Article The escape of Candida albicans from macrophages is enabled by the fungal toxin candidalysin and two host cell death pathways
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Article
The escape of Candida albicans from macrophages
is enabled by the fungal toxin candidalysin and two
host cell death pathways
Graphical abstract Authors
Françios A.B. Olivier, Volker Hilsenstein,
Harshini Weerasinghe, ..., James E. Vince,
Michael J. Hickey, Ana Traven
Correspondence
ana.traven@monash.edu
In brief
The pathogen Candida albicans uses
filamentous hyphae to escape from
immune phagocytes. By developing an
imaging assay to quantify hyphal escape,
Olivier et al. implicate three mechanisms:
Candida makes a pore-forming toxin that
permeabilizes macrophage membranes
and triggers two host lytic pathways that
further facilitate its escape.
Highlights
d A live-cell imaging assay dynamically quantifies Candida
escape from macrophages
d Candida escapes from macrophages by using multiple
mechanisms
d Candidalysin-dependent host membrane damage enables
Candida escape
d Host responses to infection, pyroptosis, and ETosis result in
Candida escape
Olivier et al., 2022, Cell Reports 40, 111374
September 20, 2022 ª 2022 The Author(s).
https://doi.org/10.1016/j.celrep.2022.111374 ll
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Article
The escape of Candida albicans from macrophages
is enabled by the fungal toxin candidalysin
and two host cell death pathways
Françios A.B. Olivier,1,2 Volker Hilsenstein,3,8 Harshini Weerasinghe,1,2 Ashley Weir,4,5 Sebastian Hughes,4,5
Simon Crawford,6 James E. Vince,4,5 Michael J. Hickey,7 and Ana Traven1,2,9,*
1Infection Program, Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton 3800
VIC, Australia
2Centre to Impact AMR, Monash University, Clayton, VIC 3800, Australia
3Monash MicroImaging, Monash University, Clayton, VIC 3800, Australia
4The Walter and Eliza Hall Institute of Medical Research, University of Melbourne, Parkville, VIC 3052, Australia
5The Department of Medical Biology, University of Melbourne, Parkville, VIC 3010, Australia
6Monash Ramaciotti Centre for Cryo-Electron Microscopy, Monash University, Clayton, VIC 3800, Australia
7Monash Centre for Inflammatory Diseases, Monash University Department of Medicine, Monash Medical Centre, Clayton, VIC 3168,
Australia
8Present address: EMBL Heidelberg, Meyerhofsrt 1, Heidelberg, Germany
9Lead contact
*Correspondence: ana.traven@monash.edu
https://doi.org/10.1016/j.celrep.2022.111374
SUMMARY
The egress of Candida hyphae from macrophages facilitates immune evasion, but it also alerts macrophages
to infection and triggers inflammation. To better define the mechanisms, here we develop an imaging assay to
directly and dynamically quantify hyphal escape and correlate it to macrophage responses. The assay re-
veals that Candida escapes by using two pore-forming proteins to permeabilize macrophage membranes:
the fungal toxin candidalysin and Nlrp3 inflammasome-activated Gasdermin D. Candidalysin plays a major
role in escape, with Nlrp3 and Gasdermin D-dependent and -independent contributions. The inactivation
of Nlrp3 does not reduce hyphal escape, and we identify ETosis via macrophage extracellular trap formation
as an additional pathway facilitating hyphal escape. Suppressing hyphal escape does not reduce fungal
loads, but it does reduce inflammatory activation. Our findings explain how Candida escapes from macro-
phages by using three strategies: permeabilizing macrophage membranes via candidalysin and engaging
two host cell death pathways, Gasdermin D-mediated pyroptosis and ETosis.
INTRODUCTION 2016; Lo et al., 1997), followed by rapid intra-phagosomal
growth of the hyphal filaments (Westman et al., 2020). Attempt-
Macrophages are important for host defenses against infec- ing to contain the growing fungal hyphae, macrophages fold
tion. They phagocytose pathogens for containment and elim- them and expand the phagosomal membrane by fusing with ly-
ination and also produce antimicrobial cytokines to amplify sosomes (Bain et al., 2021; Westman et al., 2020). Despite this,
the immune response. To counteract macrophages, microbial hyphae breach the phagosomal membrane and eventually
pathogens have evolved mechanisms to survive and even egress from macrophages concomitant with macrophage lysis
thrive inside them (Price and Vance, 2014). Furthermore, (Lo et al., 1997; Westman et al., 2018, 2020). Hyphal escape
some pathogens escape from macrophages to spread the may facilitate immune evasion and pathogen dissemination,
infection (Traven and Naderer, 2014). One of the most striking but it comes with a cost because it activates antifungal inflam-
examples of macrophage escape is that by the fungal path- mation (Joly et al., 2009; Uwamahoro et al., 2014; Wellington
ogen Candida albicans. C. albicans is a common cause of et al., 2012, 2014; Westman et al., 2020). Inflammation is crucial
mucosal infections (Denning et al., 2018) and life-threatening for antifungal defenses (Gross et al., 2009; Hise et al., 2009; van
candidiasis in immunosuppressed patients (Pappas et al., de Veerdonk et al., 2011), but heightened inflammation can
2018). cause collateral damage (Ardizzoni et al., 2021; Borghi et al.,
Macrophages are critical for controlling C. albicans (Erwig 2015; Bruno et al., 2015; Roselletti et al., 2017). Hyphal escape
and Gow, 2016; Lionakis et al., 2013). In vitro studies show therefore presents a promising therapeutic avenue through
that, once inside macrophage phagosomes, C. albicans which immune responses and pathogen clearance could be
switches from yeast to hyphal morphology (Erwig and Gow, balanced.
