Abstract
More than two million people worldwide are affected by
life-threatening, invasive fungal infections annually. Candida species
are the most common cause of nosocomial, invasive fungal infections and
are associated with mortality rates above 40%. Despite the increasing
incidence of drug-resistance, the development of novel antifungal
formulations has been limited. Here we investigate the antifungal mode
of action and therapeutic potential of positively charged, synthetic
peptide mimics to combat Candida albicans infections. Our data
indicates that these synthetic polymers cause endoplasmic reticulum
stress and affect protein glycosylation, a mode of action distinct from
currently approved antifungal drugs. The most promising polymer
composition damaged the mannan layer of the cell wall, with additional
membrane-disrupting activity. The synergistic combination of the
polymer with caspofungin prevented infection of human epithelial cells
in vitro, improved fungal clearance by human macrophages, and
significantly increased host survival in a Galleria mellonella model of
systemic candidiasis. Additionally, prolonged exposure of C. albicans
to the synergistic combination of polymer and caspofungin did not lead
to the evolution of tolerant strains in vitro. Together, this work
highlights the enormous potential of these synthetic peptide mimics to
be used as novel antifungal formulations as well as adjunctive
antifungal therapy.
Subject terms: Antifungal agents, Target identification, Target
validation
__________________________________________________________________
Fungal infections are severely underestimated as a cause of mortality,
and alternative drugs are urgently needed. Here, Schaefer et al. show
that a synthetic polymer mimicking defensins shows different, but
synergistic activity with known antifungals.
Introduction
Modern medicine often relies on invasive medical interventions or drugs
which can compromise the patient’s immune system. An unfortunate
consequence of these undeniably successful treatments for
life-threatening diseases like cancer is severe infections caused by
opportunistic pathogens^[60]1,[61]2. Among these opportunists are
fungal pathogens, including Candida, Aspergillus, Cryptococcus, and
Pneumocystis species^[62]1–[63]3. More recently, increasing numbers of
opportunistic fungal infections caused by Aspergillus, Mucorales, and
Candida species have been observed in COVID-19 patients with severe
respiratory syndromes in intensive care units^[64]4. These and other
factors result in over 2 million invasive fungal infections annually
worldwide, with alarmingly high mortality rates and more than 1.5
million deaths^[65]3.
Candida spp. is the fourth most common cause of hospital-acquired
infections, and mortality rates from systemic Candida infections exceed
40%, even with antifungal intervention^[66]3,[67]5. Among all Candida
species, Candida albicans accounts for around 50% of Candida
bloodstream infections^[68]3,[69]6. Novel pathogenic species, such as
the multi-drug-resistant Candida auris have emerged, potentially
through adaptations to higher ambient temperature due to global climate
change^[70]7,[71]8. Indeed, C. auris and C. albicans were listed as two
of the four critical group pathogens in the World Health Organization’s
first-ever fungal priority pathogens list, emphasising the need for new
treatment options^[72]9.
There are currently only four classes of antifungal drugs approved for
the treatment of invasive Candida infections – azoles (e.g.,
fluconazole), polyenes (e.g., amphotericin B), echinocandins (e.g.,
caspofungin), and flucytosine^[73]10. Their application is limited by
undesired drug-drug interactions (azoles), detrimental off-target
side-effects (polyenes), and the increasing occurrence of drug
resistance (azoles, flucytosine, and echinocandins)^[74]10,[75]11.
Resistance of Candida spp. occurs mainly due to target over-expression,
modification of the drug target, or upregulation of drug-efflux
pumps^[76]11. The urgency to find new treatment options against Candida
spp. was highlighted by the Centers for Disease Control and
Prevention’s (CDC) 2019 classification of Candida spp. as a serious
threat to human health with equal standing to multi-drug-resistant
bacteria such as Pseudomonas aeruginosa^[77]12. However, discovering
novel targets for antifungal drugs is complicated by the evolutionary
similarity of eukaryotic human and fungal cells, and the antifungal
development pipeline is dominated by compounds from established
classes, which are likely to result in similar
complications^[78]10,[79]13. Exceptions are fosmanogepix and
ibrexafungerp which are first-in-class and undergoing clinical
trials^[80]14,[81]15, but ultimately the emergence of resistance and
clinical success for these new classes remain to be seen^[82]14.
Combination therapy can decrease the development of resistance or
re-sensitise resistant strains by acting on multiple
targets^[83]16–[84]18. It can also reduce toxicity to the host by
decreasing required drug concentrations^[85]16.
In nature, antifungal peptides (AFPs) prevent and combat fungal
infections in all domains of life^[86]19. Most interact with the fungal
cell membrane, damaging the cell wall or membrane or causing
intracellular stress^[87]19. Employing those potent natural effectors
as a drug is hampered by several issues; the membrane-active AFPs are
often toxic to the expression host in biotechnological synthesis,
chemical synthesis of peptides is expensive and complicated by their
sequence specificity, and susceptibility to host proteases generally
limits the applicability of AFPs^[88]19. These issues can be
circumvented by mimicking cationic amphiphilic properties of AFPs
synthetically^[89]20,[90]21. Various polymeric synthetic structures
show promising antifungal properties, including
β-peptides^[91]22,[92]23, poly(2-oxazoline)s^[93]24,[94]25,
polycarbonates^[95]26, polyacrylamides^[96]27,[97]28, and
peptidopolysaccharides^[98]29. Owing to advances in polymerisation
techniques, particularly reversible-deactivation radical polymerisation
(RDRP), synthetic macromolecules can be produced in a facile manner
with precise control over molecular weight and composition^[99]30.
However, one of the remaining challenges for RDRP is the synthesis of
strictly homogeneous sequence-defined molecules, which would resemble
AFPs with precisely defined sequences^[100]31. Due to this limitation,
synthetic copolymers are usually synthesised with statistically
distributed monomers or as a block copolymer, which to a certain degree
provide control over sequence and dispersity^[101]31.
Our group previously synthesised and screened a library of synthetic
polyacrylamides inspired by AFP structures for activity against
C. albicans and biocompatibility^[102]27. We identified polymers that
outperformed amphotericin B in terms of their therapeutic index against
C. albicans in vitro^[103]27. In the current work, we determined the
mode of action of our most promising polymer compositions and
investigated the in vitro and in vivo therapeutic potential of
synergistic combinations of our polymers with existing antifungals with
a view to enhancing efficacy, minimising toxicity and preventing the
emergence of antifungal drug resistance.
Results and discussion
Synthesis and characterisation of amphiphilic polyacrylamides that mimic AFPs
Inspired by the physicochemical properties of antimicrobial peptides,
we previously synthesised random acrylamide copolymers using
photo-induced reversible-deactivation radical
polymerisation^[104]27,[105]30. The polymer characteristics that
conferred the highest activity against C. albicans and best
biocompatibility with mammalian host cells (measured by in vitro
therapeutic indexes) were short polymers with a degree of
polymerisation (X[n]) of 20 and an optimal balance of hydrophilic to
hydrophobic groups^[106]27. Here, we synthesised four ternary
polyacrylamides with these characteristics (Fig. [107]1 and
Table [108]1), which previously demonstrated the most promising
properties: LP (linear, pentyl), LH (linear, heptyl), CB (cyclic,
benzyl), and CX (cyclic, hexyl), named by their distinct hydrophobic
features. The successful synthesis and purification were confirmed by
^1H nuclear magnetic resonance (NMR) spectroscopy and refractive
index-based size-exclusion chromatography (SEC). We achieved nearly
complete monomer conversion of >98% and observed narrow, unimodal
molecular weight distributions with a dispersity (Ð) between 1.09 and
1.12 (Supplementary Figs. [109]S1-[110]S15).
Fig. 1. Chemical structures of polyacrylamides synthesised for this study.
Fig. 1
[111]Open in a new tab
R represents the different side chains and x indicates the targeted
number of hydrophobic residues within the molecule.
Table 1.
Composition, targeted degree of polymerisation (X[n]), calculated
molecular weight (M), experimental number-average molecular weight
(M[n]) and dispersity (Ð) of polymers employed in this study
Polymer Targeted polymer composition (% positively
charged/hydrophilic/hydrophobic functionality) Experimental polymer
composition (% positively charged/hydrophilic/hydrophobic
functionality)^a Targeted X[n] Calculated M (g/mol) ^b M[n] (g/mol) ^c
Ð ^c
LP 50/20/30 50/21/29 20 3700 6300 1.12
LH 50/25/25 50/25/25 20 3800 6400 1.09
CB 50/15/35 50/15/35 20 3900 6600 1.10
CX 50/25/25 49/26/25 20 3800 6100 1.09
[112]Open in a new tab
^adetermined by ^1H NMR spectroscopy before polymerisation.
^bcalculated by ChemDraw (version 19.0) for Boc-protected polymers
based on targeted composition and X[n], rounded to the nearest 100.
^cdetermined by SEC using poly(methyl methacrylate) standards.
Polyacrylamides are active against drug-resistant, clinical C. albicans
isolates
Our previous work showed that our polymers were active against
C. albicans and other ascomycetes including Candida glabrata
(Nakaseomyces glabratus), Candida krusei (Pichia kudriavzevii), and
Saccharomyces cerevisiae, as well as the basidiomycete Cryptococcus
neoformans^[113]27. Despite a high tolerance of C. neoformans towards
AmpB and fluconazole, and intrinsic resistance of S. cerevisiae and
C. glabrata towards fluconazole, all were susceptible to the four
candidate polymers^[114]27. This suggested a mode of action that is
different to AmpB and fluconazole. To explore this further, the minimum
inhibitory concentration (MIC, growth inhibition of >90% at 24 h) of
the polymers against antifungal drug-resistant strains of C. albicans
(Table [115]2) was assessed using slightly modified Clinical and
Laboratory Standards Institute (CLSI) guidelines^[116]32,[117]33.
