Saranna Fanning, Aaron P. Mitchell*
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States of America
Biofilms are a principal form of microbial growth and are
critical to development of clinical infection. They are responsible
for a broad spectrum of microbial infections in the human host.
Many medically important fungi produce biofilms, including
Candida , Aspergillus , Cryptococcus , Trichosporon ,
Coccidioides , and Pneumocystis . In this review we emphasize
common features among fungal biofilms, and point toward genes
and pathways that may have conserved roles.
Biofilm cell communities are more resistant to antifungal drugs
than planktonic cells. Contributing factors include biofilm
structural complexity, presence of extracellular matrix (ECM),
metabolic heterogeneity intrinsic to biofilms, and biofilm-associ-
ated up-regulation of efflux pump genes. The actual fold increase
in resistance varies with both the drug and species. Candida albicans
and Candida parapsilosis biofilms are relatively resistant to
fluconazole, amphotericin B, nystatin, voriconazole, and others.
Aspergillus fumigatus biofilms are relatively resistant to itraconazole
and, to some extent, to caspofungin. Cryptococcal biofilms are
unaffected by fluconazole and voriconazole, and biofilms of
Trichosporon asahii display elevated resistance to amphotericin B,
caspofungin, voriconazole, and fluconazole. Azole and amphoter-
icin B therapies are ineffective against Pneumocystis carinii biofilms.
Biofilm-associated resistance mechanisms have been characterized
in C. albicans and A. fumigatus and include drug binding by ECM
and production of persister cells [2,7] (see supplementary
references for this section in Text S1). Persister cells represent
only a fraction of the population, and probably reflect its metabolic
heterogeneity. These mechanisms may pertain to other fungi as
Fungal Pathogen Biofilm Architecture
Biofilms are complex surface-associated cell populations em-
bedded in an ECM that possess distinct phenotypes compared to
their planktonic cell counterparts. Nutrients, quorum-sensing
molecules, and surface contact are contributory factors. C. albicans
biofilms are comprised primarily of yeast-form and hyphal cells,
both of which are required for biofilm formation . Formation is
a sequential process involving adherence to a substrate (either
abiotic or mucosal surface), proliferation of yeast cells over the
surface, and induction of hyphal formation . ECM accumulates
as the biofilm matures, and seems to contribute to cohesion . C.
albicans biofilms form on numerous abiotic  and biotic surfaces
[10–12]. In denture stomatitis, a combination of biotic mucosal
(the host) and abiotic surface (the denture) biofilm formation exists
. Other Candida spp. including C. tropicalis, C. parapsilosis, and C.
glabrata form ECM-containing biofilms but do not produce true
Aspergillus biofilms can form both on abiotic and biotic surfaces
. The initial colonizing cells that adhere to the substrate are
conidia. Mycelia (the hyphal form) develop as the biofilm matures
. ECM that binds the biofilm together  has been observed
in vitro  and in vivo . Hyphal organisation is different in
the two forms of A. fumigatus biofilm infection: aspergilloma
infections present an intertwined ball of hyphae; aspergillosis
infections present individual separated hyphae . Hyphae of C.
albicans and of A. fumigatus can form pores or channels through
biotic surfaces [17,18].
The emerging fungal pathogen T. asahii forms biofilms
comprised of yeast and hyphal cells embedded in matrix , as
do those of Coccidioides immitis . C. neoformans forms biofilms
consisting of yeast cells on many abiotic substrates , and shed
capsular polysaccharide forms the ECM. Although C. neoformans
forms hyphae in the course of mating, no hyphae have been
observed in C. neoformans biofilms to date. Similarly, Pneumocystis
species do not produce hyphal structures as part of their biofilms
. Thus, hyphal formation is not a uniform feature of fungal
Genetic Determinants of Fungal Biofilm
Transcription factors play fundamental roles in both positive
and negative regulation of biofilm formation through regulation of
hyphal formation and cell surface proteins responsible for
adherence . Bcr1, a C2H2zinc finger transcription factor, is a
critical determinant of C. albicans biofilm formation in all
environments studied to date [10,12,13,19]. Bcr1 seems to be a
conserved regulator of biofilm formation, because the Bcr1
ortholog of C. parapsilosis is required for biofilm formation as well
. Ace2, another C2H2zinc finger transcription factor, also
contributes to C. albicans biofilm formation, probably through its
role in adherence as well as hypha formation . The C. albicans
transcription factor Efg1, a global regulator of cell surface protein
genes and hyphal formation , is required for biofilm formation
as well. The orthologs or best hits of Bcr1, Ace2, and Efg1,
including C. glabrata CAGL0E06116g, CAGL0M04323g, and
CAGL0L01771g, and C. parapsilosis CPAG00564, CPAG00148,
and CPAG00178, are good candidates for biofilm regulators in
those species. Transcription factors with analogous roles to Bcr1,
Ace2, and Efg1 of C. albicans in A. fumigatus may be identified
amongst the 124 uniquely expressed or upregulated transcription
factors identified in biofilm culture by Gibbons et al., 2011 .
