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                    Journal of

Fungi
Article

Inhibitory Effects of the Fungal Pigment Rubiginosin C
on Hyphal and Biofilm Formation in Candida albicans
and Candida auris
Haoxuan Zeng 1,2 , Marc Stadler 1,2 , Wolf-Rainer Abraham 1 , Mathias Müsken 3, * and Hedda Schrey 1,2, *
1

2

3

*

Citation: Zeng, H.; Stadler, M.;
Abraham, W.-R.; Müsken, M.; Schrey,
H. Inhibitory Effects of the Fungal
Pigment Rubiginosin C on Hyphal
and Biofilm Formation in Candida
albicans and Candida auris. J. Fungi

Department of Microbial Drugs, Helmholtz Centre for Infection Research GmbH and German Centre for
Infection Research (DZIF), Partner Site Hannover/Braunschweig, Inhoffenstrasse 7,
38124 Braunschweig, Germany; haoxuan.zeng@helmholtz-hzi.de (H.Z.);
marc.stadler@helmholtz-hzi.de (M.S.); wrabraham253@gmail.com (W.-R.A.)
Institute of Microbiology, Technische Universität Braunschweig, Spielmannstraße 7,
38106 Braunschweig, Germany
Central Facility for Microscopy, Helmholtz Centre for Infection Research GmbH, Inhoffenstrasse 7,
38124 Braunschweig, Germany
Correspondence: mathias.muesken@helmholtz-hzi.de (M.M.); hedda.schrey@helmholtz-hzi.de (H.S.);
Tel.: +49-5316181-4009 (M.M.); +49-5316181-4239 (H.S.)

Abstract: The two fungal human pathogens, Candida auris and Candida albicans, possess a variety
of virulence mechanisms. Among them are the formation of biofilms to protect yeast against harsh
conditions through the development of (pseudo)hyphae whilst also facilitating the invasion of host
tissues. In recent years, increased rates of antifungal resistance have been associated with C. albicans
and C. auris, posing a significant challenge for the effective treatment of fungal infections. In the
course of our ongoing search for novel anti-infectives, six selected azaphilones were tested for their
cytotoxicity and antimicrobial effects as well as for their inhibitory activity against biofilm and
hyphal formation. This study revealed that rubiginosin C, derived from stromata of the ascomycete
Hypoxylon rubiginosum, effectively inhibited the formation of biofilms, pseudohyphae, and hyphae
in both C. auris and C. albicans without lethal effects. Crystal violet staining assays were utilized to
assess the inhibition of biofilm formation, while complementary microscopic techniques, such as
confocal laser scanning microscopy, scanning electron microscopy, and optical microscopy, were used
to investigate the underlying mechanisms. Rubiginosin C is one of the few substances known to
effectively target both biofilm formation and the yeast-to-hyphae transition of C. albicans and C. auris
within a concentration range not affecting host cells, making it a promising candidate for therapeutic
intervention in the future.

2023, 9, 726. https://doi.org/
10.3390/jof9070726
Academic Editor: David S. Perlin

Keywords: Candida auris; Candida albicans; biofilms; pseudohyphae; hyphae; extracellular vesicles;
drug resistance; virulence

Received: 13 June 2023
Revised: 29 June 2023
Accepted: 30 June 2023

1. Introduction

Published: 5 July 2023

Antimicrobial resistance has been a significant and ongoing challenge to public health
and manifests itself when pathogenic species become able to eliminate or withstand antibiotic treatments [1,2]. Among the various factors contributing to antimicrobial recalcitrance
in bacterial and fungal diseases, biofilm populations play a critical role. According to the
National Institute of Health, biofilm formation is linked to 65% of microbial and 80% of
chronic infections [3].
Candida species are typically commensal yeasts that inhabit human skin and mucosal
surfaces. However, they can also cause both superficial and life-threatening systemic
infections in the human body, such as oral or vaginal candidiasis, as well as nosocomial
bloodstream infections [4,5]. The pathogenicity of Candida species is driven by the expression of specific virulence factors, such as biofilm formation, yeast-to-hyphae transition, or

Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).

J. Fungi 2023, 9, 726. https://doi.org/10.3390/jof9070726

https://www.mdpi.com/journal/jof

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the secretion of proteolytic and lipolytic enzymes, which are associated with high mortality
rates of infections in hospital settings [6,7].
The process of biofilm formation in Candida involves four stages: (i) attachment of
yeast cells to a solid surface (within 90 min); (ii) formation of hyphae or pseudohyphae
and involvement in biofilm formation; (iii) elongation of hyphae and growth of other
polymorphic cells to form an extracellular matrix; (iv) release of new yeast cells [8–11].
The formation of biofilms not only facilitates the attachment on polymeric surfaces, for
example, on medical devices introduced into the human body, but also promotes drug
resistance [8]. The underlying resistance mechanisms might be multifactorial, including,
e.g., extracellular matrix polysaccharides and efflux pumps [9]. While polysaccharides can
impede the activity of antifungal agents by preventing them from reaching their target
sites, an increased efflux pump activity can transport antibiotics from inside the cell to the
exterior environment [9].
Among all Candida species, C. albicans is considered to be the most commonly found
fungus, causing approximately 50–90% of candidiasis infections [7]. Compared to other
Candida species, C. albicans can produce denser biofilms [11]. Another emerging nosocomial pathogen is C. auris, which was first described in 2009 in an external ear canal in a
patient from Japan [12]. After its initial identification, numerous outbreaks of invasive
infections have been reported in hospitals across several countries and it is rapidly spreading worldwide [13–15]. Candida auris is resistant to multiple drugs, especially azoles (e.g.,
fluconazole), polyenes (e.g., amphotericin B), and echinocandins (e.g., caspofungin) [16],
and can survive in high-salt and high-temperature environments [17]. It forms biofilms
that colonize the skin and can persist on medical device surfaces for up to 14 days [18],
leading to infections such as bloodstream infections, urinary tract infections, and invasive
candidiasis with high mortality rates [19]. Due to its resistance and ability to form biofilms,
it is difficult to eradicate [9]. In general, the biofilms of C. auris are thinner than that of
C. albicans, although the ability to form biofilms varies among C. auris isolates [20,21].
Furthermore, several studies have indicated that the pathogenicity of C. auris is comparable
to, or even more virulent than, that of C. albicans [6,22].
Fungi are prolific producers of structural complex secondary metabolites with various
biological activities. Over the last century, they have provided several lead structures and
pharmacophores that benefit humans. In recent years, novel azaphilones have been identified that exhibit a wide range of biological activities, including antimicrobial, antifungal,
antiviral, antibiofilm, antitumor, cytotoxic, and anti-inflammatory activities [23–25]. Ascomycetes of the family Hypoxylaceae (Xylariales) are by far the most versatile producers
of azaphilones [26]. Due to their chemotaxonomic significance, the pigments have already
been successfully consulted to discriminate within the genera and species [27–29]. The
Hypoxylaceae currently comprises 15 genera (www.mycobank.org; accessed on 3 May
2023), such as Hypoxylon, Daldinia, or Jackrogersella, inhabiting a multitude of habitats, e.g.,
as endophytes or insect-associates, colonizing lichens, or stromata on decaying wood [26].
It was recently shown that highly complex azaphilones were present in archaeological
specimens of Hypoxylon fragiforme, estimated to be over 1000 years old [30]. Here, we
focused on rubiginosin- and rutilin-type azaphilones, obtained from the stromata of the ascomycetes Hypoxylon rubiginosum and Hypoxylon texense, which have the ability to produce
the pigments around the perithecia [30–32]. Weak to moderate antimicrobial activities of
some rubiginosin- and rutilin-type azaphilones against Bacillus subtilis and Staphylococcus
aureus were previously reported [28].
In the course of our ongoing search for novel anti-infectives, we conducted a study to
investigate the potential of the selected azaphilones (Figure 1) for their inhibitory efficacy
against (pseudo)hyphae development and their effects on biofilms. Our investigation
focused on their effectiveness against the two invasive fungal pathogens C. albicans and
C. auris. Although all pigments share the same pyronoquinone core, they differ in the type
of substituents. Thus, rubiginosin C (Rub C) and rubiginosin W (Rub W) are substituted by
a linear polyketide moiety of different lengths and substitution patterns. In contrast, the

x FOR PEER REVIEW

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J. Fungi 2023, 9, 726

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of substituents. Thus, rubiginosin C (Rub C) and rubiginosin W (Rub W) are substituted
by a linear polyketide moiety of different lengths and substitution patterns. In contrast,
the monomeric rubiginosin
(Rub A) and
rubiginosin
Z (Rub Z)
carryZ)an
orsellinic
acid acid (OA)
monomeric A
rubiginosin
A (Rub
A) and rubiginosin
Z (Rub
carry
an orsellinic
(OA) unit, while rutilin
A
(Rut
A)
and
rutilin
B
(Rut
B)
are
their
dimeric
congeners.
unit, while rutilin A (Rut A) and rutilin B (Rut B) are their dimeric congeners.