Cell Reports 40, 111374, September 20, 2022 ª 2022 The Author(s). 1
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
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2 Cell Reports 40, 111374, September 20, 2022
ll Article OPEN ACCESS Before we can do that, we need to define the underlying mech- RESULTS anism(s). So far, the clearest requirement for rapid escape is growth as hyphae (Lo et al., 1997; McKenzie et al., 2010; Tucey A live-cell imaging assay to measure the escape of et al., 2020; Uwamahoro et al., 2014). Phagosomal rupture C. albicans from macrophages caused by hyphal growth activates the host immune sensor The escape of C. albicans hyphae from macrophages is Nlrp3-caspase-1 inflammasome and leads to a lytic form of commonly inferred from data that measure host processes, spe- macrophage death called pyroptosis, which can release patho- cifically macrophage membrane permeabilization and cell lysis. gens (Uwamahoro et al., 2014; Wellington et al., 2014; Westman However, these host pathways may not be strictly correlated et al., 2020). The above-mentioned link between C. albicans to hyphal escape. For example, non-lytic escape has also escape and inflammation comes from the fact that pyroptosis been shown, albeit as a rare event (Bain et al., 2012). To gain a is highly inflammatory. Pyroptosis is triggered by proteolysis of deeper understanding of the mechanisms of C. albicans escape, the caspase-1 substrate Gasdermin D into the N-terminal we developed a live-cell imaging assay that directly and dynam- pore-forming domain (Banoth et al., 2020; He et al., 2015; Shi ically quantifies escaped hyphae in macrophage populations et al., 2015), followed by pore formation in the plasma mem- with high temporal resolution. In parallel, the assay quantifies brane, cellular death, and lysis (DiPeso et al., 2017; He et al., the permeabilization of macrophage membranes. For this, we 2015; Shi et al., 2015). Caspase-1 also cleaves pro-interleukin used murine bone marrow-derived macrophages (BMDMs) (IL)-1b into the mature inflammatory cytokine IL-1b, which is and a previously described C. albicans strain constitutively ex- released through Gasdermin D pores to mediate inflammation pressing the fluorophore dTomato (Lionakis et al., 2013). This (Evavold et al., 2018; He et al., 2015; Heilig et al., 2018; Shi fluorescent strain allowed for visualization and quantification of et al., 2015). It should be noted that C. albicans mutants that phagocytosed and total (phagocytosed + escaped) cannot make hyphae but activate pyroptosis have also been re- C. albicans, as well as normalization of the hyphal escape data ported (O’Meara et al., 2015). The same authors also showed (see below). that inflammasome activation can occur with C. albicans still in Our imaging assay is based on using two membrane-imper- the phagosome (O’Meara et al., 2018). These studies suggest meable dyes: the fungal cell wall stain calcofluor white (CFW) that, in some instances, C. albicans may escape from macro- and the DNA stain DRAQ7 (Figure 1A). Using dTomato and phages without hyphae. CFW together allowed us to distinguish between phagocytosed C. albicans can also cause macrophage death that is indepen- (dTomato+ but CFW) and escaped C. albicans (dTomato+ and dent of inflammasome-mediated processes. Following the initial CFW+) (Figures 1A and 1B). At the same time, DRAQ7 stains hyphal escape, C. albicans kills macrophages by depleting the nuclei of macrophages after membrane permeabilization glucose (Tucey et al., 2018). Furthermore, the peptide toxin (Figures 1A and 1B). The CFW concentration (10 mg mL1) was candidalysin, which is expressed at very high levels by fungal optimized so that it stains the cell wall after hyphal escape but hyphae (Moyes et al., 2016), triggers macrophage lysis does not block the growth of C. albicans hyphae (Figure S1A; (Kasper et al., 2018). Candidalysin can activate the NLRP3 in- Video S1). flammasome and IL-1b secretion (Kasper et al., 2018; Rogiers Macrophages were infected with C. albicans (yeast et al., 2019), but candidalysin-mediated macrophage lysis morphology cells) and live-cell imaging data acquired every is independent of caspase-1, suggesting that it is not py- 30 min. At 2 h post-infection, the dTomato signal of phagocy- roptosis (Kasper et al., 2018). The physical forces of the growing tosed C. albicans cells was clear, but only a minority of hyphae hyphae can further disrupt macrophage membranes (Westman was stained by CFW (Figure 1B). This shows that the majority et al., 2018, 2020). The involvement of the various macro- of C. albicans cells are still internalized in macrophages. We phage cell death mechanisms in C. albicans escape, and the dy- observed a small number of CFW-stained hyphae at 2 h post- namic aspects of their contributions remain to be precisely infection that were mostly not associated with the nuclei of per- understood. meabilized macrophages (Figures 1B; Video S1, part 1). These To provide insights into these pathways, we developed a live- could represent rare, non-lytic escape events (Bain et al., cell imaging platform that dynamically quantifies hyphal escape 2012). Alternatively, these could be remnant non-phagocytosed from macrophages. With it, we show how C. albicans uses C. albicans cells. However, microscopy during experiments several escape mechanisms enabled by candidalysin, Gasder- showed that phagocytosis was essentially complete and, min-mediated pyroptosis and a cell lysis pathway associated further, any non-phagocytosed cells were washed out before im- with macrophage extracellular trap formation termed ETosis. aging (see Method details). In some cases, DRAQ7+ macro- Our study defines the strategies used by C. albicans to escape phage nuclei were not associated with CFW-stained hyphae. immune containment and the functional relationships between This could represent low-level macrophage cell death unrelated them. to infection (generally
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By 4 h, the number of new CFW-stained hyphae increased and By measuring increases in CFW-stained hyphal area over
then by 6 h they further increased, both in number and length time, the CAF2.1 dTomato isolate was most successful in
(Figure 1B). The appearance of new CFW-stained hyphae coin- escaping from macrophages, followed by P76067, P75010,
cided with the appearance of DRAQ7-stained nuclei of proximal and then efg1D/D cph1D/D (Figure 2A). CAF2.1, followed closely
macrophages (Figures 1B; Video S1). Further imaging of these by P76067, was also most successful in inducing macrophage
events with 2-min intervals showed that 8 of 20 DRAQ7+ nuclei permeabilization as measured by DRAQ7 staining (Figure 2A;
that we counted appeared in the same frame as the CFW+ hy- see also images in Figure 2B). A plateau in the hyphal escape
phae, 10 of 20 were narrowly preceded by the CFW+ hyphae, area was observed after 8–10 h post-infection (Figure 2A), which
and 2 of 20 did not associate with CFW+ hyphae (Video S1). In coincided with hyphal overgrowth (Figure 2B). Therefore, under
this analysis, we did not observe any DRAQ7+ nuclei preceding our experimental conditions and for highly hyphal strains such
CFW+ hyphae. Collectively, our findings are consistent with hy- as CAF2.1, escape is best judged within 8–10 h of macrophage
phal egress occurring in conjunction with the permeabilization infection, for linear escape values. For these experiments, a mul-
of macrophage membranes. tiplicity of infection (MOI) of 3 Candida to 1 macrophage was
We next developed an image analysis pipeline to measure (1) used. Hyphal escape also depends on the MOI, as lower MOI re-
the total area of phagocytosed C. albicans (dTomato), (2) the to- sulted in slower hyphal escape, while a higher MOI resulted in
tal area of escaped C. albicans hyphae (CFW), and (3) the num- faster escape of the CAF2.1 dTomato isolate (Figure S2A). Linear
ber of membrane-permeabilized macrophages (DRAQ7). Un- escape values will therefore depend on the MOI and the extent of
even image illumination was corrected for, and thresholding hyphal formation by the strains under investigation.
was used to generate binary images (Figure S1B). A link to a tem- Few extracellular fungi were observed in the CFW view of
plate imaging analysis project file is detailed in the STAR P75010 and efg1D/D cph1D/D at 4 and 6 h post-infection (Fig-
Methods. Using output area values, we plotted the total area of ure 2B). The CFW signal increased beyond 8 h for these isolates
escaped C. albicans hyphae at each time point (Figure 2A). but was not correlated with increased membrane permeabiliza-
The percentage of DRAQ7+ macrophages was calculated as tion in macrophage populations (Figure 2A). This shows that
described previously (Tucey et al., 2020). We called the collec- even in the absence of hyphal morphogenesis, C. albicans can
tive of the staining protocol and imaging pipeline the ‘‘hyphal egress from macrophages. However, this occurs much more
escape assay.’’ slowly, and these escape events are rare (i.e., they occur in a small
To validate the assay, we performed infections with four proportion of macrophages) (Figure S2B). Further analyses of the
C. albicans strains known to vary in their ability to form hyphae imaging video showed that with P75010, but not efg1D/D cph1-
and kill macrophages. The CAF2.1. dTomato strain served as a D/D, these rare escape events are correlated with macrophage
positive control since this strain is derived from the highly hyphal membrane permeabilization (Figure S2C). The increase in the
clinical isolate SC5314 known to rapidly egress from macro- CFW signal that is seen after 10 h is likely due to the extracellular
phages (Tucey et al., 2020). C. albicans with deleted CPH1 and growth of C. albicans originating from these rare escape events.