Table 2.
Antifungal activity of polymers and selected antifungal drugs against
C. albicans clinical isolates
C. albicans strain Phenotype Minimum inhibitory concentration[24h, 90%]
in µg/mL (µM)
Polymers Antifungal drugs
LP LH CB CX AmpB Flu Cas
SC5314 Wild type 64–128 (24–47) 16–32 (6–11) 64 (22) 64 (23) 0.5–1
(0.5–1.1) 0.25–0.5 (0.8–1.6) 0.25–0.5 (0.2–0.5)
110.12 Gsc1-mutation ↑ 256 (95) 16 (6) ↑ 128 (44) ↑ 128 (46) 1–2
(1.1–2.2) ↑↑ >8 (>26) ↑↑ 4–8 (3.6–7.3)
EU0108 Erg11-/Erg5-mutation, enhanced Cdr-efflux 64 (24) 8–16 (3–6) ↓
16–32 (6–11) 32–64 (11–23) ↑ 2 (2.2) ↑↑ >8 (>26) 0.5–1 (0.5–0.9)
EU0012 Erg3-mutation 128–256 (47–95) 16–32 (6–11) 64–128 (22–44) 64–128
(23–46) ↑↑ 2–4 (2.2–4.3) ↑↑ >8 (>26) 0.5–1 (0.5–0.9)
EU1008 Erg3-/Erg11-mutation 32–64 (12–24) 8–16 (3v6) ↓ 32 (11) ↓ 16–32
(6–11) ↑ 2 (2.2) ↑↑ >8 (>26) 0.5–1 (0.5–0.9)
EU0136 Erg6-deficient ↑ 256 (95) 16–32 (6–11) ↑ 128 (44) 64–128 (23–46)
↑↑ 4 (4.3) ↑↑ >8 (>26) 0.5 (0.5)
EU0992 Enhanced Cdr-efflux 64–128 (24–47) 16 (6) 64–128 (22–44) 64–128
(23–46) 1–2 (1.1–2.2) ↑↑ >8 (>26) 0.5–1 (0.5–0.9)
EU0989 Enhanced Cdr-efflux 128 (47) 8–16 (3–6) 64 (22) 64–128 (23–46)
1–2 (1.1–2.2) ↑↑ >8 (>26) 0.5–1 (0.5–0.9)
EU0981 Enhanced Mdr-efflux 64 (24) 8–16 (3–6) 64 (22) 32–64 (11–23) 1–2
(1.1–2.2) ↑↑ >8 (>26) 0.25–0.5 (0.2–0.5)
EU0999 Enhanced Mdr-efflux 64–128 (24–47) 8–16 (3–6) 64–128 (22–44)
32–64 (11–23) 1–2 (1.1–2.2) ↑↑ >8 (>26) 0.25–0.5 (0.2–0.5)
[118]Open in a new tab
Minimum inhibitory concentrations were determined by the concentration
at which the respective compound inhibited >90% of fungal growth
compared to an untreated control after 24 h at 35 °C.
AmpB Amphotericin B, Flu Fluconazole, Cas Caspofungin.
↑↑ indicates resistance, ↑ indicates increased tolerance (up to
two-fold increase in MIC), and ↓ indicates a decreased MIC compared to
the wild type.
C. albicans strain 110.12 is resistant to azoles and caspofungin, the
latter due to a mutation in the echinocandin target Gsc1, an essential
β-1,3-glucan synthase subunit^[119]34. The MICs for the polymers LP, CB
and CX were only slightly increased compared to wild type, and the
clinical isolate was at least as susceptible as the wild type to
polymer LH.
AmpB-tolerant (EU0108, EU1008) or -resistant (EU0012, EU0136) strains
have various mutations in enzymes involved in the biosynthesis of
ergosterol (Erg3, Erg5, Erg6, Erg11), the target of
AmpB^[120]35,[121]36. The strains are also azole-resistant, which has
been attributed to increased drug efflux at least in strain
EU0108^[122]35,[123]36. However, all polymers were active against those
clinical isolates with even decreased MICs against the AmpB-tolerant
strains. LH was fully active against the AmpB-resistant strains. A
slight increase in MIC was observed for LP, CB, and CX.
The fluconazole-resistant C. albicans strains EU0992 and EU0989 have an
increased Cdr-mediated drug efflux, and EU0981 and EU0999 show more
Mdr-dependent export activity, while all these strains are normal in
sterol biosynthesis (O. Bader, personal communications)^[124]37. The
antifungal activity of the polymers was not affected by those
mutations, suggesting that they are not transported out by Cdr- or
Mdr-related efflux pumps.
Comparing the MICs of the most active antifungal polymer LH against
C. albicans (8–32 µg/mL, 3–11 µM) to reported MICs of antimicrobial
peptides reveals them to be active at similar concentrations. For
example, the membrane-lytic peptides LL-37 (human) and melittin (bee
venom) inhibit C. albicans growth at concentrations of 64 µg/mL (14 µM)
and 11–22 µg/mL (4–8 µM), respectively^[125]38,[126]39, while the
intracellularly acting histatin 5 (human) exhibited slightly lower MICs
against C. albicans (4–8 µg/mL, 3–5 µM)^[127]40,[128]41. Some synthetic
cationic peptides composed of 9–11 amino acids have shown MICs against
C. albicans in an equivalent range of 8–32 µg/mL (6–23 µM) and also
interfere with fungal membranes^[129]42,[130]43.
Overall, each antifungal drug-resistant C. albicans strain tested was
as susceptible to polymer LH, if not more, as the wild type. This
indicates a distinct mode of action of LH. The different activity
pattern of the other polymers against drug-resistant strains may
indicate slight differences in modes of action between the polymers. We
therefore investigated the potentially novel modes of action of our
polymers.
Transcript profiling to investigate the mode of action of the antifungal
polymers
We compared the transcriptome of C. albicans cells grown for 1 h in the
presence of sub-inhibitory concentrations of the antifungal polymers to
untreated control. A non-active polymer, poly(hydroxyethyl acrylamide)
(poly-HEA), was also included for comparison (chemical characterisation
for poly-HEA in Supplementary Figs. [131]S14 and [132]15 and
Table [133]S1) as well as the membrane-active antimicrobial peptide
LL-37^[134]38,[135]44.
An overview of the global transcriptomic differences of C. albicans in
response to the polymers and LL-37 was gained through hierarchical
clustering and principal component analyses of normalised gene
expression data. These analyses showed that the transcriptome of cells
exposed to the antifungal polymers LP, CB, and CX clustered together
(Supplementary Fig. [136]S16). Surprisingly, the hierarchical
clustering revealed that the transcriptomic patterns for these three
polymers were more similar to the non-toxic poly-HEA (Supplementary
Fig. [137]S16A), while our most active candidate LH clustered
separately after applying either of the two statistical methods
(Supplementary Fig. [138]S16A, B). The antimicrobial peptide LL-37
clustered separately from all polymers.
Next, we looked for biological functions and pathways where there was
an overrepresentation of up- or downregulated genes associated with the
function or pathway in the datasets by performing Gene Ontology (GO)
term^[139]45 and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway
enrichment^[140]46 analyses (Fig. [141]2). Similar to our clustering
analyses, we noted that the non-toxic poly-HEA differed from the four
antifungal polymers and that among the polymers, LH showed the most
distinct pattern with the highest number of differentially expressed
genes. Therefore, we focussed our studies on the effect of polymer LH
on C. albicans since it demonstrated the best activity against clinical
C. albicans isolates in comparison to the other three polymers
(Table [142]2) and triggered a distinct transcriptomic response in
C. albicans (Fig. [143]2 and Supplementary Fig. [144]S16) in the
present study. Additionally, a previous study highlighted the potential
of polymer LH due to its superior antifungal activity while showing
comparatively low damage to red blood cells and murine
fibroblasts^[145]27.
Fig. 2. Antifungal polymers cause transcriptomic responses associated with
impaired protein glycosylation, membrane stress, and cell wall damage in
C. albicans.
[146]Fig. 2
[147]Open in a new tab
A Gene Ontology (GO) term enrichment analysis, based on molecular
function^[148]45, and B KEGG (Kyoto Encyclopedia of Genes and Genomes)
pathway enrichment analysis^[149]46 of RNA microarray data after
treating C. albicans SC5314 for 1 h at 30 °C with sub-inhibitory
concentrations of polymers and LL-37. Statistically significantly up-
(red) or downregulated (green) gene groups associated with GO terms and
KEGG pathways compared to the untreated control are shown. Diameter of
circles reflects the percentage of genes differentially regulated in
the associated pathway or term and the shading represents the adjusted
p-value (calculated by hypergeometric distribution and adjusted by
Benjamini–Hochberg correction for multiple testing). GO terms are
additionally ordered by their assigned parental processes.
Functional GO term enrichment with the GO term finder tool at Candida
Genome Database^[150]45 (Fig. [151]2A) and KEGG pathway enrichment
analyses^[152]46,[153]47 (Fig. [154]2B) were performed on the sets of
differentially expressed genes. Both analyses indicated that the
amphiphilic antifungal polymers caused damage to the cell membrane and
cell wall, leading to the metabolic arrest of C. albicans. GO terms
associated with transporter activity were enriched in the downregulated
gene set, indicating general stress and metabolic arrest of the cells
(Fig. [155]2A). More specifically, oligopeptide transmembrane
transporter activity was enriched in the set of genes downregulated
following treatment with LH. Together with the overrepresentation of
genes involved in lipid and protein binding, this supports the
hypothesis of C. albicans sensing a toxic, peptide-like structure with
amphiphilic properties. The KEGG pathway mitogen-activated protein
kinase (MAPK) stress response was overrepresented in upregulated genes
upon treatment with the antifungal polymers (Fig. [156]2B and
Supplementary Fig. [157]S17 after treatment with polymer LH). An
upregulation of genes in the MAPK signalling pathway is typical for
cell wall and osmotic stress or starvation and leads to cell cycle
arrest and cell wall remodelling^[158]48,[159]49. The metabolic arrest
of the fungal cells was further indicated by the enrichment of the KEGG
pathways ribosome, biosynthesis of amino acids, and oxidative
phosphorylation in the downregulated gene set upon treatment with the
antifungal polymers.