Citation: Fanning S, Mitchell AP (2012) Fungal Biofilms. PLoS Pathog 8(4):
Editor: Joseph Heitman, Duke University Medical Center, United States of
Published April 5, 2012
Copyright: ? 2012 Fanning, Mitchell. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Funding: Our work on biofilms was funded by NIH research grant R01 AI067703,
and support from the Richard King Mellon Foundation. The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of
Competing Interests: The authors have declared that no competing interests
* E-mail: firstname.lastname@example.org
PLoS Pathogens | www.plospathogens.org1 April 2012 | Volume 8 | Issue 4 | e1002585
The A. fumigatus transcription factor LAEA, a regulator of cell type
and secondary metabolism gene clusters in A. fumigatus, is highly
upregulated in biofilms , and it remains to be seen whether it
may influence biofilm phenotypes.
Cell wall proteins are of particular interest in biofilm formation.
Besides its expected role in adherence, the cell wall may have a
sensory role that promotes adherence-induced responses .
Numerous cell wall protein genes that may function in these
capacities are upregulated early in C. albicans and A. fumigatus
biofilm formation [22,24,25]. C. albicans cell surface proteins have
been reviewed authoritatively (see Text S1). Among the upregu-
lated A. fumigatus surface proteins are the hydrophobins RodA,
RodB, RodD, and RodE. RODB is thought to play the most
crucial role with its expression increased over 4,000-fold in biofilm
versus planktonic growth conditions . Ten other putative
adhesins have been identified by Gibbons et al., 2011 . It is
possible these Aspergillus proteins have functions analogous to
known adhesins in C. albicans.
Gene Expression Portrait of Fungal Biofilms
Biofilm cells have phenotypes distinct from planktonic cells, and
this difference is reflected in greatest detail at the gene expression
level. Detailed gene expression profiling comparisons, conducted
in both C. albicans and A. fumigatus, have revealed substantial
changes in gene expression between biofilm and planktonic cells
[22,26]. Changes in transcription factor expression is characteristic
of C. albicans biofilm formation in vitro and in vivo [24–26],
suggesting biofilm formation to be a highly regulated process.
Similarly, almost 50% of the predicted transcription factors of A.
fumigatus, including many with roles in asexual and sexual
development, are upregulated in biofilms compared to planktonic
Although biofilms are thought to include dormant cells, biofilms
of C. albicans and A. fumigatus have increased expression of genes
involved in protein synthesis. These genes encode ribosomal
proteins, protein turnover, and translation factors as well as
ribosomal proteins, indicating increased protein translation and
ribosome production in biofilms to be a feature of biofilms
[22,25,26]. If indeed biofilm cells are nutrient limited, these
particular gene expression features may optimize recycling of
Upregulation of multi-drug resistance transporter genes is
common to A. fumigatus (MDR1, MDR2, MDR4) and C. albicans
(MDR1, CDR1, CDR2) biofilms in vitro . C. albicans MDR1 and
CDR2 are upregulated in in vivo biofilms, as is PDR16, which is
increased in fluconazole-resistant cells that overexpress CDR1 and
CDR2 . Phase dependency of these transporters exists in vivo
for C. albicans CDR1 and in A. fumigatus for MDR4 [25,27].
Additionally, ergosterol gene expression may account for increased
drug resistance of biofilms. Genes involved in sterol biosynthesis
are upregulated in A. fumigatus and C. albicans biofilms [22,25,26].
Increases in ERG gene expression as well as multi-drug resistance
transporters has been correlated with increased azole resistance in
C. albicans patient isolate samples, though their contribution to
biofilm-specific azole resistance has not been detected in mature
biofilms (see Text S1).
Increased expression of adherence genes is also a property of
biofilm cells. ALS1 is the most upregulated of the known adherence
genes of C. albicans under biofilm conditions. Garcia-Sanchez et al.