EFG1 genes (efg1D/D cph1D/D) does not form hyphae (Warten- The fluorophore dTomato allows for monitoring of phagocy-
berg et al., 2014) and was used as a control for poor escape. We tosed C. albicans (Figure 1B). At the early time point after infec-
included two clinical isolates P67067 and P75010, which form tion (2.5 h), C. albicans is entrapped in macrophages. Therefore,
poor hyphae in macrophages and were respectively graded the dTomato signal at that time point can be used to normalize
0.5 and 0.1 out of 5 for macrophage hyphal index (Tucey et al., CFW fluorescence to obtain phagocytosis/hyphal escape ratios
2020). While the growth rates of these strains in macrophages (Figure 2C). This normalization step helps to account for any dif-
are not known, strain P67067 showed slower doubling time ferences in Candida:macrophage infection ratios, phagocytosis
in vitro relative to SC5314, but intriguingly was able to form sub- rates, or macrophage seeding densities.
stantial hyphae in vitro, although it was very poor for hyphal
formation in macrophages (Tucey et al., 2020). Strain P75010 Loss of Gasdermin D, but not Nlrp3, reduces hyphal
had only marginally slower doubling time in vitro relative to escape from macrophages
SC5314, but a major hyphal defect in vitro and in macrophages We next applied our assay to investigate the contribution of
(Tucey et al., 2020). Nlrp3 inflammasome-mediated pyroptosis to hyphal escape.
Figure 2. The hyphal escape assay detects differences in the escape of C. albicans depending on hyphal morphogenesis
(A) Area of escaped hyphae and % DRAQ7+ nuclei measured for macrophages challenged with C. albicans CAF2.1 (a lab derivative of the highly hyphal clinical
isolate SC5314); efg1D/D cph1D/D, which cannot make hyphae; and 2 clinical isolates, which were previously shown to display reduced hyphal formation in
macrophages, P76067 and P75010 (Tucey et al., 2020). MOI = 3. Shown are the averages from 4 independent biological repeats (macrophages isolated from 4
different mice), with 3 technical repeats performed for each. Error bars represent the standard error of the mean. Bright-field images of infected macrophages,
including the rare escape events of poorly hyphal strains, are shown in Figure S2B.
(B) CFW viewfor infections detailed in (A). Little hyphal escape is seen for P75010 and efg1D/D cph1D/D. Saturation of CFW-stained hyphae is evident for CAF2.1,
reflected in the plateau in hyphal escape values seen in (A). Scale bar, 100 mM.
(C) Left panel: Escaped hyphae area of the C. albicans CAF2.1 infection described in (A). Data are from 4 independent biological replicates (macrophages isolated
from 4 different mice), with 3 technical repeats for each. Left panel: Raw mm2 values of escaped hyphae plotted as spaghetti plots. Right panel: the same mm2
values of escaped hyphae but normalized to the total area of dTomato+ C. albicans at the first time point (2.5 h post-phagocytosis). This normalization gives the
escape-phagocytosis ratio.
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Figure 3. Loss of Gasdermin D, but not Nlrp3 reduces hyphal escape from macrophages
Hyphal escape and macrophage membrane permeabilization (DRAQ7 staining) were measured following infection with the dTomato C. albicans strain.
Escape-phagocytosis ratio = area of CFW-stained C. albicans hyphae divided by total dTomato signal at 2.5 h post-phagocytosis.
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Nlrp3 inflammasome activation is dependent on fungal load, with and we observed significant hyphal escape from them
less pyroptosis observed at lower MOIs (Tucey et al., 2020; (Figures S4A and S4B; Video S2). Surprisingly, in iBMDMs, hy-
Westman et al., 2020). We therefore performed infections with phal escape did not coincide with macrophage membrane per-
MOIs of 6:1 and 3:1 (Candida to macrophages). Based on our meabilization (DRAQ7 staining) or cellular lysis (LDH release),
previous studies, these MOIs should lead to pyroptosis (Tucey as both of these measures of macrophage damage by
et al., 2020; Uwamahoro et al., 2014). Hyphal escape was C. albicans remained at the level of uninfected controls
measured for the first 7.5 h and normalized to phagocytosed (Figures S4C and S4D). Nevertheless, deletion Gasdermin D
C. albicans as in Figure 2C, using macrophages deficient for reduced hyphal escape from iBMDMs (Figures S4A and S4B).
Nlrp3 (Nlrp3/) or Gasdermin D (Gsdmd/), and for macro-
phages treated with the Nlrp3 inhibitor MCC950 (Coll et al., Macrophage extracellular trap formation provides an
2015). escape route for C. albicans
Following C. albicans infection, Nlrp3/ macrophages dis- The Nlrp3 inflammasome regulates Gasdermin D processing in
played decreased membrane permeabilization (DRAQ7 staining) response to C. albicans (Figure 3E) (Banoth et al., 2020). There-
(Figure 3A) compared to wild-type (WT) macrophages, as well fore, it was surprising that inactivation of Nlrp3 did not reduce hy-
as lower cell lysis as measured by lactate dehydrogenase phal escape. Deeper analysis of our imaging data uncovered that,
(LDH) release (Figure S3A). This was expected, as we and in addition to pyroptosis (condensed DRAQ7-stained nuclei),
others have previously shown that the inhibition of Nlrp3 C. albicans-infected macrophages were undergoing another
results in lower membrane permeabilization and cell death of form of cell death with large, more diffuse DRAQ7-stained struc-
C. albicans-infected macrophages (Tucey et al., 2016; tures (Figure 4A). These structures became more spread out as
Wellington et al., 2014). Surprisingly, no hyphal escape differ- escaped C. albicans hyphae grew (Video S3) and resembled
ence was seen between WT and Nlrp3/ macrophages (Fig- macrophage extracellular traps (METs) that have been reported
ure 3B). Similarly, treatment with the Nlrp3 inhibitor MCC950 re- to entangle and kill C. albicans hyphae (Halder et al., 2016; Liu
sulted in lower permeabilization of C. albicans-infected et al., 2014; Loureiro et al., 2019). More extensively characterized
macrophages, but hyphal escape was not affected (Figure S3C). in neutrophils, macrophages are also capable of expelling these
In contrast, a decrease in both macrophage membrane perme- networks of chromatin, embedded with histones and granule anti-
abilization and hyphal escape was observed for Gsdmd/ mac- microbial proteins (Boe et al., 2015; Doster et al., 2018). We note
rophages (Figures 3C, 3D, and S3D). Processing of Gasdermin D that the diffuse MET-like structures fade as imaging progresses,
to its N-terminal pore-forming fragment was not detected in and these structures are not always visible at later time points.
