Introduction
Candida albicans, Candida auris and Candida tropicalis are three fungal pathogens
that WHO recently reported as requiring urgent attention and additional resources
for research and development (World Health Organisation, 2022). Candida species often
form biofilms on the surfaces of tissues or medical devices, such as catheters and
heart values and these represent reservoirs of infection that are difficult to eradicate
using conventional antifungal treatment (Junqueira and Mylonakis, 2023, Nett and Andes,
2020, Ramage et al., 2023). The biofilms compromise the protective capacity of sentinel
activities of the host immune system, and they render the fungal biomass resistant
to most clinically relevant antifungal drugs. Candida albicans biofilms contain yeast,
hyphae and a complex extracellular matrix (ECM) whilst other species form biofilms
with a simpler array of cell types. The biofilm is a three dimensional structure composed
of a foundation basal layer of yeast cells that is tightly adhered to a biological
or non-biological surface from which a proliferation of branching and budding hyphae
and yeast cells are seeded and which is encased in covering of the ECM (Atiencia-Carrera
et al., 2022, Gulati and Nobile, 2016). The presence of the ECM radically alters the
physiology of the fungal cells and confers protective properties that severely compromise
the ability of immune cells and administration of antifungal drugs to kill the fungus
(Ajetunmobi et al., 2023) (Fig. 1). We review here the properties of this ECM and
how this influences the drug resistant phenotype of Candida cells in biofilms and
help protect cells against immune phagocytes.
Fig. 1
Schematic diagram showing Candida biofilm with extracellular vesicles (EV) (Black
dot) and extracellular matrix (ECM). ECM protects Candida from both antifungal drugs
by reducing access to the fungal cells and also protects against phagocytosis (Blue)
by macrophages and other phagocytes. In presence of high dose of antifungal drugs
(Red), persister Candida cells (Brown) emerge that can reseed the biofilm with viable
cells post antifungal therapy.
The drug resistance phenotype of the Candida biofilms is related to a number of properties
of the ECM – its impermeability, ability to sequester and immobilise many drugs, and
the presence of drug resistance persister cells within the biofilm. Persister cells
represent a minority of cells of the biofilm, but the proportion may increase after
exposure to high doses of antifungal drugs (Fig. 1). These are a non-growing, metabolically
quiescent subpopulation of the biofilm cells that are able to survive high doses of
antimicrobial drugs (Li et al., 2015, Wuyts et al., 2018). These dormant cells exhibit
properties that are similar to glucose-starved planktonic cells whose physiology enables
them to survive challenge with fungicidal drugs by inducing stress tolerance pathways
and protective levels of internal glycogen and trehalose (Wuyts et al., 2018). Persister
cells of the biofilm may increase the production of ECM materials and induce enzyme
activities that promote their survival. For example, the Bgl2 glucanosyltransferase,
the exoglucanase Xog1, and the signalling proteins KRE1 and SKN1 were shown to be
upregulated in persister cells. These proteins, along with Extracellular Vesicles
(EVs), are involved in ECM production (Li et al., 2015).
Biofilm composition and function in drug sensitivity
Candida biofilm matrix is composed approximately of 25 % carbohydrate, 55 % protein,
15 % lipid and 5 % nucleic acid (Mitchell et al., 2016, Pierce et al., 2017). The
carbohydrate component of the biofilm contains similar polysaccharides as found in
cell wall; however, the macromolecular structures of the cell wall and biofilm polysaccharides
are distinct in their fine structure and they may be actively modified after they
are secreted from the cell into the ECM (Mitchell et al., 2016). A major contributor
of biofilm ECM, including 45 % of the ECM protein is derived from Extracellular Vesicles
(EVs), and biofilm EVs are distinct in composition from those generated by planktonic
cells (Zarnowski et al., 2018). Mutants in the ESCRT pathway required for vesicle
biogenesis and secretion prevented EVs entering into the biofilm, and this prevented
ECM synthesis and resulted in increased sensitivity to fluconazole (Zarnowski et al.,
2018). It is interesting that AmBisome liposomal vesicles, have been shown to be able
to transit the intact fungal cell wall (Walker et al., 2018), and so vesicular carriers
seem to be important for the synthesis of the ECM and may provide opportunities in
the treatment with antifungal drugs encapsulated in liposomes.
Using NMR, it was shown that mannans and β-glucan two of the major polysaccharides
identified in Candida EVs (Zarnowski et al., 2018). The biofilm matrix contains α-1,2-
mannan, α −1,6 mannan and β-1,6 glucan which are greatly enriched in abundance compared
to the underlying cell wall. Mannan is less rigid than chitin or β1,3 glucan (Gow
& Lenardon, 2022), but makes the matrix complex antifungal drug resistant by decreasing
permeability of the biofilm (Walker et al., 2018). In addition, the β-1,3 glucan,
β −1,6 glucan, and α-1,2-branched α −1,6 mannan components of the ECM can sequester
drugs such as amphotericin B, anidulafungin, and flucytosine via non-covalent binding,
thereby reducing their efficacy (Fig. 1) (Mitchell et al., 2016, Nett and Andes, 2020).