1. Chemical
structures
of selected
rubiginosinandazaphilones
rutilin-type azaphilones
together with
Figure 1. ChemicalFigure
structures
of selected
rubiginosinand
rutilin-type
together with
farnesol (FA)
orsellinic
acid (OA);
core shown in red.
farnesol (FA) and orsellinic
acidand
(OA);
azaphilone
coreazaphilone
shown in red.

2. Materials and Methods
2.1. Isolation of Selected Azaphilones and Preparation of Pathogenic Strains

2. Materials and Methods

2.1. Isolation of SelectedIsolation
Azaphilones
Preparation
Pathogenic
Strains work by Becker et al. [28], and
of alland
azaphilones
wasofachieved
in previous
aliquots
were used
herein
(Supplementary
Material
for Becker
detailedet
information).
Isolation of all
azaphilones
was
achieved
in previous
work by
al. [28], and
Strains
C. auris [DSM 21092]
and for
C. albicans
[DSM
1665, DSM 11225] were obtained
aliquots were used herein
(Supplementary
Material
detailed
information).
from
the German
of Microorganisms
Cell
Cultures
GmbH
(DSMZ, BraunStrains C. auris
[DSM
21092] Collection
and C. albicans
[DSM 1665,and
DSM
11225]
were
obtained
schweig, Germany) as a freeze-dried sample. The organisms were cultured in Yeast Extract
from the German Collection of Microorganisms and
Cell Cultures GmbH (DSMZ, BraunPeptone Dextrose (YPD) medium (30 ◦ C, 120 rpm, 2 d). The 1 mL aliquots with 20% glycerol
schweig, Germany) as a freeze-dried
sample. The organisms were cultured in Yeast Exwere stored at −20 ◦ C for short-term use, and −80 ◦ C for long-term use. Strain C. albicans
tract Peptone Dextrose
(YPD)
medium
(30[33]
°C,was
120provided
rpm, 2 d).
mL aliquots
with
20% Centre
CAI-4 HWP1-lacZ [ZK3379]
by The
Prof.1Ursula
Bilitewski
(Helmholtz
glycerol were stored
at −20 °CResearch
for short-term
use, and −80 °C
for long-term
Strainstored
C. in 20%
for Infection
GmbH, Braunschweig,
Germany)
in 1 mLuse.
aliquots,
◦
albicans CAI-4 HWP1-lacZ
[
ZK3379]
[33]
was
provided
by
Prof.
Ursula
Bilitewski
(Helmglycerol at −20 C.
holtz Centre for Infection Research GmbH, Braunschweig, Germany) in 1 mL aliquots,
stored in 20% glycerol at −20 °C.

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2.2. Determination of Minimum Inhibitory Concentration (MIC) and Cytotoxicity
The MICs of rubiginosin- and rutilin-type azaphilones—in detail, Rub A, C, W, Z, and
Rut A and B—were determined as described previously [34] using the selected pathogens
C. albicans [DSM 11225, DSM 1665], C. albicans CAI-4 HWP1-lacZ [ZK3379] and C. auris
[DSM 21092] (see Table S1 for detailed information). All compounds were tested within
the concentration range of 250 µg/mL–2 µg/mL for C. albicans [DSM 11225, DSM 1665], C.
albicans CAI-4 HWP1-lacZ [ZK3379], C. auris [DSM 21092].
Cytotoxic effects were evaluated on human endocervical adenocarcinoma KB-3-1
[ACC 158] cells and mouse fibroblasts L929 [ACC 2] upon treatment with Rub C, W, and
Rut A and B within the concentration range of 37 µg/mL–0.63 ng/mL. The half-maximum
inhibitory concentrations (IC50 ) were determined by standard MTT assays as reported
previously [32] (see the Supplementary Material for detailed information). The IC50 values
of Rub A and Z have currently been reported [28].
2.3. Antibiofilm Assay with Crystal Violet
2.3.1. Biofilm Formation Assay of C. auris and C. albicans
The fungal pathogens C. auris [DSM 21095] or C. albicans [DSM 11225] were cultured
from stock in 25 mL YPD medium in a 250 mL flask (30 ◦ C, 100 rpm, 18 h). The turbidity of
the broth was measured at 280 nm using a spectrophotometer (Nanodrop 2000c, Thermo
Fisher Scientific, Waltham, MA, USA) and diluted to match the turbidity of a 0.5 McFarland
standard in RPMI 1640 medium (Gibco, New York, NY, USA; Thermo Fisher Scientific),
supplemented with 0.165 mM 3-(N-morpholino)propanesulfonic acid (MOPS, Carl Roth,
Karlsruhe, Germany) for C. auris [35,36], or 0.05 McFarland standard in RPMI 1640 medium
in the case of C. albicans [37–39]. Subsequently, 150 µL of the fungal dispersion were added
into each well of a 96-well microtiter plate (Falcon no. 351172, Thermo Fisher Scientific) and
further incubated (37 ◦ C, 150 rpm, 2 h [C. auris] or 90 min [C. albicans]). The supernatant
was discarded and the plate was rinsed one time by using a PBS buffer. Afterwards,
azaphilones (Rub A, C, W, Z, and Rut A and B) were serially diluted in 150 µL in fresh
medium to concentrations of 250–2 µg/mL (Rub C for C. auris: 250–0.02 µg/mL). Methanol
(2.5%) was used as a solvent control and both, nystatin (NYS; Thermo Fisher Scientific) and
farnesol (FA; Sigma Aldrich, St. Louis, MO, USA) as positive controls (250–2 µg/mL) for
C. auris and C. albicans, respectively. Plates were further incubated (37 ◦ C, 150 rpm, 24 h).
The supernatant was discarded and biofilms were washed with PBS, stained by adding
150 µL of the crystal violet (CV; Sigma Aldrich) solution (0.1%), and incubated (room
temperature, 25 min). Afterwards, the plates were washed twice with PBS buffer. A total of
150 µL ethanol (95%) were applied to dissolve the biofilm-bound CV. The absorbance was
measured by a plate reader (Synergy 2, BioTek, Santa Clara, CA, USA) at 570 nm for C. auris
or 610 nm for C. albicans. Error bars indicate SD with duplicates in two biological repeats.
2.3.2. Assay to Determine Rub C Effects on C. auris Biofilms of Various Ages
The turbidity of C. auris [DSM 21092] dispersion was measured at 280 nm and diluted
to the turbidity of a 0.5 McFarland standard. C. auris was cultured in RPMI 1640 medium
supplemented with 0.165 mM MOPS (37 ◦ C, 150 rpm, 2 h, 12 h, 24 h) in 96-well non-tissue
microtiter plates (Falcon no. 351172, Thermo Fisher Scientific) [40]. After incubation,
C. auris biofilms of various ages (2 h, 12 h, 24 h) were washed once by PBS buffer and
treated (37 ◦ C, 150 rpm, 24 h) with serial diluted Rub C (250–0.02 µg/mL) in fresh RPMI
1640 medium supplemented with 0.165 mM MOPS. Samples of each time point were further
processed and evaluated by a microtiter plate reader, as described above.
2.4. Observations of Biofilm by Confocal Laser Scanning Microscopy (CLSM)
A culture of C. auris [DSM 21092] was adjusted to the turbidity of a 0.5 McFarland
standard and cultured (37 ◦ C, 2 h) in RPMI 1640 medium supplemented with 0.165 mM
MOPS to allow for the attachment of fungal cells to µClear microtiter plates (Greiner BioOne, Kremsmünster, Austria) [41]. Afterwards, wells were gently rinsed with PBS buffer