Nlrp3/ macrophages (Figure 3E). Although Gsdmd/ macro- In addition, the irregular morphologies of MET-like structures are
phages showed reduced hyphal escape, there was no reduction usually excluded by cell-counting parameters, which are selective
in the dTomato signal (Figure 3F). These data suggest that trap- of regular, dense nuclei.
ping C. albicans inside macrophages by inactivation of Gasder- Using scanning electron microscopy (SEM), we confirmed that
min D does not reduce fungal viability. a small proportion of macrophages form METs in response to
To further confirm the requirement for Gasdermin D in hyphal C. albicans (Figure 4B and quantification in Figures 4C and
escape, we utilized immortalized bone marrow-derived macro- 4D). METs were not observed in SEM imaging of uninfected
phages (iBMDMs) where GSDMD was deleted by CRISPR- macrophages (Figure 4B). In some images, escaped hyphae
Cas9 targeting. iBMDMs phagocytosed C. albicans efficiently were ensnared in large networks of extracellular fibers, sharing
(A) Left panel: Percentage of DRAQ7+ wild-type (WT ) and Nlrp3/ macrophages infected with C. albicans (MOI = 6). Values are averages from infections with 4
independent biological repeats (BMDMs from 4 different mice), with 3 technical repeats each. Error bars represent standard error of the mean. Right panel: Paired
t test comparisons (assuming Gaussian distribution) of macrophage death percentages from 3-, 5-, and 7-h time points. A p value lower than 0.05 was considered
significant. The data for WT macrophages are the same here and in (C), as the Nlrp3/ and Gsdmd/ mutants were assayed together in 4 out of 5 experiments.
(B) Hyphal escape data for experiments in (A). Left panel: Hyphal escape-phagocytosis ratios. Error bars represent standard error of the mean. Right panel: paired
t test comparisons (assuming Gaussian distribution) of the same hyphal escape-phagocytosis ratios from 3-, 5-, and 7-h time points. A p value lower than 0.05
was considered significant. The data for hyphal escape and DRAQ7 staining following inhibition of Nlrp3 with MCC950 are shown in Figure S3C.
(C) Left panel: Percentage of DRAQ7+ macrophages (WT and Gsdmd/) infected with C. albicans (MOI = 6). Values are averages from infections with 5 inde-
pendent biological repeats (BMDMs from 5 different mice), with 3 technical repeats each. For 4 of these animals, experiments were performed in parallel with
those described for the Nlrp3/ macrophages in (A). For these 4 animals, the data for WT macrophages are the same as in (A). Another experiment comparing
Gsdmd/ with WT macrophages from the N C57BL6 mouse strain is displayed in Figure S3D and showed equivalent results. The error bars represent the
standard error of the mean. Right panel: Paired t test comparisons (assuming Gaussian distribution) of macrophage death percentages from 3-, 5-, and 7-h time
points. A p value lower than 0.05 was considered significant.
(D) Hyphal escape data for experiments in (C). Left panel: Hyphal escape-phagocytosis ratios. Error bars represent standard error of the mean. Right panel: Paired
t test comparisons (assuming Gaussian distribution) of hyphal escape-phagocytosis ratios from 3-, 5-, and 7-h time points. A p value lower than 0.05 was
considered significant.
(E) Western blot of Gasdermin D cleavage in WT and Nlrp3/ macrophages (BMDMs) challenged with C. albicans (MOI = 6), 3 h post-infection. Full length and
cleaved Gasdermin D are indicated. Nigericin treatment was used as a positive control, showing the expected cleavage of Gasdermin D. Gsdmd/ macro-
phages are shown as the negative control.
(F) The total area of hyphae, both phagocytosed and escaped, quantified for the experiments described in (C) and (D). Top panel: Total hyphae areas plotted over
time. Error bars represent standard error of the mean. Bottom panel: Paired t test comparisons (assuming Gaussian distribution) of the same total hyphae areas
quantified from 2.5 to 5 h post-infection. A p value lower than 0.05 was considered significant.
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a beaded architecture previously reported for METs formed in events were not scored if escaped hyphae were already present
response to C. albicans (Figure 4B) (Halder et al., 2016). This in the position where a MET-like structure originates. In this way,
beaded morphology is thought to represent histones and gran- we only quantified METs that form in response to phagocytosed
ular antimicrobial proteins along chromatin fibers (Brinkmann C. albicans and not extracellular hyphae. Newly formed MET-like
et al., 2004; Doster et al., 2018). MET-like structures were rarely structures associated with newly escaped hyphae in 87% of
observed in infections with the yeast-locked strain efg1D/D the macrophages counted (Table S1). These data suggest that
cph1D/D or the poorly hyphal clinical isolate P75010 (Figure S5). MET formation by a cell death pathway called ETosis (Doster
MET-like structures were observed in response to the clinical et al., 2018) is an additional escape route for C. albicans hyphae.
isolate P76067, in line with its more efficient escape (Figure S5). Escape via METs could explain, at least in part, why hyphal
Given these observations, we developed a set of criteria to escape is not reduced for Nlrp3/ macrophages.
quantify MET-like structures formed by WT, Gsdmd/, Nlrp3/,
and MCC950-treated macrophages in response to C. albicans Candidalysin ensures the rapid escape of C. albicans
(see Method details and Figure 4A for examples). Although there from macrophages, which modulates IL-1b secretion
was a decrease in DRAQ7 staining in Gsdmd/, Nlrp3/ and but does not affect fungal loads
MCC950-treated macrophages (Figures 3 and S3), the number In addition to the inflammasome, the fungal pore-forming toxin
of MET-like structures did not decrease (Figures 4C and 4D). candidalysin damages C. albicans-infected macrophages (Kas-
The proportion of MET-like structures relative to DRAQ7+ macro- per et al., 2018). To test the involvement of candidalysin in hyphal
phages (a sum of condensed and diffused MET-like DRAQ7 escape, we challenged macrophages with C. albicans lacking
staining) increased significantly for Nlrp3/ and MCC950- the candidalysin-encoding ECE1 gene (ece1D/D). Control
treated macrophages, and there was a trend toward an increase strains were its parental WT strain and the re-integrant ece1D/D +
in Gsdmd/, although it did not reach significance at p < 0.05 ECE1. The dTomato fluorophore was introduced into these
(Figures 4C and 4D). Moreover, Nlrp3/ macrophages also dis- strains to normalize hyphal escape to phagocytosed C. albicans.