Additionally, GO terms associated with glycosylation processes were
enriched in the upregulated gene set, specifically hydrolase activity
against O-glycosyl compounds and hexosyltransferase activity – a
parental gene set of mannosyltransferase activity (Fig. [160]2A).
Similarly, one significantly overrepresented KEGG pathway enriched in
upregulated genes was protein processing in the endoplasmic reticulum
(ER) (Fig. [161]2B). A more detailed look (Supplementary Fig. [162]S18)
revealed that C. albicans cells treated with LH strongly upregulated
genes associated with glycosylation and ER-associated degradation of
misfolded protein (ERAD), together suggesting a disruption in the
correct glycosylation of proteins. In support of this hypothesis, we
also observed upregulated genes in the N-glycan biosynthesis pathway
(Supplementary Fig. [163]S19).
GO terms relating to membrane stress were enriched in the upregulated
gene set (Fig. [164]2A). This included calcium ion transmembrane
transport, suggesting increased Ca^2+ influx and membrane damage. KEGG
pathway enrichment analysis also revealed a transcriptional signature
expected from damage to the cell membrane (Fig. [165]2B), particularly
with polymer LH. This includes an overrepresentation of
glycosylphosphatidylinositol (GPI)-anchor biosynthesis and glycerolipid
biosynthesis in the upregulated gene set, and steroid biosynthesis in
the downregulated gene set.
In contrast, treatment with the non-toxic poly-HEA or the antimicrobial
peptide LL-37 did not elicit a significant enrichment of these
glycosylation-related GO terms and KEGG pathways. This shows that it is
not a general response to the presence of polymers or to bioactive
peptide(-like) substances. Instead, we found an enrichment of
upregulated genes with GO terms associated with DNA, protein, and small
molecule binding. This may indicate that C. albicans reacts to small,
peptide(-like) molecules, however, they do not seem to influence
glycosylated proteins.
We found nearly no KEGG pathways to be enriched for differentially
regulated genes in the presence of poly-HEA. In the presence of LL-37,
genes involved in MAPK signalling were significantly upregulated,
indicating a fungal stress response to a toxic compound. Additionally,
the KEGG pathways ribosome, biosynthesis of amino acids, and oxidative
phosphorylation were significantly enriched in the downregulated gene
set upon treatment with LL-37, indicating an expected metabolic arrest
of the fungal cells. Those KEGG pathways were also overrepresented in
the presence of the antifungal polymers, but not the non-toxic
poly-HEA. Notably, genes in the KEGG pathway “protein processing in the
ER” were specifically upregulated upon treatment with the antifungal
polymers, but not poly-HEA or LL-37 (Fig. [166]2B and Supplementary
Fig. [167]S18, showing polymer LH vs. LL-37), again highlighting that
ER protein processing and MAPK signalling responses are specific for
polymers with antifungal activity. In addition, we found that the gene
expression profiles of C. albicans exposed to our polymers are very
distinct from published profiles under exposure to polyenes, azoles or
echinocandins^[168]50. This again suggests a unique mode of action of
our synthetic polymers.
Currently approved antifungals have specific cellular targets, and
mutations in the affected enzymes are one of the main reasons for the
development of resistance^[169]10. In contrast, antimicrobial peptides
have multiple modes of action, decreasing the likelihood of the
development of resistance^[170]51. For example, a Musca domestica
(housefly) AFP triggers responses in C. albicans that include reaction
to oxidative stress, cell wall and membrane maintenance, protein
synthesis, and energy metabolism^[171]52. A bacterium-derived,
membranolytic antifungal lipopeptide (jagaricin)^[172]53 similarly
induces a broad transcriptional response, comprising the upregulation
of cell wall organisation/biogenesis and calcium ion transmembrane
transport genes, and downregulation of transmembrane transport for
substances such as oligopeptides^[173]37. Our antifungal polymers
induced a similar transcriptional response in C. albicans. Altogether,
these data led us to hypothesise that the antifungal polymers likely
target multiple processes in C. albicans – they damage the cell wall
and permeabilise the cell membrane, and they target protein
glycosylation and thereby induce ER stress.
O-mannosylation, calcineurin, MAPK signalling, and a phosphoinositide
regulator are required for C. albicans to survive polymer exposure
To gain further insights into the target of the polymers, we screened
selected deletion mutants of C. albicans for their growth at
sub-inhibitory polymer concentrations (LP, LH, CB, CX). Mutants were
chosen to represent suspected target and resistance pathways, including
cell wall organisation and stress response, membrane composition and
inositol signalling, protein glycosylation (O- and N-linked
mannosylation), calcineurin pathway, osmotic and oxidative stress
response (MAPK signalling), drug efflux, and polyamine uptake
(Supplementary Data [174]S1). Growth curves were compared based on the
time to reach half-maximal absorption (Supplementary Data [175]S1).
This growth speed index is positive for beneficial mutations (green
shading in Supplementary Data [176]S1) and negative for detrimental
ones (red shading in Supplementary Data [177]S1).
The deletion of genes important for cell wall organisation and stress
response had little effect on growth in the presence of polymers
(Supplementary Data [178]S1), with the exception of ire1, which showed
no growth in the presence of LH. IRE1 encodes a protein kinase of the
unfolded protein response and cell wall organisation^[179]54.
Interestingly, IRE1 was also upregulated in the presence of LH
(Supplementary Fig. [180]S18).
Among the mutants for genes relevant to membrane composition and
inositol signalling (Supplementary Data [181]S1), inp51 – lacking a
phosphatase involved in the maintenance of phosphoinositide levels and
thus cell wall and membrane integrity^55 – exhibited no growth in the
presence of all polymers. INP51 deletion has been shown to increase
susceptibility to cell wall-active compounds^[182]55. The deletion of
ERG5, involved in ergosterol biosynthesis, did not change the
susceptibility to the polymers. This agrees well with the
susceptibility of clinical AmpB-tolerant isolates towards the polymers
(Table [183]2) and indicates that ergosterol biosynthesis likely does
not affect the polymers’ mode of action.
Deletion of genes contributing to O-linked protein mannosylation (PMT1,
PMT3, PMT4, PMT5)^[184]56 consistently increased susceptibility of
C. albicans (Supplementary Data [185]S1), suggesting that these
activities are involved in the response to all antifungal polymers. In
contrast, gene deletions affecting N-linked mannosylation (MNN13,
MNN14, MNN15, MNN22, MNN4, MNN9, MNS1, MNT4, OCH1)^[186]56 resulted in
no change in susceptibilities.
Calcineurin and MAPK signalling are crucial in fungal development and
response to environmental stress^[187]57. Two calcineurin pathway
deletion mutants (crz1, mid1) showed no growth with normally
sub-inhibitory polymer concentrations (Supplementary Data [188]S1), and
their genes’ expression was also upregulated in C. albicans exposed to
the antifungal polymers, but not poly-HEA. Two MAPK signalling deletion
mutants (hog1, pbs2) also showed no growth in the presence of polymers.
HOG1 similarly has shown upregulation after exposure to the antifungal
polymers, most prominently for LH and CX (Supplementary Fig. [189]S17
for LH-treatment). Hence, the antifungal polymers seem to cause stress
in C. albicans, and both calcineurin and MAPK pathways appear to be
essential for fungal survival.
In agreement with the clinical drug-efflux mutants (Table [190]2),
neither gain-of-function (in MRR1 and TAC1) nor deletion (of MRR1,
SNQ2, TAC1) of genes involved in drug efflux had a consistent impact on
susceptibility.
The AFP histatin 5 is actively transported across the fungal membrane
by polyamine transporters like Dur31 in C. albicans to exert its
intracellular antifungal effect^[191]58. Our synthetic polymers share
characteristics like cationic charge with both histatin 5 and
polyamines, and their presence led to the upregulation of DUR31.
However, deletion of DUR31 or the related DUR35^[192]59 resulted in a
slightly increased (dur31) or unchanged susceptibility (dur35)
(Supplementary Data [193]S1), suggesting that the polymers are not
primarily taken up by those transporters.
We also investigated selected C. glabrata (Nakaseomyces glabratus)
mutants (marked blue in Supplementary Data [194]S1). While none of the
tested C. albicans mutations were beneficial, the deletion of
C. glabrata ERG5 and INO2 increased tolerance to all antifungal
polymers. Both genes are associated with membrane composition and
inositol signalling. C. glabrata cdr1, lacking a drug-efflux
pump-encoding gene, also showed increased growth with polymers, except
LH. In contrast, deletion of MNN2, coding for an N-linked
mannosyltransferase, had the highest benefit for LH-treated C. glabrata
cells. Despite these slight differences, our data still suggests a very
similar mode of action against C. glabrata. Like in C. albicans,
deletion of genes encoding proteins involved in membrane composition
and inositol signalling (INP51, INP53, ISC1), O-linked protein
mannosylation (PMT1, PMT2) and MAPK signalling (PBS2) were more
susceptible to the polymers, while the deletion of genes coding for
proteins in the calcineurin pathway (CNA1, CRZ1, MID1) resulted in no
growth in the presence of antifungal polymers.