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and incubated with 150 µL of fresh medium containing Rub C in the concentrations 250
and 15.6 µg/mL, respectively. Medium with 2.5% methanol was used as a solvent control.
The plates were covered with an air-permeable breath seal cover foil (Greiner Bio-One)
and further incubated (37 ◦ C, 24 h). Afterwards, the supernatant with planktonic cells
was slowly removed with a multi-channel pipette. Biofilms were gently washed once
with PBS buffer and stained with the fluorescent dyes FUN-1 (Invitrogen, Waltham, MA,
USA; Thermo Fisher Scientific) and Calcofluor White M2R (Invitrogen, Thermo Fisher
Scientific) by incubating (37 ◦ C, 30 min) the wells with 150 µL PBS containing 10 µM
FUN-1 and 25 µM M2R in the dark [42]. Plasma membrane integrity and metabolic function of fungi are required to convert the yellow-green-fluorescent intracellular staining
of FUN 1 into the red-orange-colored intravascular structures. This contrasts with the
yellow-green fluorescence of dead cells where FUN-1 remains in the cytosol. Calcofluor
White M2R labels cell-wall chitin and beta glucoside bonds appearing as green, fluorescent signals regardless of the metabolic state of the cell. Stained biofilms were observed
using an inverted, confocal laser scanning microscope SP8 (Leica Microsystems, Wetzlar,
Germany) and acquired with the software LAS X and the following settings: Z-step size is
2 µm, green (excitation = 488 nm and emission = 530 nm) and red (excitation = 488 nm and
emission = 620 nm) fluorescence signal. Image analysis was performed with the software Image J 1.53 k (National Institute of Health, Bethesda, MD, USA) for quantification and Imaris
9.31 (Oxford, UK) for visualization.
2.5. Colony Forming Units (CFU) and the Growth Curve of Candida
2.5.1. CFU of C. auris
The preculture of C. auris [DSM 21092] was prepared and adjusted to the turbidity as
previously described in Section 2.4. After 2 h of incubation, Rub C was added to each well
to the final concentrations of 250 µg/mL and 15.6 µg/mL. CFU was tested after 12 and 24 h,
respectively. Cells were resuspended in the well 50 times. We prepared a dilution series in 1
to 10 steps (20 µL in 200 µL) down to a final dilution level of 10−6 and platted 100 µL of this
last dilution on YPD agar plates using small glass beads (5 to 10) to homogeneously spread
the liquid. Individual yeast colonies on the non-hyphal promoting agar plates were counted
after incubation at 30 ◦ C for 2 days [43]. Afterwards, CFUs were calculated considering the
dilution factors. Error bars indicate SD with duplicates in two biological repeats.
2.5.2. The Growth Curve of Candida
The preculture of C. auris [DSM 21095] and C. albicans [DSM 11225] were adjusted to a
0.1 McFarland standard, cultured (37 ◦ C, 150 rpm) in RPMI 1640 medium supplemented
with 0.165 mM MOPS and RPMI 1640 medium, respectively, and added together with
Rub C to a 96-well microtiter plate (Falcon no. 351172, Thermo Fisher Scientific) to result
in concentrations of 250 µg/mL and 62.5 µg/mL [44]. The absorbance was measured at
630 nm after 2 h, 5 h, 8 h, 14 h, and 20 h. Methanol (2.5%) was used as a solvent control.
Error bars indicate SD with duplicates in two biological repeats.
2.6. Observations of Candida Cells by Optical Microscopy
2.6.1. Visualization of C. auris and C. albicans Cells
Planktonic cells of C. auris [DSM 21092] or C. albicans [DSM 11225] were incubated with
different concentrations of Rub C in 96-well non-tissue microtiter plates (37 ◦ C, 150 rpm,
24 h; C. auris: with 250, 15.6 µg/mL of Rub C in 150 µL RPMI 1640 medium with 0.165 mM
MOPS; C. albicans: with 250, 62.5 µg/mL of Rub C in 150 µL RPMI 1640 medium with 50 nM
glucose (Sigma Aldrich) and 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES, Sigma Aldrich)) [44]. After 24 h of incubation, 30 µL of 25% paraformaldehyde
(PFA, Thermo Fisher Scientific) were added into each well. Planktonic cells were fixed
at room temperature for 30 min and the supernatant of each well was taken out and
collected in a 1.5 mL Eppendorf tube (Lot. I182541I, Eppendorf, Hamburg, Germany)
following centrifugation with 12,000 rpm for 12 min at room temperature. The supernatant

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was removed and the pellet was resuspended in 20 µL PBS buffer. Afterwards, 15 µL
resuspended cells were loaded on microscope slides (LOT 7691777, Thermo Fisher Scientific)
and covered with cover glass (24 × 50 mm Menzel-Gläser, Braunschweig, Germany).
Samples were monitored using an Axio Imager A2 light microscope equipped with a
63×/1.25 oil objective (Zeiss, Jena, Germany) and the Zen blue 3.0 software.
2.6.2. Assay to Determine the Impact of Rub C on Hyphae
C. albicans [DSM 11225] was cultured (37 ◦ C, 150 rpm, 24 h and 48 h) in 96-well nontissue microtiter plates in RPMI 1640 medium supplemented with 50 nM glucose and
50 mM HEPES to form hyphae. Afterwards, 24 h- and 48 h-old hyphae were treated with
Rub C (62.5 µg/mL) and cultured (37 ◦ C, 150 rpm, 2 h, 5 h, 10 h, and 18 h) in fresh RPMI
1640 medium supplemented with 50 nM glucose and 50 mM HEPES for various periods of
time [44]. Each sample was fixed as described in Section 2.6.1 and monitored using an Axio
Imager A2 light microscope equipped with a 63×/1.25 oil objective (Zeiss) and the Zen
blue 3.0 software.
2.7. Observations of Candida Cells by Scanning Electron Microscopy (SEM)
C. auris [DSM 21092] was incubated (37 ◦ C, 24 h) with Rub C at the concentration
of 250 and 15.6 µg/mL in 96-well non-tissue microtiter plates in RPMI 1640 medium
supplemented with 0.165 mM MOPS. Cultures were fixed with 5% formaldehyde and
2% glutaraldehyde (final concentrations) and washed twice in TE buffer (20 mM TRIS
with 1 mM EDTA, pH 6.9). A 50 µL aliquot was added to round poly-L-lysine pretreated
coverslips and incubated (room temperature, 10 min). Further processing was carried out
as previously described with slight modifications [45]. In brief, cells were fixed for 10 min
on the coverslip with TE buffer including 1% glutaraldehyde (final concentration). The
coverslips were washed twice in TE buffer and dehydrated in 10 min steps on ice with a
graded series of acetone (10%, 30%, 50%, 70%, and 90%), followed by two steps in 100%
acetone at room temperature. The coverslips were mounted onto aluminum stubs with
carbon adhesive discs; they were critical-point-dried with the automated CPD300 (Leica
Microsystems) and gold-palladium-sputter-coated (55 s at 45 mA) with a SCD500 (BalTec, Balzers, Liechtenstein). Images were acquired with a field emission scanning electron
microscope Zeiss Merlin (Zeiss, Oberkochen, Germany) using the Everhart Thornley HESE2
detector and the in lens SE detector in a 25:75 ratio with an acceleration voltage of 5 kV.
2.8. Screening and Quantification of Hyphal Inhibitory Activities with β-Galactosidase
Activity Assay
The strain C. albicans CAI-4 HWP1-lacZ was directly precultured overnight from a cryostock in a defined medium (6.7 g/L yeast nitrogen base without amino acids, 9 g/L glucose,
1 g/L maltose) at 30 ◦ C [46]. Cells were washed twice in pre-warmed hyphae-inducing
medium SLAD (1.7 g/L yeast nitrogen base without amino acids without ammonium
sulfate, 2 g/L glucose, 1 g/L maltose, 6 mg/L ammonium sulfate, buffered to pH 7.3 using
0.165 M MOPS) and resuspended in the same medium. The turbidity of fungal dispersion
was set to a 0.1 McFarland standard (280 nm). A total of 50 µL of the mixed suspension was
added to each well of a 96-well microtiter plate containing azaphilones (Rub A, C, W, Z,
and Rut A and B), serially diluted to concentrations of 100–6.3 µg/mL, and FA was used as
a positive control (100–0.8 µg/mL). Afterwards, the 96-well microtiter plate was incubated
(37 ◦ C, 150 rpm, 5 h) to induce hyphal growth.
After, hyphae induction cells were incubated (37 ◦ C, 150 rpm, 60 min) with 100 µL of zbuffer (composed of 60 mM Na2 HPO4 , 40 mM NaH2 PO4 , 10 mM KCl, 1 mM Mg2 SO4 , 1 mM
DTT, and 0.2% sodium lauroyl sarcosinate) to lyse the cells. Following this, 50 µL of 4 g/L
o-nitrophenyl beta-D-galactopyranoside (ONPG, Sigma Aldrich) supplemented with 0.1 M
potassium phosphate buffer (pH 7.0) was added, and the optical density was measured at
414 nm and 550 nm wavelengths using a microtiter plate reader (Synergy 2, BioTek), and
was measured again after 120 min incubation at 37 ◦ C to determine the hydrolysis of ONPG

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due to β-galactosidase activity. The absorption by o-nitrophenol was calculated as follows:
(OD414 nm − c × OD550 nm )120 min − (OD414 nm – c × OD550 nm )0 min . c was determined as
1.36 when using microtiter plates as shown in the study of Heintz-Buschart et al. [47].
2.9. Statistical Analysis
Differences between samples and the control group were determined by a two-tailed
Student’s t-test. Statistical significance was defined as p < 0.05. Analysis was carried out
using GraphPad Prism 9® (GraphPad Software, San Diego, CA, USA) [48].
3. Results
3.1. Determination of MIC and Cytotoxicity of Azaphilones
In accordance with previous results, no antimicrobial activity was observed for the six
selected azaphilones (Rub A, C, W, Z, and Rut A and B) against the four strains of Candida
(C. albicans [DSM 1665, DSM 11225], CAI-4 HWP1-lacZ, and C. auris) at the highest tested
concentration of 250 µg/mL (Table 1) [28].
Table 1. Antimicrobial activity of selected rubiginosin- and rutilin-type azaphilones against C. albicans
and C. auris.
MIC [µg/mL]
Tested
Organisms
C. albicans [28]
C. albicans
C. albicans
CAI-4
HWP1-lacZ
C. auris

Rub

Strain No.