played an increased formation of MET-like structures in total At 4 and 6 h post-infection, a comparable intracellular hyphal
macrophage populations (Figure 4C). Given the reduction of phenotype was observed for WT and ece1D/D C. albicans (Fig-
DRAQ7 staining and LDH release in Nlrp3/ macrophages (Fig- ure 5A, dTomato view). This is expected, as the candidalysin
ures 3 and S3A), we were surprised that MET-like structures mutant is known to form hyphae of normal morphology, including
were increased in these cells. However, previous work on in the macrophage phagosome (Kasper et al., 2018; Moyes et al.,
C. albicans-induced METs reported no accompanying increase 2016; Westman et al., 2018). Despite successful hyphal forma-
in LDH release (Liu et al., 2014). Also, as explained above, our tion inside the phagosome, hyphal escape for ece1D/D was
standard DRAQ7-staining counts condensed nuclei and not significantly slower (Figures 5A and 5B). DRAQ7 staining of mac-
MET formation, the former being reduced in Nlrp3/ macro- rophages challenged with ece1D/D was also reduced (Fig-
phages (Figure 3). ure 5C), as was LDH release (Figure S3A). These findings indi-
Next, we addressed the association of MET-like structures cate that ECE1 is required for effective escape, in conjunction
with the appearance of escaping hyphae. MET-like structures with efficient membrane permeabilization and macrophage cell
formed by Nlrp3/ macrophages were identified using an imag- lysis. In three out of four experiments, the re-integrant ece1D/D +
ing time-lapse of DRAQ7 staining. A corresponding time-lapse of ECE1 showed partial or full complementation relative to the
escaping CFW+ hyphae was used to determine whether a hyphal WT strain, while in one it behaved like the mutant (Figure S6A).
escape event corresponded with the initial appearance of a When consolidated, these results suggest that the candidalysin
macrophage nucleus that disperses into a MET-like structure re-integrant ece1D/D + ECE1 showed intermediate hyphal
within 30 min (see Method details). MET/CFW co-localization escape and macrophage permeabilization phenotypes. This
Figure 4. Macrophage extracellular trap formation in response to C. albicans
(A) Images of macrophage extracellular trap (MET)-like structures in WT and Nlrp3/ macrophages infected with C. albicans. Boxes indicate the portion within
the image that was magnified, and the magnified part of the image is shown as an inset. The red arrow (left panel: WT macrophages) in the magnified box shows
the condensed morphology of pyroptotic macrophages. The magenta arrow (right panel: Nlrp3/ macrophages) in the magnified box shows the irregular, MET-
like structures. The MOI was 6. The time point after infection is 7 h. Scale bar, 100 mM.
(B) Scanning electron microscopy images of METs formed by Nlrp3/ macrophages in response to C. albicans (MOI = 3), 3 h post-infection. Top panel, magenta
arrow shows a MET-like structure. The structure spans an area between 3 macrophages, with escaping hyphae seen from 2 (top and bottom left, green arrows).
Bottom left panel: uninfected macrophages. Bottom center panel: C. albicans hyphae escaping from a macrophage where no MET formation is seen; the green
arrow indicates an example of an escaping hypha. Bottom right panel: Individual MET fibers (white arrow) have a beaded appearance consistent with globular
protein domains previously described for neutrophil and macrophage extracellular traps. Scale bars, 5 mM.
(C) Quantification of MET-like structures from the experiments described in Figure 3. MET-like structures formed at 7 h post-infection are represented as a fraction
of total seeded macrophages (left panel) or as a fraction of macrophages that are DRAQ7+ (left panel). Six images were used per condition within a biological
repeat (2 for each technical repeat), with at least 500 macrophages estimated to be seeded in each image. Counts are therefore based on at least 12,000 seeded
macrophages per condition (WT, Gsdmd/, and Nlrp3/). Paired t test comparisons (assuming Gaussian distribution) were performed. A p value lower than
0.05 was considered significant. The Gsdmd/ data for % METs out of DRAQ7+ macrophages had a p = 0.0578.
(D) Quantification of MET-like structures from the experiments described in Figure S3 (MCC950 data). The quantification was done as described in (C). Six images
were used per condition within a biological repeat (2 for each technical repeat), with at least 500 macrophages estimated to be seeded in each image. Counts are
therefore based on C. albicans challenge of at least 9,000 seeded macrophages per condition. Paired t test comparisons (assuming Gaussian distribution) of
MET-like percentages were performed. A p value lower than 0.05 was considered significant.
Cell Reports 40, 111374, September 20, 2022 9ll
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10 Cell Reports 40, 111374, September 20, 2022ll
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may reflect expression of only one copy of the ECE1 gene in the brane permeabilization and lysis. As such, C. albicans takes
complemented strain. Macrophages rarely formed MET-like advantage of two pore-forming proteins to egress from macro-
structures in response to the ece1D/D strain, although some phages: fungal candidalysin and host Gasdermin D. These
could be observed (Figure S6B). pore-forming factors are intimately linked to the hyphal cell
By quantifying the dTomato signal, we assessed whether longer type, which explains why hyphal morphogenesis promotes
containment of the ece1D/D mutant inside macrophages reduced escape. Specifically, the candidalysin-encoding gene ECE1 is
fungal growth. There was no reduction in dTomato signal over highly expressed in hyphae but not in C. albicans yeast cells
time for the trapped ece1D/D mutant (Figures 5D and 5E), which (Moyes et al., 2016), and the processing of Gasdermin D to its
is consistent with no reduction when C. albicans was trapped in pore-forming N-terminal fragment requires the Nlrp3-caspase-
Gsdmd/ macrophages (Figure 3F). However, the ece1D/D 1 inflammasome, which is activated following hyphal invasion
mutant induced lower IL-1b secretion from macrophages (Fig- and rupture of the phagosomal membrane (Joly et al., 2009;
ure 6A). IL-1b secretion induced by C. albicans was almost Westman et al., 2020). Our conclusions on the roles of candida-
completely reduced by MCC950, showing it is dependent on lysin and Gasdermin D in C. albicans escape are supported by a
Nlrp3 (Figure 6A). The ece1D/D mutant also induced reduced manuscript published while our study was in preparation (Ding
cleavage of Gasdermin D into its pore-forming fragment (Fig- et al., 2021). Gasdermin D processing to its pore-forming frag-
ure 6B). We observed different levels of reduction across five inde- ment is reduced in macrophages infected with the ece1D/D
pendent biological repeats, from partial reduction to non-detect- mutant (our data, Figure 6B,) and Ding et al., 2021). This result
able cleaved Gasdermin D (Figure S7). Collectively, these results is consistent with previous reports showing that candidalysin ac-
show that trapping C. albicans inside macrophages does not tivates the Nlrp3-caspase-1 inflammasome (Kasper et al., 2018;
cause increased fungal killing within the time frame of this assay. Rogiers et al., 2019), and demonstrates that candidalysin and
Instead, the primary consequence of trapping C. albicans inside Gasdermin D are in the same pathway that facilitates hyphal
macrophages is reduced inflammatory activation as a conse- escape. However, candidalysin also has Gasdermin D- and in-
quence of loss of candidalysin. flammasome-independent roles since ece1D/D displayed
We further addressed the functional relationship between can- reduced escape from Gsdmd/ and Nlrp3/ macrophages.