In sum, the mutant screening agreed with our analyses of the
transcriptome and suggested a mode of action connected to protein
glycosylation and general stress of the polymers against C. albicans.
Some similarities (involvement of MAPK and calcineurin signalling) were
detected in a previous C. albicans mutant screen with the antifungal
lipopeptide jagaricin, for which disruption of membrane integrity was
suggested as the primary mode of action^[195]37,[196]53. This suggests
a similar effect of the synthetic polymers, especially as our gene
expression analyses also showed possible interference with the plasma
membrane (Fig. [197]2).
Polymer LH lyses C. albicans cell membranes
The polymers are inspired by antimicrobial peptides, which lyse
bacterial and fungal membranes^[198]60. Membrane damage by the
synthetic polymers was also suggested by gene expression analyses
(Fig. [199]2) and mutant screening (Supplementary Data [200]S1). We
therefore investigated the membranolytic potential of polymer LH with a
C. albicans strain constitutively expressing GFP in the cytosol
(Fig. [201]3A–G). Two antifungal compounds were included – AmpB, which
has membranolytic activity^[202]61,[203]62, and tunicamycin, which
inhibits N-glycosylation of proteins, leading to cell death without
membrane lysis^[204]63. Additionally, tethered membranes isolated from
C. albicans and erythrocytes were incubated with polymer LH and
non-toxic poly-HEA to investigate their potential to lyse synthetic
membranes (Fig. [205]3I, J).
Fig. 3. Polymer LH causes membrane lysis in C. albicans.
[206]Fig. 3
[207]Open in a new tab
Detection of fluorescence of C. albicans expressing cytoplasmic GFP
(ADH1-GFP) after 6 h at MIC assay conditions for (A) untreated
C. albicans cells, B cells treated with 1× MIC Amphotericin B (AmpB), C
1× MIC tunicamycin, D 0.25× MIC polymer LH, E 0.5× MIC polymer LH, and
F 1× MIC polymer LH to investigate lytic activity of the compounds.
Scale bars in (A–F) represent 10 µm. G The GFP signal was quantified
from at least 50 C. albicans-GFP cells per biological replicate
(n = 3), and then averaged and normalised to the respective untreated
control. H Colony forming units (CFU) were determined by backplating
and normalised to the inoculum (n = 3 biological replicates).
Statistical significance in (G) and (H) was determined by Dunnett’s
ordinary one-way ANOVA multiple comparisons analysis (compared to
untreated (100%) in (G) or inoculum (100%) in (H), *p < 0.05
[G: p = 0.0148, H: p = 0.0296], **p < 0.01 [G: p = 0.0051],
***p < 0.0005 [H: p = 0.0002], ****p < 0.0001) with ns indicating
non-significant [H: p = 0.4987]. Average conductance (G[m]) of tethered
membranes isolated from C. albicans yeast (black) or hyphae (grey), or
from erythrocytes (red) after the addition of increasing concentrations
(c) of (I) antifungal polymer LH and (J) non-toxic poly-HEA in RPMI
medium at 37 °C (n = 3 biological replicates). Error bars in (G–J)
represent the standard deviation (SD) around the mean. Source data are
provided as a Source Data file.
Untreated C. albicans-GFP cells showed a prominent intracellular GFP
signal (Fig. [208]3A), which was absent in AmpB-treated cells
(Fig.[209]3B, G) where only 0.3% were viable as determined via
backplating (Fig. [210]3H). The loss of GFP signal was due to membrane
lysis, and not cell death per se, as tunicamycin-treated cells showed a
less prominent loss of intracellular GFP (Fig. [211]3C), even though
only 3% of cells were viable. The polymer LH (Fig. [212]3D–F) led to
membranolytic activity, as seen by GFP signal loss, in a
concentration-dependent manner with a proportional decrease in cell
viability (36% at 0.5× MIC, 3% at 1× MIC at the assay conditions).
Interestingly, at concentrations below MIC, we observed cell
aggregates, which suggest a change in the surface properties of
C. albicans cells.
To confirm the permeabilisation of membranes by polymer LH as part of
its mode of action against C. albicans, tethered membranes composed of
lipid mixtures isolated from C. albicans yeast or hyphae, or from
erythrocytes, were incubated with polymer LH (Fig. [213]3I) or
non-toxic poly-HEA (Fig. [214]3J). Polymer LH led to a
concentration-dependent increase in membrane conductance, indicating
membrane permeabilisation by pore formation (Fig. [215]3I and
Supplementary Fig. [216]S20A). The increase in membrane conductance was
highest in the case of lipids from C. albicans yeast (Fig. [217]3I,
black) and weaker for those from C. albicans hyphae (grey). Membranes
composed of erythrocyte lipids were more stable and showed lower
conductance before the addition of polymer LH and also at low
concentrations of polymer LH, compared to membranes composed of
C. albicans membranes. In contrast to polymer LH, poly-HEA did not lead
to an order of magnitude differences in membrane conductance when
increasing its concentration in any of the three lipid mixtures
(Fig. [218]3J), indicating no lytic effect. As a control, the same
experiment was performed with antimicrobial peptide LL-37
(Supplementary Fig. [219]S20B), for which membrane permeabilisation has
been reported as the primary mode of action against
C. albicans^[220]38,[221]44. Like polymer LH, LL-37 permeabilised
C. albicans membranes at a concentration of 8 µg/mL. However, it also
strongly affected tethered membranes isolated from erythrocytes,
indicating a lower selectivity of LL-37 compared to polymer LH.
LH damages mannans attached to cell wall proteins, enhances phagocytosis, and
affects yeast-to-hypha transition
Mutant screening and gene expression analyses suggested that
glycosylated proteins might be primarily affected by the polymers. A
major group of glycosylated proteins in C. albicans are the cell wall
proteins. These are both O- and N-mannosylated as they pass through the
ER and Golgi on their way to the wall. Once attached to the wall
through GPI anchors or Pir linkages, the short O-mannan chains remain
buried in the inner cell wall layer, whereas the long N-mannan chains
protrude out from the cell surface forming an outer fibrillar layer of
the cell wall (Fig. [222]4A)^[223]64. To investigate the effects of LH
on the cell wall structure of C. albicans, fungal cells were incubated
at sub-inhibitory concentrations for 6 h and analysed by transmission
electron microscopy (TEM, Fig. [224]4A–D). Compared to the no-treatment
control (Fig. [225]4A), treatment with the non-toxic poly-HEA resulted
in no major ultrastructural changes to the cell wall (Fig. [226]4B). A
reduction of the outer cell wall N-mannan layer was observed after
treatment with tunicamycin, an inhibitor of N-glycosylation
(Fig. [227]4C)^[228]63. Treatment with a sub-inhibitory concentration
of LH (Fig. [229]4D) disrupted the arrangement of the N-mannan fibrils,
supporting our transcriptome and mutant screen data. At these
sub-inhibitory concentrations, LH did not cause any obvious disruption
of the membrane.
Fig. 4. Cell wall changes in C. albicans and immune cell response to
LH-treated C. albicans.
[230]Fig. 4
[231]Open in a new tab
Transmission electron microscopy (TEM) micrographs of the C. albicans
cell wall after 6 h incubation in SD medium at 30 °C with (A) no
additives, B with poly-HEA, C with tunicamycin, or D antifungal polymer
LH, at sub-inhibitory concentrations of the antifungal compounds. The
scale bars in (A–D) represent 100 nm. E The amount of glucan and mannan
in cell walls isolated from C. albicans wildtype (SC5314) and och1
mutant cells after 6 h of incubation in SD medium (untreated) and with
sub-inhibitory concentrations of polymer LH, poly-HEA, tunicamycin, and
caspofungin was determined by HPLC. Glucose (from glucan) and mannose
(from mannan) represented 55% and 45% of the dry weight of the cell
wall in wild-type untreated cells. The graph shows the proportions of
glucan (grey bars) and mannan (striped bars) relative to those observed
in untreated C. albicans wild-type cells. F Proportion of C. albicans
cells not taken up over 15–30 min by human monocyte-derived
macrophages. C. albicans cells were pre-treated with LH for one hour
before putting them into contact with the macrophages (n = 6 biological
replicates). Statistical significance in (E) was determined by
Dunnett’s repeated measures ANOVA multiple comparisons analysis
(*p < 0.05 [0 vs 8 µg/ml: p = 0.0173; 0 vs 16 µg/ml; p = 0.0192]).
Error bars in (F) represent the standard deviation (SD) around the
mean. Source data are provided as a Source Data file.
To confirm the transcriptome and TEM observations quantitatively, cell
walls from C. albicans were isolated after 6 h incubation, and after
acid hydrolysis, mannose (from mannans) and glucose (from glucans) were
separated and detected by high-performance liquid chromatography (HPLC)
(Fig. [232]4E). Exemplary HPLC spectra are shown in Supplementary
Fig. [233]S21. In accordance with the TEM observations (Fig. [234]4D),
treatment with sub-inhibitory concentrations of polymer LH decreased
the proportion of mannan relative to untreated cells. In contrast,
poly-HEA treatment had no effect on the relative proportion of glucan
and mannan. Consistent with our observations in the TEM images
(Fig. [235]4C), treatment with the N-glycosylation inhibitor
tunicamycin reduced the mannan content relative to untreated cells. The
proportion of mannan in C. albicans och1 mutant cells was reduced
relative to wild-type cells, as expected for an N-mannosylation mutant
and consistent with previous observations^[236]62. Finally, the
β-glucan synthase-inhibiting antifungal drug caspofungin had the
expected effect of decreasing the glucan and increasing mannan,
relative to the untreated control^[237]63. Overall, our measurements
were therefore in good agreement with the expected outcome four our
controls and supported our notion of a defective protein glycosylation
in C. albicans after treatment with polymer LH.