Rut

Nystatin

A

C

W

Z

A

B

DSM 1665
DSM 11225

–
–

–
–

–
–

–
–

–
–

–
–

8.3
8.3

ZK3379

–

–

–

–

–

–

8.3

–

–

–

–

–

–

31.3

DSM 21092

–: no activity.

Only the dimeric rutilin-type azaphilone derivatives Rut A and Rut B exhibited
significant cytotoxic activity with IC50 values in a range of 1.1–2.2 µM (0.9–1.8 µg/mL)
against murine fibroblasts (L929) and human endocervical adenocarcinoma cells (KB-3-1)
(Table 2). Furthermore, moderate cytotoxicity against these two cell lines with IC50 values
between 3.2 and 5.4 µM (2.6–4.5 µg/mL) has been reported for monomeric OA-carrying
Rub A and Rub Z in a previous study by Becker et al. [28].
Table 2. Cytotoxic activity of selected rubiginosin- and rutilin-type azaphilones against two cell lines.
IC50 [µM]
Cell Lines

KB-3-1
L929

Strain No.

ACC 158
ACC 2

Rub

Rut

Epothilone B

A [28]

C

W

Z [28]

A

B

5.2
3.2

–
–

–
–

5.2
4.7

1.1
1.2

1.5
2.2

5.3 × 10−5 /2.8 × 10−5 [27]
1.7 × 10−4 /3.1 × 10−5 [27]

–: no activity.

3.2. Inhibitory Effects of Azaphilone Pigments against Candida Biofilm Formation
In addition to MIC assays, we also assessed the efficacy of the six azaphilone derivatives (Rub A, C, W, Z, and Rut A and B) towards biofilms of several pathogens (C. albicans,
C. auris, Pseudomonas aeruginosa, and S. aureus) by a CV assay. Rub C, W, and Rut A and
B exhibited moderate inhibitory effects against the biofilm formation of S. aureus, while
none of the tested rubiginosin- or rutilin-type azaphilones was active against P. aeruginosa
(Table S2). Interestingly, moderate activities against the biofilms of S. aureus have also
been reported for the known hybridorubrins, which are structurally related to Rub C and
W [29]. Furthermore, promising inhibitory effects were observed for Rub A, C and W

J. Fungi 2023, 9, 726

exhibited moderate inhibitory effects against the biofilm formation of S. aureus, while none
C. auris, Pseudomonas aeruginosa, and S. aureus) by a CV assay. Rub C, W, and Rut A and B
of the tested rubiginosin- or rutilin-type azaphilones was active against P. aeruginosa (Ta
exhibited moderate inhibitory effects against the biofilm formation of S. aureus, while none
ble S2). Interestingly, moderate activities against the biofilms of S. aureus have also been
of the tested rubiginosin- or rutilin-type azaphilones was active against P. aeruginosa (Tareported for the known hybridorubrins, which are structurally related to Rub C and W
ble S2). Interestingly, moderate activities against the biofilms of S. aureus have also been
8 ofW
19 agains
[29]. Furthermore, promising inhibitory effects were observed for Rub A, C and
reported for the known hybridorubrins, which are structurally related to Rub C and W
the biofilm formation of C. auris and C. albicans at sub-inhibitory concentrations (Figures
[29]. Furthermore, promising inhibitory effects were observed for Rub A, C and W against
2 and 3).
the biofilm
formation of C. auris and C. albicans at sub-inhibitory concentrations (Figures
against the biofilm formation of C. auris and C. albicans at sub-inhibitory concentrations
2 and
3). 2 and 3).
(Figures

Figure 2. Effect on the biofilm formation of C. auris after 24 h treatment with Rub C and Rub W. (A
Figure 2. Effect on the biofilm formation of C. auris after 24 h treatment with Rub C and Rub W.
Image of CV-stained wells of a microtiter plate. (B) Efficacy of Rub C and Rub W on the formation
(A)2.
Image
of CV-stained
wells formation
of a microtiter
(B) Efficacy
C and Rub
W on
theCformation
Figure
Effect
on the biofilm
ofplate.
C. auris
after 24ofhRub
treatment
with
Rub
and Rub W. (A)
of C. auris biofilms. Nystatin (NYS) was used as positive control, methanol as solvent control. Erro
of
C.
auris
biofilms.
Nystatin
(NYS)
was
used
as
positive
control,
methanol
as
solvent
control.
Error
Image of CV-stained wells of a microtiter plate. (B) Efficacy of Rub C and Rub W on the
formation
bars indicate SD of duplicates in two biological repeats; p values: * p < 0.05, ** p < 0.01, *** p < 0.001.
indicate
SD of
duplicates
in twowas
biological
repeats;
p values:
* p <methanol
0.05, ** p < as
0.01,
*** p < control.
0.001.
of C.bars
auris
biofilms.
Nystatin
(NYS)
used as
positive
control,
solvent
Error
bars indicate SD of duplicates in two biological repeats; p values: * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure
3. Effect
biofilmformation
formation of
after
24 h24
treatment
with rubiginosinand rutilinFigure
3. Effect
onon
biofilm
ofC.C.albicans
albicans
after
h treatment
with rubiginosinand rutilin
type
azaphilones.
FA
was
used
as
positive
control,
methanol
as
solvent
control.
Error
bars
indicate
type azaphilones. FA was used as positive control, methanol as solvent control. Error
bars indicate
Figure
on biofilm
formation of C. palbicans
24 h**treatment
rubiginosin- and rutilinSD 3.
of Effect
duplicates
two biological
*after
p < 0.05,
p < 0.01, ***with
p < 0.001.
SD of
duplicates
inintwo
biologicalrepeats;
repeats; values:
p values:
* p < 0.05,
** p < 0.01,
*** p < 0.001.
type azaphilones. FA was used as positive control, methanol as solvent control. Error bars indicate
Rub C and
showed repeats;
prohibitive
activity*against
the**formation
of C.
biofilms
SD of duplicates
in Rub
two W
biological
p values:
p < 0.05,
p < 0.01, ***
p <auris
0.001.
Rub C2),and
Rub monomeric
W showedand
prohibitive
activity against
the formation
of C.
auris bio
(Figure
whereas
dimeric OA-carrying
azaphilones
(Rub A, Z and
Rut
A (Figure
and B) did
affect the
formationand
(Table
S2). Regarding
the effects
of Rub C,(Rub
moreA, Z and
films
2), not
whereas
monomeric
dimeric
OA-carrying
azaphilones
Rub C and was
Rub W showed
prohibitive
activity against
the formation
of C. auris bioabove
the concentration
125 µg/mL.
At effects
a concentration
Rutbiofilm
A and mass
B) did notobserved
affect the
formation
(Table S2).ofRegarding
the
of Rub C, more
filmsof(Figure
2), whereas
monomeric
and dimeric
azaphilones
A, Z and
250 µg/mL,
absorption
was almost
doubled OA-carrying
compared to the
control. At(Rub
Rub C
biofilm
mass wasthe
observed
above
the concentration
of 125 µg/mL.
At a concentration
o
Rut concentrations
A and B) did between
not affect
the
formation
(Table
S2).
Regarding
the
effects
of
Rub
0.5 and 62.5 µg/mL, we could detect significant inhibitory effectsC, more
of ca.mass
50% against
the formation
of biofilms.
Rub W, which
structurally
to Rub C,
biofilm
was observed
above
the concentration
ofis125
µg/mL.related
At a concentration
of

J. Fungi 2023, 9, 726

was slightly less inhibitory. Interestingly, we could not detect a stro
biomass for Rub W at higher concentrations.
As shown in Figure 3, all tested pigments inhibited the format
9 of 19
films when applied at the highest test concentration of 250 µg/mL.
O
Rub A and C, exhibited activity below this concentration. While Rub
was slightly
less inhibitory.
Interestingly, we could
not detect
strong increase
biofilm
effects
starting
at concentrations
above
31.3a µg/mL,
Rubin C
exhibited
biomass for Rub W at higher concentrations.
fectsAsof
ca. in
50–80%
biofilm
reduction
attheconcentrations
between
shown
Figure 3, all
tested pigments
inhibited
formation of C. albicans
biofilms 7.8 µ
when applied
the highest
test concentration
of 250
Only twoactivity
rubiginosins,
A
(Figure
3);athence,
even
exceeding
theµg/mL.
inhibitory
ofRub
FA.

and C, exhibited activity below this concentration. While Rub A showed significant effects
starting at concentrations above 31.3 µg/mL, Rub C exhibited strong inhibitory effects of ca.
50–80%
biofilm
reduction at
concentrations
between
7.8 µg/mL andStages
250 µg/mL
(Figure
3); Biofilm
3.3.
Rub
C Activity
against
Different
Maturation
of C.
auris
hence, even exceeding the inhibitory activity of FA.