didalysin and the Nlrp3-inflammasome pathway in the context of As such, candidalysin could be mediating the residual escape
hyphal escape. The ece1D/D mutant displayed lower escape from macrophages lacking Gasdermin D.
and membrane permeabilization than WT C. albicans in both The initial model posited that the physical force of hyphal
Gsdmd/ and Nlrp3/ macrophages (Figures 7A–7D). LDH growth leads to macrophage membrane damage and hyphal
release was also lower (Figures S3A and S3B). In immortalized escape (Lo et al., 1997). We and Wellington et al. challenged
iBMDMs, inactivation of both Gasdermin D and candidalysin this model by showing that hyphae damage macrophages by
produced the greatest effect on lowering hyphal escape (Fig- activating Nlrp3 inflammasome-dependent pyroptotic lysis
ure S4). There was no effect of these mutations on membrane (Uwamahoro et al., 2014; Wellington et al., 2014). Here, we
permeabilization or cell lysis of iBMDMs, which were low used macrophages lacking the ‘‘executioner’’ of pyroptosis,
even in WT infections (Figure S4). Collectively, these results sug- Gasdermin D (Man and Kanneganti, 2015), to demonstrate
gest that, in addition to contributing to Nlrp3-dependent Gasder- directly that pyroptosis enables hyphal escape. It should be
min D processing, candidalysin has Nlrp3 and Gasdermin noted, however, that our results do not exclude contributions
D-independent roles in hyphal escape from macrophages. from hyphal force, as hyphal growth causes rupture of the phag-
osomal membrane, which in turn activates the Nlrp3 inflamma-
DISCUSSION some (Westman et al., 2018, 2020). However, distinction must
be made between hyphal escape into the extracellular space
Our findings establish that the escape of C. albicans hyphae from measured by us here and hyphal force-dependent phagosomal
macrophages is facilitated by a combination of Gasdermin escape characterized by others (Westman et al., 2018, 2020).
D-mediated pyroptosis, ETosis, and candidalysin-driven mem- In fact, we argue that our data with the candidalysin mutant
Figure 5. The requirement for candidalysin in hyphal escape and fungal growth during macrophage infections
Hyphal escape and macrophage membrane permeabilization were measured using WT (BWP17), ece1D/D, and ece1D/D + ECE1 C. albicans. Escape-
phagocytosis ratio = area of CFW-stained C. albicans hyphae divided by total dTomato signal at 2.5 h post-phagocytosis.
(A) dTomato (phagocytosed) and CFW (escaped)-positive C. albicans at 4 and 6 h post-infection (MOI = 3). dTomato images were overlaid with brightfield. Scale
bar, 100 mM.
(B) Hyphal escape-phagocytosis ratios. Values are averages from infections with 4 independent biological repeats (BMDMs from 4 different mice), with 3
technical repeats for 3 animals and 2 technical repeats for 1 animal. Left panel: Entire live-cell imaging time course. Error bars represent standard error of the
mean. Right panel: Paired t test comparisons (assuming Gaussian distribution) of hyphal escape-phagocytosis ratios from 3-, 5-, and 7-h time points. A p value
lower than 0.05 was considered significant. The independent experiments are plotted individually in Figure S6A.
(C) Left panel: Percentages of DRAQ7+ macrophages for the experiments described in (A). Error bars are standard error of the mean. Right panel: Paired t test
comparisons (assuming Gaussian distribution) of macrophage death percentages from 3-, 5-, and 7-h time points. A p value lower than 0.05 was considered significant.
(D) The total area of hyphae, both phagocytosed and escaped, quantified for C. albicans WT, ece1D/D, and ece1D/D + ECE1 strains (same experiments as in [B]
and [C]), from 2.5 to 5 h post-infection. Error bars are standard error of the mean.
(E) Paired t test comparisons (assuming Gaussian distribution) of total hyphae areas described in (D), quantified at 2.5 and 5 h post-infection. A p value lower than
0.05 was considered significant.
Cell Reports 40, 111374, September 20, 2022 11ll
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Figure 6. Candidalysin contributes to in-
flammasome activation outputs: IL-1b
secretion and Gasdermin D processing
(A) Release of IL-1b quantified by ELISA from su-
pernatants of BMDMs challenged with C. albicans
WT, ece1D/D, and ece1D/D + ECE1 strains. MOI
was 6 and supernatants were collected 3 h after
challenge. BMDMs were primed with 50 ng/mL
lipopolysaccharide (LPS) 3 h before infection,
except for uninfected controls. Treatment with
10 mM MCC950 also commenced 3 h before
infection. As a positive control, BMDMs were
treated with 10 mM nigericin at the time of infection.
Ratio paired t tests (assuming Gaussian distribu-
tion) performed for selected comparisons, with a p
value lower than 0.05 considered as significant.
Shown are averages and standard error of the
mean of 4 independent experiments.
(B) Western blot of Gasdermin D cleavage in
response to C. albicans (MOI = 6), 3 h post-infec-
tion. Macrophages were infected with WT or
ece1D/D C. albicans (as indicated by the asterisk).
Depending on the experiment, infection with ece1D/D C. albicans resulted in either reduced cleavage (as seen here) or no detectable cleavage of Gasdermin D
(shown in Figure S7). Nigericin-treated and Gsdmd/ macrophages (BMDMs) were used as positive and negative controls, respectively. All independent ex-
periments are shown in Figure S7.
suggest that hyphal force is not sufficient to drive macrophage Gasdermin D is cleaved to its pore-forming domain by
escape. First, consistent with previous work (Kasper et al., caspase-1, downstream of Nlrp3 inflammasome activation.
2018; Westman et al., 2018), we observed normal intra-phago- Given the role of Gasdermin D, it was surprising that the inactiva-
somal hyphal morphogenesis of ece1D/D. Second, hyphal force tion of Nlrp3 did not reduce hyphal escape from macrophages.
is presumably not significantly affected by the lack of candidaly- Deeper analysis of our imaging data detected an increase in
sin, given a minor contribution to phagosomal membrane dam- another form of death in Nlrp3/ macrophages, which resem-
age measured with the ece1D/D mutant (Westman et al., bles ETosis. ETosis is a form of cell death that involves the expul-
2018). In spite of this, our results indicate that macrophage per- sion of DNA to form extracellular traps, which then engulf and
meabilization and hyphal escape are reduced in ece1D/D infec- inhibit microbes. ETosis is best known to occur with neutrophils,
tions. We thus conclude that candidalysin is an essential medi- but macrophages and monocytes can also form extracellular
ator of C. albicans’ escape from macrophages, and that the traps (i.e., METs) in response to C. albicans with some evidence
physical forces exerted by the expanding hyphae alone are not of antifungal activity (Halder et al., 2016; Liu et al., 2014; Loureiro
sufficient in mediating escape. et al., 2019). From the perspective of the host, METs function to
We could correlate macrophage membrane permeabilization trap and kill pathogens. Our findings now suggest that this
(measured by DRAQ7 staining), cellular lysis (measured by LDH pathway may provide an additional route for hyphal escape
release), Gasdermin D processing (detected by western blot), from immune containment. Indeed, in our experiments, MET-
and hyphal escape (measured by CFW staining). These linkages like structures were associated with escaped hyphae in the
are particularly evident with the ece1D/D mutant, which dis- vast majority of cases (87%).