Components of the C. albicans cell wall are also recognised by innate
immune cells^[238]65. We hypothesised that the LH-induced changes to
the cell wall structure (Fig. [239]4D) could also impact immune
recognition and clearance by human macrophages. To test this, we
challenged C. albicans cells that were preincubated with or without
polymer LH at sub-inhibitory concentrations with primary human
monocyte-derived macrophages (hMDMs). Primary immune cells such as
hMDMs show donor-dependent differences in their uptake efficiency. We
therefore chose the time point (15 or 30 min) for each donor when
30–50% of untreated C. albicans cells were not yet phagocytosed by the
specific macrophages. We found that LH pre-treatment significantly
increased clearance of C. albicans by primary hMDMs, even at
sub-inhibitory concentrations between 4–16 µg/mL (Fig. [240]4F).
Concentrations above 16 µg/mL of polymer LH, however, resulted in
decreased clearance (Supplementary Fig. [241]S22A). These are most
likely due to toxic effects on the hMDMs, which became apparent at
128 µg/mL after 30 min (Supplementary Fig. [242]S22B, measured by LDH
release).
It has been previously reported that C. albicans mutants with N- or
O-linked glycosylation defects are more efficiently phagocytosed than
the wild type^[243]66,[244]67. With our TEM, HPLC, and transcriptome
data, this suggests that LH disrupts the mannan layer and induces cell
wall remodelling which increases phagocytosis by macrophages. Thus, in
vivo, the application of polymer LH could potentiate fungal clearance
by innate immune cells.
The cytokine release by primary human peripheral blood mononuclear
cells (PBMCs) to pre-treated C. albicans was characterised by the
pro-inflammatory cytokines IL-1β, IL-6, and TNF (Supplementary
Fig. [245]S23). The increased clearance by hMDMs (Fig. [246]4F), was
reflected by significantly decreased IL-1β, IL-6, and TNF responses of
PBMCs at an LH concentration of 4 µg/mL (Supplementary Figs. [247]S23).
Higher LH concentrations resulted in no significant changes in the
release of IL-1β and TNF and a significant increase of IL-6. The
decrease in TNF agrees with previous observations on C. albicans
N-mannosylation defective mutants with impaired recognition by immune
cells^[248]68,[249]69. As mannoproteins in C. albicans cause a
pro-inflammatory immune response^[250]56,[251]65, these data support
our notion that polymer LH can act on protein glycosylation.
The cell wall of C. albicans is adaptive and constantly remodels,
including when C. albicans’ transitions from yeast to hyphae, a process
that is commonly associated with virulence^[252]65. Hence, we tested
C. albicans under hypha-inducing conditions (37 °C and 5% CO[2]) in the
presence of antifungal polymers at sub-inhibitory concentrations over
4, 6 (to measure hypha length), and 24 h (to measure microcolony
diameter) (Supplementary Fig. [253]S24). Treatment with each polymer,
especially LH, reduced the hyphal length and the diameter of
microcolonies. Besides lytic activity against synthetic membranes
isolated from C. albicans hyphae (see Fig. [254]3I, grey line), a
reduced speed of hyphae formation could be beneficial for clearance and
protection of epithelial cells, as the formation of hyphae drives cell
invasion and infection of human epithelial cells by
C. albicans^[255]70, however, further studies are necessary to confirm
this correlation for polymer LH.
Polymer LH prevents in vitro infection of human epithelial cells by
C. albicans synergistically with caspofungin or fluconazole
During systemic infection, C. albicans hyphae invade the human
epithelial barrier and allow it to spread via the bloodstream to distal
organs^[256]71. The yeast-to-hypha transition is also important for
common superficial infections, such as those of the vaginal mucosa,
which affect approximately 75% of women worldwide at least once in
their lifetime^[257]72,[258]73. To investigate the therapeutic
potential of polymer LH, we used an in vitro human epithelial cell
model (HECM). Monolayers of vaginal epithelial cells (A-431) were
infected with C. albicans, after the addition of different drug
dilutions with minimum preincubation time. After 24 h, epithelial
damage to A-431 cells was assayed by lactate dehydrogenase (LDH)
release. Despite being a rather simple model, it has been routinely
used to simulate vaginal candidiasis in vitro^[259]74,[260]75.
First, we examined the biocompatibility of polymer LH in the HECM in
the absence of C. albicans (Supplementary Fig. [261]S25, light-grey
bars). Only at concentrations above 128 µg/mL (4–8× MIC against
C. albicans) we observed more than 50% damage. Previously, polymer LH
has similarly been reported to cause more than 50% damage against
murine fibroblasts at 128 µg/mL or higher^[262]27. Based on the
therapeutic index – the ratio of cytotoxic concentration to MIC –
polymer LH, with its therapeutic index of 4–8, outperformed AmpB
(therapeutic index 2–4)^[263]27. For the HECM in the present study,
this would theoretically present a therapeutic window for polymer LH of
up to 128 µg/mL. However, in the HECM at concentrations between 16 and
128 µg/mL, polymer LH did not prevent damage by C. albicans to the
vaginal epithelial cells (Supplementary Fig. [264]S25, dark-grey bars).
One possibility for the unexpected failure of LH to prevent damage in
the HECM could be the bioavailability. To test this hypothesis, we
pre-treated C. albicans cells with the polymer for 1 h at
concentrations between 16 and 512 µg/mL before infection. Damage by
C. albicans was strongly reduced with this altered protocol in a
concentration-dependent manner up to 256 µg/mL (34% of untreated
infection control at 256 µg/mL, Supplementary Fig. [265]S26A). This
supports the hypothesis that poor bioavailability in the HECM reduces
the antifungal properties of the polymer. We next took supernatants
from uninfected vaginal epithelial cells, treated with LH, and added
them to C. albicans in a conventional MIC assay. These were unable to
inhibit C. albicans growth in vitro, even when the initial
concentration of polymer LH exceeded the MIC (Supplementary
Fig. [266]S26B). This suggests that the presence of human cells reduces
the concentration of polymer in the supernatant. In contrast, the
common antifungal drugs AmpB, caspofungin, and fluconazole (among
others) successfully reduced damage by C. albicans to the epithelial
cells in the HECM (Supplementary Table [267]S2).
Since the polymer LH alone unexpectedly did not inhibit damage by
C. albicans in the HECM, we studied combinations of LH with established
antifungal compounds, first at MIC assay conditions and without human
cells (Supplementary Figs. [268]S27 and [269]28, and Table [270]S3).
Synergy was defined as a minimum two-fold decrease in MIC in the
presence of the other drug (fractional inhibitory concentration (FIC)
index ≤0.5). Antagonistic drug combinations result in an FIC index of
at least 4, i.e., a MIC increase of at least two-fold. Indifferent (FIC
index = 1), and additive (FIC index between 0.5 and 1) effects were
also considered.
The compounds selected for our synergy studies differ in their targets:
cell wall (Calcofluor White, Congo Red, caspofungin, nikkomycin Z);
cell membrane (AmpB, cetyltrimethylammonium bromide (CTAB),
dodecyltrimethylammonium bromide (DTAB), sodium dodecyl sulfate (SDS));
or intracellular processes (fluconazole, cycloheximide, tunicamycin,
FK506, geldanamycin), which in turn could again affect cell membrane or
wall composition. Seven out of these thirteen antifungal compounds
showed a synergistic or strong additive effect (FIC index below 0.6)
with LH in vitro and were therefore tested in the HECM. In addition to
the LDH release assay, propidium iodide was used to visualise dead
epithelial cells after 24 h of infection (Fig. [271]5, Supplementary
Fig. [272]S29).
Fig. 5. Synergistic effects of LH in combination with selected antifungal
drugs against C. albicans SC5314 during vaginal epithelial cell infections.
[273]Fig. 5
[274]Open in a new tab
Damage to vaginal epithelial (A-431) cells (infected by C. albicans and
uninfected) after treatment with polymer LH and A caspofungin (Cas) or
B fluconazole (Flu) and their respective combinations was measured by
LDH release (n = 4 biological replicates, SD around the mean). Damage
to A-431 cells was normalised to untreated infection control (for
infected samples) or a Triton-X-treated 100% lysis control (uninfected
samples; each indicated by a dotted line). Source data are provided as
a Source Data file. (C–J) show fluorescence microscopy images of the
scenarios represented in (A) and (B) to visualise morphological changes
and the viability of vaginal epithelial cells by staining with 1 µg/mL
propidium iodide. Scale bars represent 100 µm.
Although LH alone did not prevent damage by C. albicans (Supplementary
Fig. [275]S26), it did so in combination with the antifungal drugs
caspofungin or fluconazole, even at normally sub-inhibitory
concentrations (Fig. [276]5). Strikingly, only 0.03 µg/mL of
caspofungin (i.e., 8× less than its MIC of 0.25 µg/mL in the HECM)
combined with 4 µg/mL LH was required to reduce host cell damage to 2%
with no visible cytotoxicity (Fig. [277]5A). Similarly, a low dose of
0.03 µg/mL fluconazole (again 8× less than its MIC in the HECM)
combined with 16 µg/mL LH reduced vaginal epithelial cell damage to
13%, while remaining biocompatible (Fig. [278]5B). Without LH, these
low antifungal drug concentrations did not prevent infection with over
50% epithelial cell damage. This finding was supported by fluorescence
microscopy (Fig. [279]5C–J), where the same combinations resulted in
healthy vaginal epithelial cells and low doses of antifungal drugs did
not significantly protect the epithelial cells from damage at up to
0.25 µg/mL. Combination with LH therefore reduced the MIC for
established antifungal drugs up to eight-fold, showing its potential as
a synergistic agent for antifungal applications.