To investigate the efficacy of Rub C towards different develop
auris biofilm formation, the cells were grown for 2 h (attachment p
To investigate the efficacy of Rub C towards different developmental phases of C.
(maturating
biofilms),
medium
exchange
auris biofilm formation,
the cellsrespectively,
were grown for 2 before
h (attachment
phase), 12
h, and 24 h and t
(maturating
biofilms),
respectively,
before
medium
exchange
and
treatment
with
Rub C for
for 24 h. The drug nystatin was used as the positive control
(Table
24 h. The drug nystatin was used as the positive control (Table S3). The CV assay revealed
vealed
Rub
activity
was
carriedbiofilm
out with
a progressing
biofilm
that Rub Cthat
activity
wasC
carried
out with
a progressing
state (Figure
4).
3.3. Rub C Activity against Different Maturation Stages of C. auris Biofilm

Figure 4. Effect on biofilm formation of pre-grown C. auris biofilms of different developmental
Figure
4. Effect on biofilm formation of pre-grown C. auris biofilms of d
stages (2 h, 12 h, and 24 h) after 24 h treatment with Rub C. The solvent methanol served as solvent
stages
(2 h,bars
12indicate
h, and
h) afterin24
treatment
with
Rub
The
met
control. Error
SD 24
of duplicates
twohbiological
repeats;
p values:
* p C.
< 0.05,
** psolvent
< 0.01,
*** p < 0.001.Error bars indicate SD of duplicates in two biological repeats; p valu
control.
***
< 0.001. of the Effect of Rub C on C. auris Biofilms via CLSM
3.4. pVisualization

To address the inhibitory effects of Rub C towards C. auris, CLSM was used to visualize
the three-dimensional structure in the early phase of biofilm development. Analysis of
3.4.
Visualization of the Effect of Rub C on C. auris Biofilms via CLSM
CLSM exhibited morphological variances in the biofilm structures treated with two different
concentrations
of Rub C
(Figure
5). At 250 effects
µg/mL, the
of the biofilm
was
To address
the
inhibitory
ofmorphology
Rub C towards
C. auris,
CL
flatter but with densely packed single cells compared to untreated biofilms while less
alize
theactivity
three-dimensional
structure
the early
ofRub
biofilm
d
metabolic
was observed. In contrast,
biofilmsin
exposed
to 15.6phase
µg/mL of
C
appeared
more
porous andmorphological
the cells were aggregated
and more
size
of
CLSM
exhibited
variances
in heterogeneous
the biofilminstructures
(Figure 5). We also saw differences in the overall biofilm biomass (250 µg/mL > solvent
ferent
ofinRub
control orconcentrations
15.6 µg/mL), as shown
FigureCS2.(Figure 5). At 250 µg/mL, the morp

was flatter but with densely packed single cells compared to untreat
metabolic activity was observed. In contrast, biofilms exposed to 1
appeared more porous and the cells were aggregated and more h
(Figure 5). We also saw differences in the overall biofilm biomass (
control or 15.6 µg/mL), as shown in Figure S2.

J. Fungi 2023, 9, x FOR PEER REVIEW
J. Fungi 2023, 9, 726

10 of 19

10 of 19

Figure
5. Three-dimensional
projections
of C.
C. auris
aurisbiofilms
biofilms(24
(24h hafter
after exFigure
5. Three-dimensional
projectionsand
andorthogonal
orthogonal sections
sections of
posure
to
the
Rub
C
of
attached
cells
[2
h]).
The
samples
were
stained
with
FUN
1
dye
and
Calcoflour
exposure to the Rub C of attached cells [2 h]). The samples were stained with FUN 1 dye and
White
M2R. Regarding
green-yellow
fluorescence
represents
deaddead
cells, and
Calcoflour
White M2R.intensive
Regardingoverlapping,
intensive overlapping,
green-yellow
fluorescence
represents
intracellular
red
fluorescence
represents
metabolically
active
cells.
Treatments
were
carried
cells, and intracellular red fluorescence represents metabolically active cells. Treatments were carriedout at
concentrations
of 250 and
15.6and
µg/mL
at 37 °Catin37RPMI
1640 (supplemented
withwith
0.165
mMmM
MOPS).
◦ C in RPMI
out at concentrations
of 250
15.6 µg/mL
1640 (supplemented
0.165
(a) Calcafluor
White
M2R.
(b)
FUN-1.
(c)
Multichannel.
(d)
Three-dimensional
projections.
ExemMOPS). (a) Calcafluor White M2R. (b) FUN-1. (c) Multichannel. (d) Three-dimensional projections.
plary
images
of
2
independent
experiments
are
shown.
Exemplary images of 2 independent experiments are shown.

Candida
GrowthIsIsPromoted
Promoted with
with High
3.5.3.5.
Candida
Growth
HighRub
RubCCConcentrations
Concentrations
In addition to CLSM, a CFU assay was conducted from resuspended biofilms in the
In addition to CLSM, a CFU assay was conducted from resuspended biofilms in the
same experimental setup to determine the effects of Rub C at concentrations of 250 µg/mL
same
experimental
to determine
thefor
effects
of Rub C attime
concentrations
250
and
62.5 µg/mL setup
on biofilms
of C. auris
two incubation
points: 12 h of
and
24µg/mL
h
and(Figure
62.5 µg/mL
on biofilms
of C. auris the
for concentration
two incubation
points:Rub
12 hCand
24 h (Figure
6A). After
12 h of treatment,
of time
250 µg/mL
resulted
in

6A). After 12 h of treatment, the concentration of 250 µg/mL Rub C resulted in more than
double the number of colonies compared to the control, while the sample treated with 15.6
µg/mL Rub C showed similar values to the control. This ratio changed after 24 h: while
the control nearly doubled in colony numbers, biofilms exposed to 250 µg/mL Rub C

J. Fungi 2023, 9, 726
R PEER REVIEW

11 of 19
11 of 19

more than double the number of colonies compared to the control, while the sample treated
µg/mL
Rub C showed
values
to the no
control.
This ratiochange
changedcomafter 24 h:
dropped in numberwith
and15.6
those
exposed
to 15.6similar
µg/mL
showed
significant
while
the
control
nearly
doubled
in
colony
numbers,
biofilms
exposed
to
250
µg/mL
pared to 12 h incubation. These observations are in line with the time-dependent treat- Rub
C dropped in number and those exposed to 15.6 µg/mL showed no significant change
ment of Rub C within
the CV assay, where the strongest effects on the 2 h-old biofilms of
compared to 12 h incubation. These observations are in line with the time-dependent
C. auris were observed
afterofa Rub
24 hCtreatment
(Figure
S1). the strongest effects on the 2 h-old biofilms
treatment
within the CV
assay, where
of C. auris were observed after a 24 h treatment (Figure S1).