played a reduction in all of these processes. As such, our Although hyphal escape was normal from Nlrp3/ macro-
data provide evidence that, under standard conditions, hyphal phages, it was reduced from Gsdmd/ macrophages. One
escape occurs in conjunction with macrophage lysis. However, explanation is that Gasdermin D pores facilitate MET formation,
our data with immortalized iBMDMs shows that hyphal escape similar to what has been shown for neutrophil extracellular traps
is not always associated with terminal macrophage membrane (NETs) (Chen et al., 2018; Silva et al., 2021; Sollberger et al.,
disruption. In iBMDMs, we detected little evidence of mem- 2018). However, this is not likely because Nlrp3 is required for
brane permeabilization or cellular lysis of macrophages in the Gasdermin D processing in response to C. albicans (Figure 3E).
first 7 h of imaging, although hyphal escape was occurring Collectively, based on our data, we propose the following model.
and Gasdermin D and candidalysin were involved (Figure S4). In a WT situation, Nlrp3 is involved in hyphal escape upstream of
Non-lytic escape of C. albicans from macrophages has been re- Gasdermin D by enabling the activation of caspase-1 and Gas-
ported in the J774-mouse macrophage-like cell line, but as a dermin D cleavage. However, C. albicans can overcome Nlrp3
rare event (Bain et al., 2012). In iBMDMs, non-lytic escape inactivation, possibly by utilizing MET formation (i.e., ETosis).
was common likely because these cells are immortalized. It is How METs form in the absence of active Nlrp3 inflammasome
possible that, in some circumstances, membrane repair path- has yet to be defined, but it may involve alternate cell death
ways could play a part in preventing macrophage lysis associ- proteins, such as caspase-8 or Gasdermin E (Weir and Vince,
ated with hyphal escape, possibly via the ESCRT pathway 2022). Candidalysin could also be involved in the mechanism
(Ru€hl et al., 2018). because the ece1D/D strain displayed reduced escape from
12 Cell Reports 40, 111374, September 20, 2022ll
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Cell Reports 40, 111374, September 20, 2022 13ll
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Nlrp3/ macrophages and low MET formation. Future work will The experiments presented in this study were performed with
need to address how ETosis facilitates hyphal escape. mouse macrophages (BMDMs and iBMDMs). We have previ-
What could be the consequence of slowing down hyphal ously shown that the hallmarks of C. albicans-macrophage inter-
escape from macrophages? The following scenarios can be actions are recapitulated in human monocyte-derived macro-
imagined: (1) the speed of hyphal escape determines how well phages (Tucey et al., 2020). However, it should be kept in mind
C. albicans is killed or its growth supressed by macrophages that tissue-specific antifungal defenses are mediated by tissue
or (2) the speed of hyphal escape determines the extent of in- resident macrophages, with ontogeny that is distinct from
flammatory activation as macrophages lyse and activate the bone marrow-derived macrophages (Ginhoux and Guilliams,
Nlrp3 inflammasome. Our data support the second scenario. 2016; Netea et al., 2015; Xu and Shinohara, 2017). On the path-
Inactivation of Gasdermin D or candidalysin trapped C. albicans ogen’s side, there is further diversity to be taken into consider-
in macrophages for longer, but it did not result in loss of the dTo- ation. Our study here and that of Ding et al. (2021) show that
mato signal, indicating that fungal viability was maintained. C. albicans hyphal formation is important for rapidly escaping
Instead, the candidalysin mutant induced lower levels of IL-1b macrophages via Gasdermin D and candidalysin-dependent
secretion from macrophages (Figure 6A), and this result is sup- processes. However, clinical isolates of C. albicans differ widely
ported by previous work in mouse and human macrophages in their ability to form hyphae, leading to distinct kinetics of
(Kasper et al., 2018; Rogiers et al., 2019). As such, our data macrophage damage and distinct mechanisms and extent of
suggest that inhibiting hyphal escape should dampen inflamma- Nlrp3 inflammasome activation (Tucey et al., 2020). We show
tion rather than increase pathogen killing, although increased here that lower hyphal formation causes lower escape, but
fungal containment may be beneficial beyond killing. Indeed, even isolates with low hyphal formation eventually escape from
Ding et al. reported reduced growth of C. albicans due to trap- macrophages, albeit with less frequency and later in infection
ping inside Gsdmd/ macrophages (Ding et al., 2021). Reduced (this study and Tucey et al., 2020). A limitation of our study
inflammation could be sufficient to improve patient outcomes, here and all previous work is that the mechanistic studies of hy-
even if fungal loads remain unchanged (Borghi et al., 2015; Bruno phal escape were performed with only one, highly hyphal isolate
et al., 2015; Roselletti et al., 2017). Supporting this possibility, of C. albicans (the favorite strain in the medical mycology com-
while our manuscript was in preparation, it was reported that munity, SC5314). To fully understand the mechanisms and path-
Gsdmd/ mice are less susceptible to systemic C. albicans ogenic importance of fungal escape from macrophages, future
infection (Ding et al., 2021). In conclusion, our study reports on studies need to extend to diverse C. albicans isolates and
the mechanisms behind the spectacular egress of C. albicans macrophage types.
hyphae from macrophages. Inhibiting hyphal escape reduces
the inflammatory response of macrophages to C. albicans infec- STAR+METHODS
tion, which could be beneficial for reducing immunopathology.
Detailed methods are provided in the online version of this paper
Limitations of the study and include the following:
We developed a widefield, live-cell imaging assay to directly
visualize and quantify hyphal escape from macrophages, d KEY RESOURCES TABLE
dynamically over time and in large macrophage populations. d RESOURCE AVAILABILITY
This is an advantage over previous work, in which conclusions B Lead contact
on hyphal escape were mostly made indirectly by measuring B Materials availability
macrophage membrane permeabilization and lysis (which are B Data and code availability
not always correlated to escape) or by scoring escape events d EXPERIMENTAL MODEL AND SUBJECT DETAILS
manually in a relatively small number of macrophages. Our assay B C. albicans strains and growth conditions
is suitable for the quantification of population-level effects, but B Mammalian cell types and culture
other imaging methods with higher spatial and temporal resolu- d METHOD DETAILS
tion will be needed to precisely determine, at the single-cell level, B Generation of dTomato C. albicans strains
the cause/consequence relationships between hyphal escape B CRISPR/Cas9 deletion of Gasdermin D in immortalised
and macrophage membrane damage (e.g., does macrophage mouse bone marrow-derived macrophages (iBMDMs)
lysis facilitate escape or does hyphal escape cause macrophage B Macrophage infection and live cell imaging
lysis?). B Image analysis
Figure 7. Candidalysin has Gasdermin D and Nlrp3-dependent and -independent roles in hyphal escape from macrophages
(A) Hyphal escape-phagocytosis ratios of WT and ece1D/D C. albicans from Nlrp3/ macrophages (MOI = 3). Values are plotted separately for 4 independent
biological repeats (BMDMs from 4 different mice), with 3 technical repeats each. Error bars represent standard error of the mean.