In addition, mutants in endo-β-D-glucosidase (Sun41), that degrades ECM increases
matrix, affects the sensitivity of the fungus to antifungal drugs such as caspofungin
(Norice et al., 2007). A range of other genes that are key to the synthesis of β-1,3
glucan all have a common phenotype of enhanced susceptibility to fluconazole (Mitchell
et al., 2013, Taff et al., 2012).
In Candida auris amplification of ALS4 copy number enhances biofilm and adherence
(Bing et al., 2023) – two aspects of the fungus which is blocked by treatments that
inhibit amyloid protein function (Malavia-Jones et al., 2023). Thus both cell wall,
ECM polysaccharides and proteins all contribute to the structure of the ECM and to
its antifungal drug retarding properties.
To date, it is not clear if there are any changes to the cell wall component in the
persister cells although it is highly likely since growth rate affects cell wall composition
(Gow & Lenardon, 2022). Supporting this, it is known that components of the cell-wall
integrity pathways (XOG1, BGl1, SUN41, SCW11 and PSA2) are up-regulated in persister
cells in the presence of Amphotericin B induced oxidative stress (Li et al., 2015).
A genome wide screen identified six “master transcriptional regulators” -Efg1, Tec1,
Bcr1, Ndt80, Brg1 and Rob1, for biofilm formation (Cavalheiro and Teixeira, 2018,
Nobile et al., 2012). Each regulator plays a visible roles in regulating biofilm structure.
For example, only the master regulator Bcr1 and downstream cell wall proteins (Als1,
Als3 and Hwp1) are required for the first crucial attachment step of biofilm formation
to surfaces. However, Bcr1 is not required for hyphal formation but it is required
for hyphal attachment in biofilms associated with oropharyngeal candidiasis (Fanning
et al., 2012).
Advances in the treatment of fungal biofilms
A range of advances have focussed on treating fungal biofilms to improve drug efficacy
(Junqueira & Mylonakis, 2023). A promising antifungal anti-biofilm drug, turbinmicin,
has been shown to disrupt the extracellular vesicles production and eliminate the
extracellular matrix of Candida biofilm. Turbinmicin is likely to target the Sec14p-a
phosphatidylinositolphosphatidyl-choline transfer protein involved in the vesicle
trafficking, and thereby biofilm formation (Zhao et al., 2021).
Administration of membrane active antimicrobial peptides such as gH625 (Galdiero et
al., 2020), and a scorpion venom ToAP2 peptide (do Nascimento Dias et al., 2020) inhibit
ECM formation in biofilm, reduced the number of persister cells and increased antifungal
drug susceptibility to a number of antifungals (Galdiero et al., 2020).These peptides
increased the permeability of cell membrane and penetration through the ECM. Surfaces
can also be created or treated to impede biofilm establishment, for example using
surface functionalization with antifungals and the use of nanoparticles that incorporate
inhibitory polymers or antifungals. These methods have shown promising results in
disruption and dispersing biofilms (Vera-Gonzalez & Shukla, 2020). Combinational therapies
in which agents that improve penetration and permeability of membranes may also have
useful applications in the treatment of Candida biofilms. Similarly, do Nascimento
Dias et al (2020) reported that when ToAP2 peptide was used in combination with fluconazole
and Amphotericin B, there was increase in efficacy of both molecules (do Nascimento
Dias et al., 2020). Furthermore, the membranotropic peptide-gH625 used in combination
with fluconazole and 5-fluorocytosine was able to efficiently eradicate biofilms and
persistor cells (Galdiero et al., 2020). Analogues of diazaspiro-decanes have also
been shown to be bioactive biofilm inhibitors (Pierce et al., 2015). Even low micromolar
concentrations of compounds with a common biaryl amide structure inhibited C. albicans
biofilm formation and filamentous growth.
Conclusions
In summary, Candida biofilms remain a significant clinical problem because of their
ability to restrict access to both antifungal drugs and immune cells. It is clear
that the ECM that encases growing and non-growing persister cells is distinct in composition
to the cell wall and has important clinically relevant properties. These studies also
demonstrate that the cell wall of fungi is not the only outer structure that effects
drug permeability and sensitivity (Casadevall & Gow, 2022). The ECM can bind and sequester
drugs and represent a permeability barrier. It also provides a protective microenvironment
in which metabolically quiescent persister cells can survive periods of antifungal
drugs administration, only to emerge and proliferate when drug levels in the bloodstream
subside.
Future studies must focus on understanding the role of each component of the biofilm
structure and how they influence antifungal drug resistance. This knowledge will be
pivotal in treating fungal infections and the roles of key components of fungal walls
and matrices.
CRediT authorship contribution statement
Sumita Roy: Writing – original draft, Investigation, Conceptualization. Neil A.R.
Gow: Writing – original draft, Supervision, Funding acquisition, Conceptualization.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
Given his role as Editor-in-Chief, Neil Gow had no involvement in the peer review
of this article and has no access to information regarding its peer review. Full responsibility
for the editorial process for this article was delegated to Wenxia Fang.