Figure 6. (A). Effects Figure
of Rub
onEffects
the biofilm
formation
of C.formation
auris (CFU).
Methanol
was usedwas
as used
6. C
(A).
of Rub C
on the biofilm
of C. auris
(CFU). Methanol
solvent control. Error as
bars
indicate
SD with
biological
p values:repeats.
** p < 0.01,
solvent
control.
Error duplicates
bars indicatein
SDtwo
with
duplicatesrepeats.
in two biological
p values:
** p < of
0.01,
*** pC< 0.001.
(B).growth
Effects ofof
Rub
on theand
growth
C. auris planktonic
and C. albicanscells.
planktonic
*** p < 0.001. (B). Effects
Rub
on the
C.Cauris
C. of
albicans
The cells.
The absorption
(OD 630 after
nm) was
measured
after
h, 520
h, 8h.h,Methanol
14 h, 20 h. Methanol
wasas
used
absorption (OD 630 nm)
was measured
2 h,
5 h, 8 h,
142 h,
was used
theas the
control. Error
bars indicate
SD with
duplicates
in two repeats.
biological repeats.
solvent control. Errorsolvent
bars indicate
SD with
duplicates
in two
biological
In order to further identify the effects of Rub C on the planktonic cells of C. auris and

In order to further
identify
the effects
ofwas
Rubmeasured
C on theinplanktonic
ofCC.at auris
and
C. albicans,
the optical
density
the presence cells
of Rub
concentrations
of
250
µg/mL
and
62.5
µg/mL
over
a
period
of
20
h.
The
optical
density
of
C.
auris
C. albicans, the optical density was measured in the presence of Rub C at concentrations of and
C. µg/mL
albicans cultures
faster
remained
up to
1.5-foldof
higher
in theand
presence
250 µg/mL and 62.5
over a increased
period of
20 and
h. The
optical
density
C. auris
C. of
250 µg/mL Rub C, while both the untreated cultures as well as cultures, which are exposed
albicans cultures increased faster and remained up to 1.5-fold higher in the presence of 250
to 62.5 µg/mL, showed more slowly increasing OD values (Figure 6B).
µg/mL Rub C, while both the untreated cultures as well as cultures, which are exposed to
3.6.
Rub Cslowly
Induces increasing
MorphologicalOD
Changes
of C.(Figure
auris and 6B).
C. albicans Cells
62.5 µg/mL, showed
more
values
To further characterize the underlying effects of Rub C on C. auris and C. albicans, planktonic cells were analyzed via light microscopy after incubation in hyphal−growth−promoting
3.6. Rub C Induces Morphological Changes of◦ C. auris and C. albicans Cells
medium RPMI 1640 at 37 C and in the presence of 15.6 µg/mL and 250 µg/mL Rub C.
To further characterize
the underlying
effects of
Rub C on C. no
auris
and C. albicans,
While the control
exhibited the formation
of pseudohyphae,
pseudohyphal
development
was observed
for Rub via
C-treated
cellsmicroscopy
(Figure 7). At both
concentrations,
mainly
planktonic cells were
analyzed
light
after
incubation
inaggregated
hycells could be found.
phal−growth−promoting medium RPMI 1640 at 37 °C and in the presence of 15.6 µg/mL
Furthermore, the dimorphic fungus C. albicans also has the ability to grow either
and 250 µg/mL Rub
While yeast
the control
exhibited
the formation
of pseudohyphae,
noto C.
as C.
unicellular
or in filamentous
pseudohyphal
or hyphal
forms. Comparable
pseudohyphal development
was observed
Rub C-treatedthe
cells
(Figure 7).differences
At both conauris—but significantly
morefor
pronounced—were
morphological
between
cellscells
and the
control
centrations, mainlyincubated
aggregated
could
be (Figure
found.7). When Rub C was applied at 250 µg/mL and
62.5 µg/mL, the hyphal growth was inhibited and only aggregated unicellular yeast cells
could be observed.
In order to evaluate whether Rub C could address the morphological switch from the
hyphal form of C. albicans to its yeast form, preformed hyphae (24 h and 48 h) were treated
with 62.5 µg/mL Rub C and monitored after 2 h, 5 h, 10 h, and 18 h. Already after 2 h
treatment with Rub C, aggregated yeast cells could be detected in both 24 h- and 48 h-old
hyphae. The most pronounced effect was achieved after 18 h incubation, with Rub C being
more effective on 24 h-old hyphae than on 48 h-old ones (Figure 8).

J. Fungi 2023, 9, 726

planktonic cells were analyzed via light microscopy after incubation in h
phal−growth−promoting medium RPMI 1640 at 37 °C and in the presence of 15.6 µg/m
and 250 µg/mL Rub C. While the control exhibited the formation of pseudohyphae,
pseudohyphal development was observed for Rub C-treated cells (Figure 7).
At both co
12 of 19
centrations, mainly aggregated cells could be found.

J. Fungi 2023, 9, x FOR PEER REVIEW

12 of 19

Figure 7. Effects of Rub C on the planktonic cells of C. auris and C. albicans. Both pathogens were
incubated with Rub C in RPMI 1640 (supplemented with 0.165 mM MOPS [C. auris] or 50 nM glucose and 50 mM HEPES [C. albicans]) at 37 °C for 24 h. All samples were monitored with an optical
microscope using a 63× oil objective. Exemplary images of 2 independent experiments are shown.

Furthermore, the dimorphic fungus C. albicans also has the ability to grow either as
unicellular yeast or in filamentous pseudohyphal or hyphal forms. Comparable to C. auris—but significantly more pronounced—were the morphological differences between incubated cells and the control (Figure 7). When Rub C was applied at 250 µg/mL and 62.5
µg/mL, the hyphal growth was inhibited and only aggregated unicellular yeast cells could
be observed.
In order to evaluate whether Rub C could address the morphological switch from the
hyphal form of C. albicans to its yeast form, preformed hyphae (24 h and 48 h) were treated
with 62.5 µg/mL Rub C and monitored after 2 h, 5 h, 10 h, and 18 h. Already after 2 h
Figure 7. Effects of Rub C on the planktonic cells of C. auris and C. albicans. Both pathogens were
treatment with Rub C, aggregated yeast cells could be detected in both 24 h- and 48 h-old
incubated with Rub C in RPMI 1640 (supplemented with 0.165 mM MOPS [C. auris] or 50 nM glucose
hyphae. The most pronounced effect was achieved after 18 h incubation, with Rub C being
and 50 mM HEPES [C. albicans]) at 37 ◦ C for 24 h. All samples were monitored with an optical
more effective on 24 h-old hyphae than on 48 h-old ones (Figure 8).
microscope using a 63× oil objective. Exemplary images of 2 independent experiments are shown.

Figure
−growth-promoting medium
Figure8.8.C.
C.albicans
albicanscultured
culturedininhyphal
hyphal−growth-promoting
mediumRPMI
RPMI1640
1640(supplemented
(supplementedwith
with
◦ C for 24 h or 48 h and treated with 62.5 µg/mL Rub C for
50
nM
glucose
and
50
mM
HEPES)
at
37
50 nM glucose and 50 mM HEPES) at 37 °C for 24 h or 48 h and treated with 62.5 µg/mL Rub C for
22h,h,55h,h,1010hhand
and1818h.h.Conversion
Conversionofofhyphae
hyphaeinto
intoyeasts
yeastswas
wasmonitored
monitoredunder
underaalight
lightmicroscope
microscope
using
× oil
usingaa63
63×
oil objective.
objective. Exemplary images of
of 22 independent
independent experiments
experiments are
are shown.
shown.

In
Inthe
thesame
samesetup,
setup,we
wealso
alsoprepared
preparedsamples
samplestotostudy
studyC.
C.auris
auriscells
cellsvia
viaSEM.
SEM.When
When
Rub
RubCCwas
wasapplied
appliedatatconcentrations
concentrationsofof250
250µg/mL
µg/mLand
and15.6
15.6µg/mL,
µg/mL, pseudohyphal
pseudohyphaldeveldevelopment
wewe
could
observe
more
extracellular
vesicles
(EVs)(EVs)
in thein
opmentwas
wasinhibited.
inhibited.Moreover,
Moreover,
could
observe
more
extracellular
vesicles

the treated samples, which were especially visible after the treatment with 250 µg/mL Rub
C; the size of the surface-bound vesicles was clearly larger (Figure 9).

J. Fungi 2023, 9, 726

13 of 19

, x FOR PEER REVIEW

13 of 19

treated samples, which were especially visible after the treatment with 250 µg/mL Rub C;
the size of the surface-bound vesicles was clearly larger (Figure 9).

