W. H. et al. Exposing the three-dimensional biogeography and metabolic states of pathogens in cystic fibrosis sputum via hydrogel embedding, clearing, and rRNA labeling. mBio 7, e00796-16 (2016).
Yang, B. et al. Single-cell phenotyping within transparent intact tissue through whole-body clearing. Cell 158, 945–958 (2014).
Chung, K. et al. Structural and molecular interrogation of intact biological systems. Nature 497, 332–337 (2013).
Ballard, E. et al. In-host microevolution of Aspergillus fumigatus: a phenotypic and genotypic analysis. Fungal Genet. Biol. 113, 1–13 (2018).
Gago, S., Denning, D. W. & Bowyer, P. Pathophysiological aspects of Aspergillus colonization in disease. Med. Mycol. 57, S219–S227 (2018).
Morales, D. K. et al. Control of Candida albicans metabolism and biofilm formation by Pseudomonas aeruginosa phenazines. mBio 4, e00526–00512 (2013).
Beattie, S. R. et al. Filamentous fungal carbon catabolite repression supports metabolic plasticity and stress responses essential for disease progression. PLoS Pathog. 13, e1006340 (2017).
Szewczyk, E. et al. Fusion PCR and gene targeting in Aspergillus nidulans. Nat. Protoc. 1, 3111–3120 (2006).
Grahl, N. et al. In vivo hypoxia and a fungal alcohol dehydrogenase influence the pathogenesis of invasive pulmonary aspergillosis. PLoS Pathog. 7, e1002145 (2011).
Willger, S. D. et al. A sterol-regulatory element binding protein is required for cell polarity, hypoxia adaptation, azole drug resistance, and virulence in Aspergillus fumigatus. PLoS Pathog. 4, e1000200 (2008).
Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).
Basenko, E. Y. et al. FungiDB: an integrated bioinformatic resource for fungi and oomycetes. J. Fungi (Basel) 4, 39 (2018).
Wu, T. D., Reeder, J., Lawrence, M., Becker, G. & Brauer, M. J. GMAP and GSNAP for genomic sequence alignment: enhancements to speed, accuracy, and functionality. Methods Mol. Biol. 1418, 283–334 (2016).
Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).
Friedman, J. H., Bentely, J. & Finkel, R. A. An algorithm for finding best matches in logarithmic expected time. ACM Trans. Math. Softw. 3, 209–226 (1977).
Treweek, J. B. et al. Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping. Nat. Protoc. 10, 1860–1896 (2015).
Misharin, A. V., Morales-Nebreda, L., Mutlu, G. M., Budinger, G. R. & Perlman, H. Flow cytometric analysis of macrophages and dendritic cell subsets in the mouse lung. Am. J. Respir. Cell Mol. Biol. 49, 503–510 (2013).
Danhof, H. A. & Lorenz, M. C. The Candida albicans ATO gene family promotes neutralization of the macrophage phagolysosome. Infect. Immun. 83, 4416–4426 (2015).
Liao, P. S., Chew, T. S. & Chung, P. C. A fast algorithm for multilevel thresholding. J. Inf. Sci. Eng. 17, 713–727 (2001).
Shepardson, K. M. et al. Hypoxia enhances innate immune activation to Aspergillus fumigatus through cell wall modulation. Microbes Infect. 15, 259–269 (2013).
Burghardt, R. C. & Droleskey, R. Transmission electron microscopy. Curr. Protoc. Microbiol. 3, 1–39 (2006).
We thank J. Obar (Dartmouth) for his helpful comments, A. Lavanway and L. Howard (Dartmouth) for their microscopy expertise, T. J. Smith for graphical model design and construction, D. Limoli for funPACT assistance, S. Dhingra for tool development (plasmids), D. Carter-House for assistance with DNA extraction for whole genome sequencing and S. Lockhart, S. Howard, and D. Hagiwara for sharing A. fumigatus isolates. This work was supported by the efforts of R.A.C. through funding by NIH National Institute of Allergy and Infectious Diseases (NIAID) (grant nos. R01AI130128 and 2R01AI081838). R.A.C holds an Investigators in Pathogenesis of Infectious Diseases Award from the Burroughs Wellcome Fund. C.H.K. was supported by the Molecular and Cellular Biology Training Grant at Dartmouth (no. 5T32 GM 8704-20, principal investigator: D. Compton) from the National Institute of General Medical Sciences from July 2016 to June 2018 and the NIH NIAID Ruth L. Kirschstein National Research Service Award (no. F31AI138354) from July 2018. C.D.N. is supported by the National Science Foundation (grant no. MCB 1817342), a Burke Award from Dartmouth College, a pilot award from the Cystic Fibrosis Foundation (grant no. STANTO15RO) and NIH grant no. P20-GM113132 to the Dartmouth BioMT COBRE. Data analyses were performed on the UC Riverside High-Performance Computational Cluster supported by grant nos. NSF DBI-1429826 and NIH S10-OD016290.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Figs. 1–16, video legends, methods and references.
Supplementary Tables 1–9
AF293 biofilm at 21% O2.
AF293 biofilm at 0.2%.
EVOL20 biofilm at 21% O2.
EVOL20 biofilm at 0.2% O2.
funPACT, AF293, lesion, day 5 post inoculation.
funPACT, EVOL20, lesion day 5 post inoculation.
About this article
Cite this article
Kowalski, C.H., Kerkaert, J.D., Liu, KW. et al. Fungal biofilm morphology impacts hypoxia fitness and disease progression. Nat Microbiol 4, 2430–2441 (2019). https://doi.org/10.1038/s41564-019-0558-7
A Heterogeneously Expressed Gene Family Modulates the Biofilm Architecture and Hypoxic Growth of Aspergillus fumigatus
Applied Microbiology and Biotechnology (2021)
Medical Mycology (2021)
Algal Research (2021)
Heat Shock Protein 60, Insights to Its Importance in Histoplasma capsulatum: From Biofilm Formation to Host-Interaction
Frontiers in Cellular and Infection Microbiology (2021)