The other antifungal compounds with strong additive or synergistic
behaviour with LH in the in vitro pre-screen (nikkomycin Z,
cycloheximide, tunicamycin, FK506, geldanamycin), did not reduce damage
to epithelial cells to less than 45% of the no-drug control, and in
some cases showed no significant synergism (Supplementary
Fig. [280]S29). Some of those compounds are cytotoxic at elevated
concentrations, and indeed, the combination of LH with cycloheximide or
geldanamycin caused damage to uninfected human cells.
Combination of polymer LH and caspofungin prolongs survival in an
invertebrate model of C. albicans infection
Next, we assessed whether the synergism of polymer LH with caspofungin
protected against fungal infection in vivo. For this, we used the
well-established Galleria mellonella (greater wax moth) model of
systemic candidiasis by injecting larvae with an infectious dose of
C. albicans followed by treatment with LH and
caspofungin^[281]76–[282]79. Even though G. mellonella is an
invertebrate organism, the model has several advantages for testing the
virulence of Candida spp. and antifungal activity of candidate
antifungals, such as ease-of-use, growth at 37 °C, and its many
similarities to the mammalian innate immune system^[283]76–[284]79.
We first determined the acute toxicity of polymer LH, caspofungin, and
AmpB in larvae. Lethal effects of LH on larvae were observed at
500 mg/kg or higher (Supplementary Fig. [285]S30A). No toxicity was
observed for caspofungin at doses up to 100 mg/kg (Supplementary
Fig. [286]S30B, blue line), AmpB showed toxicity at 100 mg/kg after 4 d
(Supplementary Fig. [287]S30B, purple line). Therefore, both LH and
caspofungin outperformed AmpB in terms of toxicity, and we determined
that doses of LH up to 250 mg/kg and caspofungin up to 100 mg/kg could
be used to treat larvae. The synergistic combination of LH with
fluconazole was not tested, because it was not active against
C. albicans in the presence of 50% (v/v) or more of foetal bovine serum
in MIC assays, in contrast to the combination of LH and caspofungin
which remained active in the presence of serum (Supplementary
Table [288]S4).
To simulate systemic candidiasis, we infected each larva with 1 × 10^5
C. albicans cells in one proleg, treated them in another proleg, and
monitored survival over 14 days (Fig. [289]6). All uninfected,
water-treated G. mellonella larvae survived for 14 d. In contrast,
untreated and water-mock treated C. albicans-infected larvae died after
2 d (difference not significant at p = 0.674).
Fig. 6. Treatment with polymer LH in combination with caspofungin (Cas)
prolongs survival of C. albicans-infected Galleria mellonella larvae.
[290]Fig. 6
[291]Open in a new tab
G. mellonella larvae were infected with 1 × 10^5 C. albicans cells
(except uninfected control, grey) and treated after 2 h with
caspofungin (Cas; 5 mg/kg: blue; 100 mg/kg: blue dotted), polymer LH
(250 mg/kg, green dashed), and the combination of 5 mg/kg Cas and
250 mg/kg LH (green and blue dashed). Untreated, infection controls are
shown in black (C. albicans only) or black dashed (C. albicans,
injected with water). Survival of G. mellonella was monitored over
14 d. Nineteen larvae per condition were tested. Statistical
significance was determined for infected larvae treated with the
combination (5 mg/kg Cas + 250 mg/kg LH) compared to infected larvae
treated with the respective dose of a single drug by Log Rank
(Mantel–Cox) pairwise comparison (**p < 0.01 [here: p = 0.003],
***p < 0.001). At the concentrations used here, the single compounds
exhibited no toxicity against G. mellonella, as shown in Supplementary
Fig. [292]S30. Source data are provided as a Source Data file.
Treatment with 100 mg/kg caspofungin alone protected 94.7% of the
larvae from death, whereas only 10.5% of larvae survived to 14 d at
5 mg/kg (Fig. [293]6). All infected larvae treated with polymer LH
alone at 250 mg/kg succumbed by day 3, however, the same dose in
combination with low-dose caspofungin (5 mg/kg) significantly increased
the survival of C. albicans-infected larvae (5 mg/kg caspofungin vs.
combination: p = 0.003; 250 mg/kg LH vs. combination: p < 0.001; water
vs. combination: p < 0.001). Compared to low-dose caspofungin
treatment, the survival after 14 d increased four-fold to 42.1%.
Notably, 100% of infected larvae treated with the synergistic
combination survived until day 6 post-infection. This therefore
demonstrates in vivo synergy between polymer LH and caspofungin and
emphasises the in vivo potential of the synthetic polymer LH in
combination therapy. Such combination therapy approaches of compounds
which are individually inactive or only marginally active in vivo have
received more attention recently. Examples include an AFP mimic
(brilacidin) potentiating caspofungin and a small organic molecule
(imidazopyrazoindole) synergising with azoles^[294]17,[295]18. Notably,
the possibility of re-sensitising drug-resistant fungi by the
combination of active compounds is promising^[296]17,[297]18,[298]20.
The key advantages of synthetic polymers are stability and
cost-effective production at scale^[299]20,[300]30, which could solve
many issues, especially in low-income countries^[301]9.
In vitro evolution leads to tolerance of C. albicans to LH, but not to the
combinations of LH with caspofungin or fluconazole
A major obstacle in the development of antifungal drugs is the
emergence of resistance. In this study, we follow the definition of
resistance and tolerance suggested by Berman and Krysan^[302]80; where
resistance is the clinically observed ineffectiveness of a drug against
a fungal pathogen and tolerance is the slow growth of a less
susceptible fungal strain at normally inhibitory concentrations in
vitro^[303]80. An in vitro evolution assay (Fig. [304]7A) was used to
determine whether prolonged exposure to polymer LH, caspofungin,
fluconazole or their synergistic combinations results in the emergence
of tolerant C. albicans variants. Growth at 1× MIC over 14 d
(Fig. [305]7B) showed an increasing tolerance for the single drug
treatments after 9–10 d incubation. A less pronounced tolerance
developed for the combination of fluconazole and LH, while essentially
no change in growth was seen for the combination of caspofungin and LH.
Fig. 7. In vitro evolution experiment of C. albicans challenged with 1× MIC
of caspofungin (Cas), fluconazole (Flu), polymer LH, and the synergistic
combinations.
[306]Fig. 7
[307]Open in a new tab
A Experimental setup of the in vitro evolution experiment. B Growth of
C. albicans was monitored by absorbance over 14 d and normalised to the
untreated controls. Filled circles highlight strains that were selected
for whole genome sequencing and empty circles highlight strains
additionally analysed for their MIC against antifungal compounds in
Supplementary Table [308]S5. Source data are provided as a Source Data
file. C Genomes of the isolated strains with the highest tolerance to
antifungal drugs were sequenced and analysed for their relative copy
number for Flu- and LH-evolved strains. Each point in (C) represents
the mean normalised read depth compared to wild-type C. albicans strain
SC5314 at t = 0 for a gene (Y-axis) on its chromosome position
(X-axis), colour-coded by allele. Positions of the centromeres are
indicated by red circles. The MICs for Flu and LH are indicated on the
right, where MIC in SC5314 is depicted in green and higher values scale
to red.
After 14 d, an aliquot of the cells was incubated at 2× MIC for
24–48 h. If growth was observed, the sample was plated, single colonies
were isolated and their MIC to the antifungal polymers (LP, LH, CB, and
CX) and antifungal drugs (AmpB, fluconazole, caspofungin, tunicamycin)
was determined (Supplementary Table [309]S5). Isolates with an
increased MIC were selected for further analysis (Fig. [310]7B, filled
circles – increased tolerance to LH, empty circles – no increase in LH
tolerance). In agreement with their growth pattern over 14 d, we did
not find any stably tolerant isolates after treatment with combinations
of LH and caspofungin or fluconazole (MICs in Supplementary
Table [311]S5).
To identify the genetic basis for LH tolerance, we sequenced the
genomes of three independent LH-evolved strains with high MIC (evo-LH1,
evo-LH2, and evo-LH3, filled black circles in Fig. [312]7B), and a
fluconazole-evolved strain (evo-Flu1) as control. Evo-LH1 showed an
increase in MIC against LH to 64–128 µg/mL and was also more tolerant
to the polymers LP, CB, and CX. The MICs towards established antifungal
drugs (AmpB, caspofungin, fluconazole, tunicamycin) remained unchanged.
Interestingly, evo-Flu1 not only showed increased tolerance to
fluconazole (MIC 2 µg/mL) but also to caspofungin and the polymers. The
caspofungin-evolved strain (evo-Cas1) developed an increased MIC for
caspofungin (2 µg/mL), but we did not observe any cross-tolerance.
To investigate the genomic mechanisms of C. albicans adaption to LH or
fluconazole, we examined the gene counts for the evolved strains
compared to wild-type C. albicans SC5314 (Fig. [313]7C, Supplementary
Fig. [314]S31). In all strains, including the parental wild-type
SC5314, loss of heterozygosity was detected in the left arm of
chromosome 2 (Supplementary Fig. [315]S31). Analysis of the evolved
strains revealed major ploidy changes in some cases: In evo-Flu1, we
observed aneuploidy of chromosome R and trisomy of chromosome 5. Both
are associated with fluconazole resistance^[316]81,[317]82, and the
chromosome 5 trisomy has been shown to be driven by amplification of
the ERG11 and TAC1 genes^[318]83. For evo-LH1 and evo-LH2, we found
aneuploidy in chromosome R, and trisomy in chromosome 2, respectively.