Effects
of Rub C oncells
the planktonic
of C. auris,
shown
by SEM micrographs.
Cells
Figure 9. Effects ofFigure
Rub C9.on
the planktonic
of C. auris,cells
as shown
by as
SEM
micrographs.
Cells
◦ C for 24 h
were
incubated
with
Rub
C
in
RPMI
1640
(supplemented
with
0.165
mM
MOPS)
at
37
were incubated with Rub C in RPMI 1640 (supplemented with 0.165 mM MOPS) at 37 °C for 24 h
and
to Rub C concentrations.
with indicated concentrations.
with
to cell morphology and
and exposed to Rub
Cexposed
with indicated
Differences Differences
with regard
to regard
cell morphology
vesicles in
arethe
observed
in the
samples
treated
with 250
µg/mL
Rub Carrows).
(yellow arrows).
and vesicles are observed
samples
treated
with
250 µg/mL
Rub
C (yellow

3.7. Quantification of Inhibitory Activities of Azaphilones against C. albicans

3.7. Quantification of Inhibitory
Activities of Azaphilones against C. albicans
To evaluate the efficacy of selected rubiginosin- and rutilin-type azaphilones as inTo evaluate hibitors
the efficacy
of selected
rubiginosinand expression
rutilin-type
azaphilones
as in-the CAI-4
for hyphal
induction,
hyphae gene
was
quantified using
HWP1-lacZ
reporter
straingene
[44,45].
In this construct,
the HWP1using
promotor
combined with
hibitors for hyphal
induction,
hyphae
expression
was quantified
the isCAI-4
the
lacZ
gene.
The
HWP1
cell
wall
protein
is
exclusively
produced
during
hyphae
HWP1-lacZ reporter strain [44,45]. In this construct, the HWP1 promotor is combined with growth,
thus,cell
the lacZ
(under
control of the
HWP1 promotor)
is only expressed
the lacZ gene. Theand
HWP1
wallgene
protein
is exclusively
produced
during hyphae
growth, under the
same conditions. With the cleavage of the β-galactosidase substrate o-nitrophenyl beta-Dand thus, the lacZ gene (under control of the HWP1 promotor) is only expressed under
galactopyranoside and the resulting photometrically measurable product o-nitrophenol,
the same conditions.
With the cleavage of the β-galactosidase substrate o-nitrophenyl
hyphae gene expression can be quantified [46,47]. Consequently, higher concentrations
beta-D-galactopyranoside
and the
resulting photometrically
measurable
productWe
o-nitroof o-nitrophenol
corresponded
to greater levels of
hyphal expression.
determined the
phenol, hyphae gene
expression
can
be
quantified
[46,47].
Consequently,
higher
concenβ-galactosidase activity after 5 h of treatment of the strain C. albicans CAI-4 HWP1-lacZ with
trations of o-nitrophenol
corresponded
to greater
levelscontrol.
of hyphal
deter- inhibitory
all azaphilones;
FA was used
as the positive
The expression.
reporter assayWe
exhibited
effects for all
azaphilones
at the
test concentration
of 100 µg/mL.
mined the β-galactosidase
activity
after 5when
h ofapplied
treatment
ofhighest
the strain
C. albicans CAI-4
Among
all
pigments,
Rub
C
and
Rub
W
exhibited
significant
inhibitory
effects.
HWP1-lacZ with all azaphilones; FA was used as the positive control. The reporter assay Rub C
showed
thefor
most
effectsat
onthe
hyphae
gene
expression
of up to 87%
exhibited inhibitory
effects
allpronounced
azaphilonesinhibitory
when applied
highest
test
concentrareduction compared to the control when applied at 100 µg/mL (Figure 10). In addition,
tion of 100 µg/mL. Among all pigments, Rub C and Rub W exhibited significant inhibitory
Rub C was able to effectively inhibit the gene expression of hyphae with 71% efficacy even
effects. Rub C showed
the most pronounced inhibitory effects on hyphae gene expression
at the concentration of 6.3 µg/mL (Figure 10).
of up to 87% reduction compared to the control when applied at 100 µg/mL (Figure 10).
In addition, Rub C was able to effectively inhibit the gene expression of hyphae with 71%
efficacy even at the concentration of 6.3 µg/mL (Figure 10).

Fungi 2023, 9, x FOR PEER REVIEW
J. Fungi 2023, 9, 726

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Figure 10. Inhibitory effects of selected rubiginosin- and rutilin-type azaphilones on the hyphal

Figure
10. Inhibitory
effects
of selected
rubiginosinand rutilin-type
azaphilones
development
of C. albicans.
Production
of o-nitrophenol
by β-galactosidase
expression
as a measureon the h
of HWP1 expression
after 5Production
h of incubation.
FA was used as the
positive control andexpression
methanol as a m
velopment
of C. albicans.
of o-nitrophenol
by β-galactosidase
as
solvent
control.
Error
bars
indicate
SD
with
duplicates
in
three
biological
repeats;
p values:
HWP1 expression after 5 h of incubation. FA was used as the positive control
and metha
*** pcontrol.
< 0.001. Error bars indicate SD with duplicates in three biological repeats; p values: **
vent
4. Discussion

4. Discussion
The discovery of novel therapeutic agents is crucial for combating the growing problem

of antibiotic resistance in fungal infections. In this context, azaphilones, a group of fungal
The discovery of novel therapeutic agents is crucial for combating the growi
secondary metabolites, have been studied extensively for their antimicrobial and cytotoxic
lem
of antibiotic
in fungal
infections.
Intherapeutic
this context,
a
properties,
gaining resistance
increasing attention
for their
potential as
agentsazaphilones,
[24].
this study, we
investigated the
activity
selected rubiginosinand
fungalInsecondary
metabolites,
have
beenofstudied
extensively
forrutilin-type
their antimicro
azaphilones
against
biofilm-related
bacterial
and
fungal
pathogens
(S.
aureus,
P.
aeruginosa
cytotoxic properties, gaining increasing attention for their potential as therapeut
as well as C. albicans [DSM 1665, DSM 11225], CAI-4 HWP1-lacZ, and C. auris) and their
[24].
cytotoxicity against two cell lines. The fungal pigments did not exhibit any inhibitory
In this
study,
westrains
investigated
the
of selectedbearing
rubiginosinactivity
against
all tested
of C. albicans
andactivity
C. auris. Azaphilones
OA moiety,and rut
such as Rub A
and Z, Rut
A, and B, demonstrated
cytotoxic
properties,
while azaphilones
azaphilones
against
biofilm-related
bacterial
and fungal
pathogens
(S. aureus, P. a
substituted
by
an
aliphatic
moiety
instead
of
the
OA
motif,
like
Rub
C
and
W,
did not
as well as C. albicans [DSM 1665, DSM 11225], CAI-4 HWP1-lacZ, and
C. auris) a
exhibit such effects. Additionally, the dimeric OA-carrying Rut A (1.2 µM; 1.1 µM) and
cytotoxicity
two cell lines.
The
fungal pigments
exhibitlines
any inhib
B (2.2 µM; 1.5against
µM) demonstrated
stronger
cytotoxicity
against L929did
andnot
KB-3-1cell
tivity
all tested
strains azaphilones
of C. albicans
C. µM;
auris.
OA
than against
the monomeric
OA-carrying
Ruband
A (3.2
5.2Azaphilones
µM) and Z (4.7bearing
µM;
5.2
µM)
[28].
These
cytotoxic
effects
might
be
attributed
to
the
presence
of
an
OA
moiety
such as Rub A and Z, Rut A, and B, demonstrated cytotoxic properties, while aza
within the molecule [28], although no cytotoxicity could be observed for OA alone [49].
substituted
by an aliphatic moiety instead of the OA motif, like Rub C and W
In addition to the antimicrobial properties of the selected azaphilones, we also tested
exhibit
such effects.
Additionally,
the dimeric
Rut
A (1.2 µM;
the inhibitory
activity against
biofilm formation.
AmongOA-carrying
all pigments, Rub
C exhibited
the 1.1 µM
most
pronounced
against the biofilm
formation
of C. albicans
and C. auris
(2.2
µM;
1.5 µM)effects
demonstrated
stronger
cytotoxicity
against
L929without
and KB-3-1
being
lethal
(MIC
>
250
µg/mL)
or
cytotoxic
against
the
tested
cell
lines
in
the
tested
than the monomeric OA-carrying azaphilones Rub A (3.2 µM; 5.2 µM) and Z (4.7
range (37 µg/mL–0.63 ng/mL). Rub C exhibited promising inhibitory effects against the
µM)
[28].
Theseofcytotoxic
effects
might
be attributed
to the
biofilm
formation
C. auris (between
2 and
62.5 µg/mL)
and C. albicans
(>7.8presence
µg/mL) of of
ca. an OA
within
theca.molecule
[28], although
noRub
cytotoxicity
could be observed
for OA alo
50% and
80%, respectively.
In contrast,
W and A demonstrated
weaker activities
against
the biofilmtoformation
of C. auris and
C. albicans, of
respectively.
At the
highest test we al
In addition
the antimicrobial
properties
the selected
azaphilones,
concentration (250 µg/mL), all azaphilones showed effects towards C. albicans biofilm forthe inhibitory activity against biofilm formation. Among all pigments, Rub C e
mation. Tentative structure–activity relations let us assume that an increase in lipophilicity
theaffected
most the
pronounced
effects
against
the
biofilm formation
of C. albicans
and C. au
inhibitory effects
of the
selected
azaphilones.
Thus, azaphilones
with a long
out
being lethal
(MIC
> 250stronger
µg/mL)
or cytotoxic
againstbiofilm
the tested
celland
lines in t
lipophilic
side chain
exhibited
inhibitory
effects against
formation
hyphal
development
than
those
carrying
an
OA
moiety.
In
line
with
this
observation,
Rub
range (37 µg/mL–0.63 ng/mL). Rub C exhibited promising inhibitory effects ag
W, which differs in the number of carbon atoms in the side chain, was less effective than
biofilm
formation of C. auris (between 2 and 62.5 µg/mL) and C. albicans (>7.8 µ
Rub C. It is likely that more lipophilic compounds can penetrate biological membranes
ca.more
50%easily
and[50].
ca. 80%,
respectively.
In contrast,
Rub
andaccess
A demonstrated
As a result,
lipophilic compounds
may
haveW
better
to their target weak
sites
within the
leadingformation
to enhanced activity
[51,52].and
In the
of Rub C,
the structure At th
ties
against
thecell,
biofilm
of C. auris
C.case
albicans,
respectively.
of
the
side
chain
resembles
to
some
degree
that
of
FA.
Interestingly,
Rub
C
demonstrated
test concentration (250 µg/mL), all azaphilones showed effects towards C. albican

formation. Tentative structure–activity relations let us assume that an increase
philicity affected the inhibitory effects of the selected azaphilones. Thus, azaphilo
a long lipophilic side chain exhibited stronger inhibitory effects against biofilm fo
and hyphal development than those carrying an OA moiety. In line with this obs