(B) Percentage DRAQ7 staining for experiments described in (A). LDH release assays for the same cells, performed in separate experiments, are shown in
Figure S3A.
(C) Hyphal escape-phagocytosis ratios of WT and ece1D/D C. albicans from Gsdmd/ macrophages (MOI = 3). Values are plotted separately for 3 independent
biological repeats (BMDMs from 3 different mice), with 2 technical repeats for biological repeat 1, and 3 technical repeats for biological repeats 2 and 3.
(D) Percentage DRAQ7 staining for experiments described in (C). LDH release assays for the same cells, performed in separate experiments, are shown in
Figure S3B.
14 Cell Reports 40, 111374, September 20, 2022ll
Article OPEN ACCESS
B Quantification of macrophage permeabilization, hyphal Banoth, B., Tuladhar, S., Karki, R., Sharma, B.R., Briard, B., Kesavardhana, S.,
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B IL-1b ELISA, LDH and Western blot supernatants and masome activation and pyroptosis, apoptosis, and necroptosis (PANoptosis).
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B IL-1b ELISA Boe, D.M., Curtis, B.J., Chen, M.M., Ippolito, J.A., and Kovacs, E.J. (2015).
Extracellular traps and macrophages: new roles for the versatile phagocyte.
B Lactate dehydrogenase (LDH) assays
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B Western blots
Borghi, M., De Luca, A., Puccetti, M., Jaeger, M., Mencacci, A., Oikonomou,
B Scanning electron microscopy (SEM) of C. albicans- in-
V., Pariano, M., Garlanda, C., Moretti, S., Bartoli, A., et al. (2015). Pathogenic
fected BMDMs NLRP3 inflammasome activity during Candida infection is negatively regulated
d QUANTIFICATION AND STATISTICAL ANALYSIS by IL-22 via activation of NLRC4 and IL-1Ra. Cell Host Microbe 18, 198–209.
https://doi.org/10.1016/j.chom.2015.07.004.
SUPPLEMENTAL INFORMATION Brinkmann, V., Reichard, U., Goosmann, C., Fauler, B., Uhlemann, Y., Weiss,
D.S., Weinrauch, Y., and Zychlinsky, A. (2004). Neutrophil extracellular traps
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Bruno, V.M., Shetty, A.C., Yano, J., Fidel, P.L., Jr., Noverr, M.C., and Peters,
ACKNOWLEDGMENTS B.M. (2015). Transcriptomic analysis of vulvovaginal candidiasis identifies a
role for the NLRP3 inflammasome. mBio 6, e00182-15. https://doi.org/10.
This work was supported by funding from the Australian National Health and 1128/mBio.00182-15.
Medical Research Council (NHMRC) project grant APP1158678 (to A.T.) and Chen, K.W., Monteleone, M., Boucher, D., Sollberger, G., Ramnath, D., Con-
an Australian Research Council (ARC) Future Fellowship (FT190100733 to don, N.D., von Pein, J.B., Broz, P., Sweet, M.J., and Schroder, K. (2018). Non-
A.T.). J.E.V. was funded by NHMRC project grants 1145788 and 1101405, canonical inflammasome signaling elicits gasdermin D-dependent neutrophil
Ideas grant 1183070, and Fellowship 1141466. M.J.H. was supported by an extracellular traps. Sci. Immunol. 3, eaar6676. https://doi.org/10.1126/sciim-
NHMRC Senior Research Fellowship (ID 1042775). F.A.B.O. was funded by munol.aar6676.
an RTP PhD scholarship. We thank Bernhard Hube and Mihalis Lionakis for
Coll, R.C., Robertson, A.A.B., Chae, J.J., Higgins, S.C., Muñoz-Planillo, R., In-
sharing C. albicans strains with us, Seth Masters (Walter and Elisa Hall Insti-
serra, M.C., Vetter, I., Dungan, L.S., Monks, B.G., Stutz, A., et al. (2015). A
tute) for the Nlrp3 mutant mice, and Avril Robertson (University of Queensland)
small-molecule inhibitor of the NLRP3 inflammasome for the treatment of in-
for providing MCC950. The clinical isolates of C. albicans are from BEI Re-
flammatory diseases. Nat. Med. 21, 248–255. https://doi.org/10.1038/nm.
sources. We thank Tricia Lo for advice regarding C. albicans transformation
3806.
experiments and acknowledge the Monash MicroImaging Facility and the
Monash Ramacioti Centre for Electron Microscopy for expert assistance. Denning, D.W., Kneale, M., Sobel, J.D., and Rautemaa-Richardson, R. (2018).
Global burden of recurrent vulvovaginal candidiasis: a systematic review.
Lancet Infect. Dis. 18, e339–e347. https://doi.org/10.1016/s1473-3099(18)
AUTHOR CONTRIBUTIONS
30103-8.
Conceptualization, F.A.B.O. and A.T. Methodology, F.A.B.O. and V.H. Soft- Ding, X., Kambara, H., Guo, R., Kanneganti, A., Acosta-Zaldı́var, M., Li, J., Liu,
ware, F.A.B.O. and V.H. Formal analysis, F.A.B.O. and H.W. Investigation, F., Bei, T., Qi, W., Xie, X., et al. (2021). Inflammasome-mediated GSDMD acti-
F.A.B.O., H.W., J.E.V., A.W., and S.C. Visualization, F.A.B.O. Resources, vation facilitates escape of Candida albicans from macrophages. Nat. Com-
J.E.V., A.W., and S.H. Writing – original draft, F.A.B.O. and A.T. Writing – re- mun. 12, 6699. https://doi.org/10.1038/s41467-021-27034-9.
view & editing, F.A.B.O. and A.T. Supervision, J.E.V., S.C., M.J.H., and A.T. DiPeso, L., Ji, D.X., Vance, R.E., and Price, J.V. (2017). Cell death and cell lysis
Project administration, A.T. Funding acquisition, A.T. are separable events during pyroptosis. Cell Death Discov. 3, 17070. https://
doi.org/10.1038/cddiscovery.2017.70.
DECLARATION OF INTERESTS Doerflinger, M., Deng, Y., Whitney, P., Salvamoser, R., Engel, S., Kueh, A.J.,
Tai, L., Bachem, A., Gressier, E., Geoghegan, N.D., et al. (2020). Flexible usage
The authors declare no competing interests. and interconnectivity of diverse cell death pathways protect against intracel-
lular infection. Immunity 53, 533–547.e7. https://doi.org/10.1016/j.immuni.
Received: December 12, 2021 2020.07.004.
Revised: June 15, 2022
Doster, R.S., Rogers, L.M., Gaddy, J.A., and Aronoff, D.M. (2018). Macro-
Accepted: August 26, 2022
phage extracellular traps: a scoping review. J. Innate Immun. 10, 3–13.
Published: September 20, 2022
https://doi.org/10.1159/000480373.
Erwig, L.P., and Gow, N.A.R. (2016). Interactions of fungal pathogens with
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