Notably, chromosome 2 trisomy and tetrasomy have been linked to
adaption to the ER stressor tunicamycin^[319]84. For the strain
evo-LH3, which showed only a minor change in MIC, we found no
large-scale ploidy changes.
We did not find mutations or copy number variations on the gene level
that could explain the drug tolerance phenotype, although some
differences were observed between our isolated C. albicans strains on
the nucleotide level (Supplementary Data [320]S2). We conclude that
major chromosomal aberrations probably drive tolerance to polymer LH in
C. albicans. Together with our transcriptomics and chemical-genetic
screening evidence, we therefore postulate that polymer LH has multiple
targets, which differ from those of known antifungal drugs.
Importantly, in our in vitro evolution experiment we found no
development of genetically stable tolerance by combinatorial treatment
of LH with caspofungin or fluconazole, in further support of our
hypothesis of distinct targets. The strong synergistic action with
caspofungin against C. albicans on human epithelial cells in vitro and
G. mellonella larvae in vivo, together with the reduced tolerance
development suggests that the amphiphilic polymer LH is a promising
antifungal lead for combination therapy with well-established drugs.
In summary, we investigated four synthetic polymers, which were
inspired by amphiphilic antimicrobial peptides and which kill
drug-resistant clinical C. albicans isolates. Our findings reveal that
the most promising polymer, LH, exerts its activity on C. albicans by a
putatively novel mode of action. We found evidence that it targets
protein glycosylation, and also interferes with the fungal membrane,
which together leads to fungal cell death. The combination of LH with
caspofungin is particularly promising in its therapeutic potential
since it inhibits infection of human epithelial cells by C. albicans at
otherwise sub-inhibitory concentrations and additionally increases
fungal uptake by human macrophages. Moreover, the synergistic
combination of polymer LH and caspofungin prolonged survival in an
in vivo model of systemic candidiasis. In addition to these promising
synergistic effects, which prevent C. albicans infection in vitro and
protect in vivo, the combination of polymer LH and caspofungin did not
lead to any tolerant C. albicans strains after prolonged exposure in
vitro, highlighting the therapeutic potential of polymer LH as an
antifungal lead, particularly for combination therapy. Future
experiments may allow to further optimise polymer LH, e.g., by
investigating the effect of monomer sequence or block order within the
polymer on its activity. This way, it may be possible to retain the
promising antifungal activity even as a stand-alone formulation, which
would then also be envisioned to be tested in vertebrate in vivo
infection models.
Methods
Ethics statement
The research presented here complies with all relevant ethical
regulations. The blood donation procedure was approved by the Jena
institutional ethics committee (Ethik-Kommission des
Universitätsklinikums Jena, Permission No 2207–01/08). All donors gave
written informed consent and did not receive any compensation. Age, sex
or gender of the donors was not taken into account.
Materials for polymer synthesis
Ethylenediamine (Sigma-Aldrich, ≥99%), N-amylamine (Sigma-Aldrich,
99%), N-benzylamine (Sigma-Aldrich, 99%), N-heptylamine (Sigma-Aldrich,
99%), N-cyclohexanemethylamine (Sigma-Aldrich, 98%), di-tert-butyl
dicarbonate (Sigma-Aldrich, 99%), N-hydroxyethyl acrylamide
(Sigma-Aldrich, 97%), triethylamine (TEA) (Scharlau, 99%),
trifluoroacetic acid (TFA) (Sigma-Aldrich, 99%), chloroform (Merck),
dichloromethane (DCM) (Merck), tetrahydrofuran (THF) (Merck), diethyl
ether (Merck), hexane (Merck), dimethyl sulfoxide (DMSO) (Merck),
dimethylacetamide (DMAc) (Sigma-Aldrich), thionyl chloride
(Sigma-Aldrich, 99%), acrylic acid (Sigma-Aldrich), deuterated DMSO
(Cambridge Isotope Laboratories, Inc.),
2-(butylthiocarbonothioylthio)propanoic acid (BTPA, Boron Molecular)
and 5,10,15,20-tetraphenyl-21H,23H-porphine zinc (ZnTPP)
(Sigma-Aldrich) were used as received.
Acryloyl chloride synthesis
Acryloyl chloride was synthesised according to the previously reported
procedure^[321]85, with slight changes^[322]27. Briefly, acrylic acid
(41.2 mL, 1.2 equiv) was added dropwise to 36.3 mL of thionyl chloride
at 0 °C over 45 min under nitrogen. The mixture was stirred for 12 h at
40 °C. The product was collected by in situ distillation under
atmospheric pressure.
Synthesis of monomers
Cationic monomer - tert-butyl (2-acrylamidoethyl)carbamate
tert-Butyl (2-acrylamidoethyl)carbamate was prepared according to the
previously reported procedure^[323]27,[324]86,[325]87 Ethylenediamine
(0.33 mol) was dissolved in chloroform (400 mL). Di-tert-butyl
dicarbonate (0.03 mol) was dissolved in 100 mL of chloroform and was
added dropwise to the ethylenediamine solution over 4 h at 0 °C while
stirring and continued overnight at room temperature. After filtering
the white precipitate, the organic phase was washed with 200 mL of
Milli-Q water six times and then dried using MgSO[4]. Solids were
separated by filtration, and chloroform was evaporated, resulting in a
pale yellow oil. THF (100 mL) was added to dissolve the obtained oil.
TEA (1.2 equiv) and acryloyl chloride (1.1 equiv) were added dropwise
to the solution at 0 °C with N[2] bubbling. The reaction mixture was
stirred at room temperature for 2 h. Afterwards, THF was removed by
rotary evaporation. The crude product was dissolved in chloroform
(150 mL) and washed with 0.1 M HCl solution (1 × 75 mL), saturated
NaHCO[3] (1 × 75 mL), brine (1 × 75 mL), and water (1 × 75 mL). The
organic phase was dried using MgSO[4] and filtered, and the remaining
solvent was removed by rotary evaporation. The product was further
purified by repeated precipitation steps in hexane to yield the
Boc-protected monomer as a fine white powder, which was dried in vacuo.
Synthesis of hydrophobic monomers
A standard procedure, as previously reported^[326]27,[327]87 was
employed for the synthesis of four hydrophobic monomers
((N-pentylacrylamide, N-heptylacrylamide, N-cyclohexanemethyl)acrylamid
e, and N-benzylacrylamide) from their corresponding amines
(N-amylamine, N-heptylamine, N-cyclohexanemethylamine, or
N-benzylamine) using acryloyl chloride: The specified amount of amine
was dissolved in THF with a ratio of 6 mL of THF per 1 mmol amine. TEA
(1.2 equiv) and acryloyl chloride (1.2 equiv) were added in a dropwise
manner at 0 °C with N[2] bubbling and further stirred overnight at room
temperature. The by-products were filtered, and the solvent was removed
by rotary evaporation. The crude product was dissolved in chloroform
(1.5× THF volume), washed sequentially with 0.1 M HCl, saturated
NaHCO[3], brine, and water using half of the chloroform volume for each
wash. The organic phase was dried with MgSO4 and basic Al2O3 and
filtered to remove solids. Finally, the solvent was removed by rotary
evaporation and dried in vacuo to yield the acrylamide monomer.
Random copolymerisation by photo-induced electron/energy transfer-reversible
addition-fragmentation chain transfer (PET-RAFT) polymerisation
The linear, random copolymers were synthesised using a slight
modification of the general one-pot protocol reported
previously^[328]88 Briefly, stock solutions of the monomers were
prepared with a concentration of 33% (w/w) in DMSO. ZnTPP was dissolved
in DMSO at a concentration of 1 mg/mL. The RAFT agent BTPA was added to
a 4 mL glass vial in an amount corresponding to the targeted X[n] of 20
and dissolved in DMSO. Monomer stock solutions were added into the vial
to a final monomer concentration of 25% (w/w) in DMSO, corresponding to
the targeted ratios. The photocatalyst (ZnTPP) was added at 100 ppm
relative to the monomers. The vial was sealed with a rubber septum, and
the headspace was degassed with N[2] for 10 min. The vial was then
placed under a green light-emitting diode light (λ = 530 nm) for 20 h
to produce the Boc-protected copolymers. The copolymers were analysed
by size-exclusion chromatography (SEC) and ^1H nuclear magnetic
resonance (NMR) to examine the monomer conversion, polymer composition,
and molecular weight distribution. Then, the polymer was purified by
precipitating in a diethyl ether/hexane mixture (3:7), followed by
centrifugation (5000 × g for 5 min, 0 °C). The precipitate was
dissolved in acetone or methanol and reprecipitated twice more. The
polymer was then dried in vacuo prior to Boc-group removal.
Polymer deprotection
TFA was used to remove Boc-protecting groups based on our group’s
previously reported protocol^[329]88 Briefly, the polymer was dissolved
in DCM (∼7% (w/w) polymer), followed by the addition of TFA (20 mol
equivalent with respect to Boc groups). The mixture was stirred at room
temperature for 3 h and precipitated into diethyl ether. The
precipitate was isolated by centrifugation, dissolved in acetone, and
reprecipitated twice more. The polymer was then dried in vacuo,
and ^1H NMR analysis was used to determine the removal of
Boc-protective groups and to examine the targeted X[n].
Polymer characterisation
^1H NMR spectra were obtained using a Bruker AVANCE III spectrometer
(300 MHz, 5 mm BBFO probe) or a Bruker AVANCE III 400 spectrometer
(400 MHz, 5 mm BBFO probe). Deuterated DMSO was used as a solvent to
determine the polymer composition and conversion at concentrations of
∼10–20 mg/mL. All experiments were run with a gas flow across the
probes at 535 L/h with sample spinning and at a temperature of 25 °C.