J. Fungi 2023, 9, 726

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similar, or even stronger, activity than FA, which was used as the positive control. FA is a
known inhibitor of hyphal growth and the biofilm formation of C. albicans [53–55].
As the treatment of different maturation stages of C. auris biofilms showed, Rub C
prevented biofilm formation during the first stage of biofilm development with the highest
efficacy when given shortly after the attachment phase. This efficacy was reduced when
biofilms were treated after 12 h and 24 h of growth. Since the hyphae/pseudohyphae
of C. albicans and C. auris are essential to attach to certain surfaces and to form robust
biofilms [56,57], we hypothesized that Rub C might inhibit the formation of biofilms by
affecting the growth of pseudohyphae or hyphae. Pseudohyphae in C. auris are known
to adapt to stressful living conditions, such as high concentrations of salts [17,58]. In
addition, the yeast-to-hyphae transition of C. albicans occurs during infections and allows
for the pathogen to escape from macrophages by destroying the cell membrane via polarized growth [59]. This invasive growth can also cause damage to tissues by invading
epithelial cells and causing bloodstream infections, resulting in larger damages in human
hosts [60–63].
According to visualization via CLSM analysis, Rub C caused changes in the morphology of C. auris biofilms. Especially high concentrations lead to a very dense and compact
surface-bound structure without visible hyphae, while a Rub C concentration of 62.5 µg/mL
resulted in a more aggregated and porous-appearing biofilm structure. Furthermore, the
results of CFU analysis revealed a significant increase in cell growth following 12 h of
treatment with a concentration of 250 µg/mL of Rub C. However, after 24 h of incubation,
the growth was inhibited compared to the control. This finding is consistent with the
observations from CLSM images acquired at the same concentration of 250 µg/mL Rub
C after 24 h of treatment, which also showed more biomass compared to the control; this
indicates an increased growth along with reduced metabolic activity of the cells. This
could be attributed to a strong, initial promotion of non-hyphae growth in the presence
of 250 µg/mL Rub C, followed by a decrease in metabolically active cells due to nutrient
limitations within the batch system after 24 h. This dense and thick cell layer at the surface
also explains the increase in biomass in the CV assay of C. auris at high concentrations
(>125 µg/mL).
Indeed, we could visualize that Rub C inhibited the pseudohyphae and hyphae
formation of C. auris and C. albicans via light microscopy. Additionally, we performed
a reporter gene assay which confirmed the inhibition of the hyphae development of C.
albicans upon treatment with 12.5 µg/mL Rub C. The construct of the hyphal wall protein
HWP1 promoter and the enzyme ß-galactosidase showed a reduction in enzymatic activity
by 87% for Rub C and 39% for Rub W at concentrations above 12.5 µg/mL, while the
other tested azaphilones only displayed inhibitory activity at the highest concentration
(100 µg/mL). Based on these results, we assume that the biofilms of C. auris (treated with
0.5 µg/mL to 62.5 µg/mL Rub C) and C. albicans (treated with 7.8 µg/mL to 250 µg/mL
Rub C) could be removed more easily by washing steps in the CV assays because they
lacked pseudohyphae and hyphae—factors which are impeding the disturbance of C.
albicans biofilms by external forces (e.g., vortexing, sonification) [56]. Similarly, biofilms
containing pseudohyphae in C. auris possessed enhanced structural integrity compared to
those lacking pseudohyphae [57].
EVs produced by C. auris are known to play crucial roles in various cellular processes [64]. Interestingly, in the SEM images of C. auris, we found that more and larger Evs
were present after the treatment with high concentrations of Rub C (250 µg/mL) compared
to the control. Thus, we not only observed growth-promoting effects on vegetative yeast
cells along with inhibitory effects on pseudohyphae formation upon treatment with high
concentrations of Rub C for C. auris, but also a release of EVs, in line with reports from C.
albicans [65,66]. Due to shared biological processes in biofilm and hyphae regulation, C.
albicans and C. auris might exhibit similar characteristics in EV production, as the study
by Zamith-Miranda et al. let assume [64]. This multifactorial process might explain the
differences in the structural architecture observed in the CLSM images. While high con-

J. Fungi 2023, 9, 726

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centrations of Rub C promoted the growth of single yeast cells and resulted in a more
compact and flattened biofilm structure, lower concentrations of Rub C did not stimulate
vegetative growth, but still effectively inhibited the pseudo/hyphae formation of C. albicans
and C. auris.
5. Conclusions
Rub C, a fungal pigment derived from the stromata of H. rubiginosum, has demonstrated significant potential in inhibiting biofilm formation and yeast-to-hyphae transition
against the two opportunistic fungal pathogens C. albicans and C. auris. This inhibition
occurs in a concentration range below cytotoxic or lethal effects. Both biofilms and hyphae
formation are critical virulence factors of these pathogens and likely strongly linked to each
other. The encouraging aspect of our findings is that Rub C effectively targets these virulence factors without exhibiting cytotoxic effects on the tested mammalian cell lines, making
it a promising candidate for therapeutic use in future. Potential applications could be the
pre-therapeutical coating of medical devices; this is similar to the already investigated
known biofilm inducer filastatin, which inhibited hyphal morphogenesis and the adhesion
of C. albicans to polystyrene and human cells [67,68]. To gain a deeper understanding about
the mechanisms of Rub C on both biofilm and hyphal inhibition, further investigations are
needed to decipher the molecular mechanism.
Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/jof9070726/s1, Figure S1: Effects on biofilm formation of 2 h
C. auris biofilms after different treatment time (8 h and 24 h) with Rub C. Methanol served as
solvent control. Error bars indicate SD of duplicates in two biological repeats; p values: ** p < 0.01,
*** p < 0.001. Figure S2: Volume of biomass for C. auris biofilms (24 h after exposure to Rub C of
attached cells [2 h]) calculated by Imaris 9.31; Table S1: MIC assay experiment parameters. Table S2:
Selected azaphilones were tested against the formation of biofilm of S. aureus, P. aeruginosa as well
as C. auris and dispersal effect on preformed biofilm of S. aureus compared to solvent control (=0%),
respectively. SDs are shown as ±SD. Table S3: Biofilm attachment of different developmental stages
(2 h, 12 h, and 24 h old) of C. auris biofilm after treatment with positive control NYS compared to
solvent control (=0%). SDs are shown as ±SD.
Author Contributions: Conceptualization, H.Z., H.S. and M.M.; methodology, H.Z., H.S. and M.M.;
software, H.Z. and M.M.; validation, H.Z., M.M. and H.S.; formal analysis, H.Z.; investigation, H.Z.;
data curation, H.Z., M.M. and H.S.; writing—original draft preparation, H.Z.; writing—review and
editing, H.Z., M.S., W.-R.A., M.M. and H.S.; visualization, H.Z. and M.M.; supervision, M.M. and
H.S.; project administration, M.S.; funding acquisition, M.S. and M.M. All authors have read and
agreed to the published version of the manuscript.
Funding: This research was funded by personal PhD stipend from the “Drug Discovery and Cheminformatics for New Anti-Infectives (iCA)” and is financially supported by the Ministry for Science &
Culture of the German State of Lower Saxony (MWK no. 21—78904-63-5/19).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: All data generated are in the manuscript or the Supplementary Materials.
Acknowledgments: We express our gratitude to Kevin Becker for his contribution in isolation of the
azaphilones, Wera Collisi for providing technical support in conducting MIC and cytotoxicity assays,
Ina Schleicher for technical support in EM sample preparation, Valentina Lember for assisting in the
establishment of CLSM, and Ursula Bilitewski for providing C. albicans CAI-HWP1-lacZ stem cells.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.

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