(2004)  highlight that the ALS genes are differentially expressed
in biofilms and have autonomous contributions in the biofilm
transcriptome. Nett et al. (2009)  observed differential
expression of ALS genes at different stages of biofilm formation
and potential for overlap of function in vivo. A similar pattern of
differential adhesin expression is seen in vitro in the A. fumigatus
biofilm environment . The inducing signal for biofilm
adherence genes is clearly an area of interest as a basic biological
question as well as a direction for prospective therapeutic
A significant number of primary metabolism genes, including
those for amino acid synthesis, in particular sulfur amino acid
biosynthesis, and nucleotide synthesis, are upregulated in C.
albicans biofilms in vitro [24,26] and in vivo , relative to in
planktonic cells in vitro. Many are regulated by GCN4, a
transcriptional activator required for biofilm formation .
Genes involved in amino acid metabolism are also upregulated
in A. fumigatus biofilms including amino acid permeases, trans-
porters, and amino peptidases. Secondary metabolism gene
upregulation is significant in A. fumigatus biofilms, possibly due to
upregulation of LAEA, a secondary metabolism regulator .
Altered metabolic gene expression may reflect nutrient limitation,
but the rapid kinetics of induction (in C. albicans at least ) may
reflect a different regulatory signal.
Many cell wall biogenesis genes are induced in the biofilm
environment. Altered expression of genes for b-1,3 glucan
synthesis and modification are features of in vivo C. albicans
biofilms including FKS1, BGL2, and XOG1 . Given the
connection between the b-glucan pathway and biofilm matrix
production, these may also contribute to ECM production. Nett et
al. (2009)  highlight downregulation of b-1,3 glucan degrading
enzymes in 24-hour biofilms and suggest this functions in glucan
conservation for matrix production. In contrast, altered expression
of a- and b-1,3 glucan synthesis genes is not observed in A.
fumigatus biofilms. Although it is not directly reflected by the
expression of polysaccharide synthase genes, the presence of a-1,3
glucan, galactosaminogalactan, and galactomannan in the myce-
lial extacellular matrix is correlated to the aerial growth of the
mycelium of A. fumigatus . Expression of more than 50% of cell
wall genes investigated in A. fumigatus is, however, altered in the
biofilm habitat, including upregulation of the ROD genes. Thus,
these two organisms both restructure their cell surfaces in biofilms,
though they may use different mechanisms to achieve that
Mating Type and Fungal Biofilms
Genetic exchange is a feature of bacterial biofilms, mediated in
part by extracellular DNA. Although extracellular DNA has been
detected in C. albicans biofilms , the main mechanism of
biofilm-associated genetic exchange involves mating and cell
fusion. Most biofilm studies have been conducted with nonmating
a/a cells, but biofilm formation of the mating-capable cell types,
a/a and a/a, has revealed a unique regulatory pathway intimately
tied to pheromone signalling. In order to mate, C. albicans must go
through a switch from the white to opaque cell type. Upon
switching, a/a opaque cells release a mating pheromone that
induces a mating response in a/a opaque cells and vice versa.
Pheromone release also induces an adhesive phenotype among the
mating-incompetent a/a white cells , leading to mixed biofilm
formation and ultimately mating .
Notably, genes upregulated specifically in white cells in response
to pheromone exposure specify primarily cell wall and surface
proteins . Several of these genes contribute to a/a-a/a biofilm
formation . The configuration of the mating type locus also
seems to affect global biofilm properties , which may result
from distinct signalling pathways . If C. albicans has distinct
ways to make a biofilm, it seems likely that other fungi will as well.
PLoS Pathogens | www.plospathogens.org2 April 2012 | Volume 8 | Issue 4 | e1002585
Fungal biofilms reflect a range of architectures. Regulators of
biofilm formation may be conserved even among disparate biofilm
architectures. From detailed analysis in C. albicans and A. fumigatus,
there are numerous candidate genes that could be investigated in
other biofilm-forming fungi. In addition to hyphal gene expres-
sion, characteristic biofilm gene expression patterns include
increased expression of transcription factors and protein synthesis
genes. Differential adhesin expression, upregulation of cell wall
genes, and increased primary metabolism are features of the
biofilm environment. Studies of mating pheromone effects on
adherence highlight how a small portion of biofilm constituents
can have a significant impact on biofilm formation. The presence
of highly drug tolerant persister cells in biofilms (discussed above) is
another illustration of the contribution of cell heterogeneity to
overall biofilm properties. How other heterogeneous properties
among biofilm cells may contribute to the overall development
and integrity of pathogenic fungal biofilms will be an interesting
question for future research.
We are grateful to past and present lab members for many helpful
discussions about biofilms, and to Joe Suhan for insight into biofilm with
electron microscopy. We thank Stephanie Guadagnini, Anne Beauvais,
and J.P. Latge for providing the A. fumigatus biofilm image in Figure 1, and
for critical reading of the manuscript. We apologize to our colleagues
whose papers we could not cite due to space limitations.
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