All chemical shifts were stated in parts per million (ppm) relative to
tetramethylsilane.
SEC analysis was performed using a Shimadzu liquid chromatography
system equipped with a Shimadzu refractive index detector and three MIX
C columns operating at 50 °C. DMAc (containing 0.03% (w/v) LiBr and
0.05% (w/v) 2,6-dibutyl-4-methylphenol) was used as the eluent at a
flow rate of 1 mL/min. The system was calibrated using narrow
poly(methyl methacrylate) (PMMA) standards with molecular weights from
200 to 10^6 g/mol.
Media and buffers for biological experiments
Phosphate-buffered saline (pH 7.4)
A 10× phosphate-buffered saline (PBS) stock (1.37 mol/L sodium
chloride, 0.027 mol/L potassium chloride, 0.08 mol/L disodium hydrogen
phosphate, 0.02 mol/L potassium dihydrogen phosphate) was prepared by
dissolving 80.06 g sodium chloride, 2.01 g potassium chloride, 11.36 g
disodium hydrogen phosphate, and 2.72 g potassium dihydrogen phosphate
in 900 mL of double-distilled water and the pH was adjusted to 7.4,
before adding double-distilled water up to a final volume of 1 L. The
solution was autoclaved for sterilisation. A 1× PBS solution was
obtained by dissolving 100 mL of 10× PBS in 900 mL of sterile
double-distilled water.
Yeast extract peptone dextrose medium
The yeast extract peptone dextrose (YEPD; 1% (w/v) yeast extract, 2%
(w/v) mycological peptone, and 2% (w/v) d-glucose) broth was prepared
by dissolving 4 g yeast extract and 8 g mycological peptone in
double-distilled water up to a total volume of 360 mL. After
autoclaving, 40 mL of filter-sterilised 20% (w/v) d-glucose was added.
For YEPD plates, 8 g agar was added to the solution before autoclaving.
Synthetic defined medium
Synthetic defined (SD) medium was prepared by dissolving 6.7 g yeast
nitrogen base (YNB) without amino acids, and 0.395 g complete
supplement mixture were dissolved in 900 mL double-distilled water,
adjusted pH to 6.0 with HCl and NaOH, and autoclaved. Afterwards,
100 mL of filter-sterilised 20% (w/v) d-glucose was added. When
required, 5 mL of a filter-sterilised 5 mg/mL uridine solution was
added, too.
Modified Roswell Park Memorial Institute (RPMI)-1640 medium for C. albicans
studies
To 1 L of RPMI-1640 medium (with l-glutamine, without bicarbonate),
18 g d-glucose and 34.53 g 3-(N-morpholino)propane-1-sulfonic acid
(MOPS) were added. Afterwards, the pH was adjusted with HCl and NaOH to
4.0 and filter-sterilised.
Polymer and antifungal stock solutions
Polymer stock solutions were prepared at a concentration of 10 mg/mL
(3.4–3.7 mM, depending on the respective polymer composition) in
sterile distilled water and stored at 4 °C. Before use, the stock
solutions were sonicated for approx. 3 min.
Antifungal drug stocks were prepared at different stock solutions in
sterile distilled water or DMSO, as summarised in Table [330]3.
Table 3.
Concentrations, medium and storage temperature of antifungal stocks
Antifungal compound Stock concentration Medium Storage temperature
mg/mL mM
Amphotericin B (AmpB) 1–2 1.1–2.2 DMSO 4 °C
Calcofluor white 50 5.5 DMSO −20 °C
Caspofungin 1 0.9 Distilled water −20 °C
Cetyltrimethylammonium bromide (CTAB) 10 27.4 Distilled water Room
temperature
Congo red 6 8.6 Distilled water 4 °C
Cycloheximide 5 17.8 Distilled water 4 °C
Dodecyltrimethylammonium bromide (DTAB) 10 32.4 Distilled water Room
temperature
FK506 5 6.2 DMSO −20 °C
Fluconazole 1–2 3.3–6.5 DMSO 4 °C
Geldanamycin 5 8.9 DMSO −20 °C
LL-37 1 0.2 Distilled water −20 °C
Nikkomycin Z 5 10.1 Distilled water 4 °C
Sodium dodecyl sulfate (SDS) 10 34.7 Distilled water Room temperature
Tunicamycin 5 5.9 DMSO 4 °C
[331]Open in a new tab
Culture conditions of fungal strains
The yeasts were routinely streaked on YEPD agar and incubated for
1–2 days at 30 °C (37 °C for clinical isolates). Cultures on agar
plates were stored for up to two weeks at 4 °C. Long-term stocks were
stored at −80 °C in 50% (v/v) sterile glycerol from an overnight
culture. Overnight cultures were prepared by inoculating colonies from
a YEPD plate in YEPD broth and shaking overnight at 30 °C at 180 rpm
(37 °C for clinical isolates).
Fungal strains
A complete list of used fungal strains is attached in Supplementary
Data [332]S3. For most assays, the C. albicans reference strain SC5314
was used, unless otherwise indicated.
Minimum inhibitory concentration (MIC) assay and evaluation of drug
interactions
The MICs of polymers against different strains of C. albicans were
determined via the broth microdilution method according to Clinical and
Laboratory Standards Institute (CLSI) guidelines for fungal
susceptibility testing, with slight modifications^[333]32,[334]33.
Briefly, the C. albicans strains were grown on YEPD plates for 48 h at
30 °C (37 °C for clinical isolates). One colony was emulsified in 1 mL
of sterile Milli-Q water. Cells were counted using a haemocytometer and
adjusted to 2–5 × 10^6 cells/mL. The cell suspension was diluted 1:1000
in the modified RPMI-1640 medium (supplemented with d-glucose and MOPS,
pH 4.0) to obtain the 2× concentrated stock suspension. A two-fold
dilution series of the 100 μL polymer solution was added into 96-well
microplates (final concentration between 4 and 512 μg/mL), followed by
the addition of 100 μL of fungal cell suspension. The 96-well plates
were incubated for 24 h at 35 °C in a humidified chamber, wells were
resuspended, and the absorbance was measured at 405 nm with a
microtiter plate reader. Additionally, AmpB, fluconazole, and
caspofungin were tested at final concentrations between 0.125 and 16
(AmpB) and 0.06–8 μg/mL (fluconazole and caspofungin). DMSO controls at
the respectively used final concentrations, no-polymer and no-cell
controls were included in all experiments. The MIC value was defined as
the lowest concentration of the respective polymer that showed growth
inhibition of >90% compared to the untreated control. Three independent
biological replicates were carried out (unless otherwise indicated).
To evaluate interactions of the polymer LH with selected antifungal
compounds (see Table [335]3), C. albicans cells were treated and
prepared as described above. The drug-dilution plates were prepared
separately for each drug before combining them. For that, a four-fold
dilution series of the respective antifungal drug was added into a
96-well microplate along the rows. The same was performed with polymer
LH, diluting it in a 96-well microplate along the columns. Then, 50 µL
of antifungal were combined with 50 µL of polymer LH, resulting in a
two-fold dilution series of the respective compounds. Thereby, one row
and one column acted as controls only containing one compound. Drug
interactions were classified according to their fractional inhibitory
concentration (FIC) indices. The FIC index was calculated as described
in the following Eq. ([336]1), where c[A/B] are the concentrations of
compounds A or B, respectively, in combination resulting in growth
inhibition >90%, and MIC[A] and MIC[B] are the MICs of compound A or B,
respectively, alone:
[MATH: FIC
index=cAMICA+cBMICB :MATH]
1
A combination was called synergistic if the FIC index was below 0.5,
antagonistic for an FIC index above 4 and values in between as additive
(between 0.5 and 1.0) or indifferent (between 1 and 4). Three
independent biological replicates were carried out.
The MICs of polymer LH against C. albicans SC5314 in all used media are
displayed in Supplementary Table [337]S6.
RNA isolation, microarray and KEGG pathway/GO term enrichment analysis
For RNA isolation, C. albicans SC5314 was grown in YEPD broth
overnight, diluted 1:50 in YEPD broth and subcultured for 4 h (30 °C,
180 rpm). The cells were washed three times in PBS (2500 × g, 1 min)
and adjusted to approx. 5 × 10^7 cells/mL in SD broth. The cell
suspension was added to a final concentration of approx.
5 × 10^6 cells/mL into sterile glass flasks containing 6 mL SD medium
supplemented with no polymer (for the untreated control) or 32 (LH,
LL-37), 64 (CB and CX), or 128 µg/mL (LP and poly-HEA) polymer,
corresponding to their MICs (Table [338]2) and being sub-inhibitory at
the conditions of this assay (Supplementary Table [339]S6). The flasks
were incubated for 1 h at 30 °C and 180 rpm. Three biological
replicates were performed for each condition. Cell viability was
ascertained by backplating on YEPD agar. Fungal cells were harvested
for subsequent RNA isolation (2500 × g, 2 min, 4 °C) and handled on ice
from that step onwards. RNA was then isolated using an RNeasy mini kit
(QIAGEN) by mechanical disruption with acid-washed glass beads
following instructions in the manual. The concentration and quality of
RNA were checked by Nanodrop ND-1000 (ThermoScientific) and Bioanalyzer
2100 (Agilent). The Quick Amp Gene Expression Labeling Kit (Agilent)
was used to synthesise Cy5-labelled cRNA. A common reference (RNA from
a mid-log-phase-grown C. albicans SC5314^[340]89) was labelled with Cy3
following the same procedure. The dye incorporation was assured by
spectrophotometric measurement using a NanoDrop ND-1000. Samples and
common references were cohybridised on Agilent arrays (AMADID 026869)