INTRODUCTION
Aspergillus fumigatus is a saprotrophic fungus found in a wide range of ecological niches, which can cause allergic, invasive, and chronic diseases in humans that are difficult to diagnose and treat (
1). Recent estimates indicate that between one and two million people are diagnosed with life-threatening invasive aspergillosis annually, where mortality rates can exceed 40% (
2–4). The first-line treatment for invasive infections is the triazoles; however, resistance to this class of compounds is increasing globally and is associated with poorer treatment outcomes [25% increase in mortality (
2)]. Major gaps remain in our understanding of
A. fumigatus pathobiology, virulence, treatment and evolution.
Molecular research on filamentous fungi such as
A. fumigatus relies on the development of novel genetic tools and techniques, which have been mainly adapted from model organisms such as
Aspergillus nidulans and
Neurospora crassa (
5,
6). In the past 20 years, multiple fluorescent proteins (FPs) have been developed that can be used in filamentous fungi, with sGFP, a synthetic GFP variant comprising a serine to threonine substitution at position 65 of the protein sequence, being the first one specifically designed for this task (
7). Since then, in addition to GFP S65T, other fluorophores have been used in
A. fumigatus covering blue, green, yellow, orange, and red FPs (
8–15). These have enabled investigations into the developmental biology of this pathogen as well as the subcellular dynamic in response to specific treatments, including antifungals (
16–18). These FPs originate from different sources; namely
Aequorea victoria and
Discosoma species and have different dimerization properties and different predicted brightness. This can lead to complications when visualizing proteins that are expressed at low levels, often leading to a need to overexpress the proteins of interest, which may not be physiologically relevant.
Significant advances in FP engineering have led to increased brightness and photostability, together with reduced oligomerization and maturation times (
19–21). Photostability is a major consideration in live-cell fluorescence microscopy. Photobleaching measurements
in vivo have been found to be consistent with measurements
in vitro, but stability can be affected by the culture medium or microscopy technique: laser-scanning confocal microscopy, used in this work, bleaches FPs faster than widefield microscopy (
22,
23). However, many FPs perform differently across different organisms. While many FPs have been developed for and evaluated in other fungal species such as
Candida albicans (
24),
Cryptococcus neoformans (
25)
N. crassa (
26), and
Saccharomyces cerevisiae (
27,
28)
, no extensive direct comparisons of FPs have been carried out in
A. fumigatus. Furthermore, characterizing a diverse set of FPs covering a wide range of wavelengths allows for multicolor imaging experiments and the potential to design Förster resonance energy transfer (FRET) systems (
29,
30).
In this work, we characterize the brightness of cytoplasmic FPs that are expressed using a common
A. nidulans promoter,
PgpdA (
31), from single-copy integrations at a defined locus within an isogenic strain of
A. fumigatus. We describe a strain labelled with four different FPs, which was generated using four endogenous counter-selectable markers, enabling the simultaneous visualization of the mitochondria, peroxisomes, vacuoles, and the cell membrane. We use this strain to monitor the effects of voriconazole, amphotericin B, olorofim, and manogepix on these subcellular compartments at the population level. Finally, we examine the temporal response to manogepix, a first-in-class glycosylphosphatidylinositol biosynthesis inhibitor (
32) at the individual level.
DISCUSSION
There are currently >1,000 entries of FPs on the open-source community database
https://www.fpbase.org/ (27 August 2024). An ever-increasing choice of FPs with increased brightness, an increasing number of monomeric fluorophores, and longer lifetime properties are becoming more available. However, a well-performing fluorophore in one organism or experimental design may not translate to a different system (
54). Therefore, we sought to explore a palette of next-generation FPs in
A. fumigatus, the major mould pathogen of humans, to be used for multicolor imaging, allowing for temporal imaging of responses to antifungals.
First, we determined the
in vivo brightness of 18 FPs in both hyphae and spores. The majority of FPs investigated have markedly improved brightness compared to the widely used fluorophore, GFP S65T. The brightest blue we found was mTagBFP2. The brightest green was mNeongreen, derived from lanYFP of
Branchiostoma lanceolatum (
55). This fluorophore has recently been used in
A. fumigatus integrated into the genome to tag a protein, although under high expression levels (
56). The next brightest green FP was mGreenlantern, derived from avGFP of
A. victoria. Citrine and mTurquoise2 have also been derived from this FP, potentially highlighting that certain fluorophore lineages or sources might perform better than others in
A. fumigatus. Similarly, mTagBFP2, Katushka2S, and mMaroon1 are all derived from eqFP578 of
Entacmaea quadricolor. The brightest orange mKO2 is the only FP derived from KO of
Verrillofungia concinna. While we did not attempt different codon optimization algorithms, in
C. albicans, there is not one clear strategy to improve FP characteristics by codon optimization (
24). Improvements to codon optimization may lead to even further improvements to FP performance.
These next-generation FPs can be used in microscopy to investigate cell biological phenomenons in general and aid antifungal drug discovery in particular, by identifying protein-protein interaction inhibitors, assessing drug effects in subcellular structures, or studying drug targets (
17,
57–59). Using brighter FPs can be used at lower expressed proteins broadening their range of applications to include fluorescent genetic barcoding, gene expression reporters, and host-pathogen interactions. Overcoming oligomerization issues with FPs, using monomeric FPs can allow for more robust use of a split-FP system, overcome protein aggregation issues, and reduce background noise. In addition, we have characterized far-red shifted proteins in
A. fumigatus; this may allow for deeper tissue imaging in infection models such as zebrafish, which have been previously described using
C. albicans (
60).
We used FPs with minimal spectral overlap to generate the tetrachrome strain to visualize the cell membrane, mitochondria, peroxisomes, and vacuoles simultaneously. Four-color imaging has previously been described in
N. crassa (
26), and
S. cerevisiae (
61), but to our knowledge, this is the first time, four FPs have been used simultaneously in a human pathogenic fungus. We expanded the genetic marker toolbox with the new endogenous counter-selectable marker
cntA, to achieve in combination with the previously described markers
fcyB,
fcyA, and
uprt, the insertion of four different FP-fusion cassettes. Importantly, the simultaneous loss of
fcyB,
fcyA,
uprt, and
cntA did not compromise
A. fumigatus growth and development (Fig. S3d and h).
We investigated the changes in the subcellular compartments in response to two well-characterized antifungal drugs with different mechanisms of action: amphotericin B and voriconazole. Our data suggest a generalized response to both drugs, in which the distance between peroxisomes decreases, indicating an increase in peroxisomes. Peroxisomes are important in
A. fumigatus to overcome oxidative stress (
62), which has been known to be induced by the azoles and amphotericin B (
63,
64). Mitochondria became fragmented in response to both antifungals. This switch from tubular to clustered and fragmented mitochondria has previously been observed in response to oxidative stress, cell death,and azole resistance (
62). A sustained increase in vacuole size was only seen upon amphotericin B exposure. Vacuole disruption has been observed in response to amphotericin B in
C. albicans (
65) and
S. cerevisiae (
66). Our results highlight that the mechanism of action correlating with effects on subcellular structures could be elucidated via analysis of our tetrachrome strain.
We further characterized the response to the novel antifungals olorofim and manogepix, both of which are currently in the antifungal pipeline (
67). In line with previous findings, we found vacuoles to become larger upon olorofim exposure (
17). Upon manogepix exposure, we could not find any statistical differences in the morphology of peroxisomes, mitochondria, or vacuoles after 2 h of exposure. However, a clear biphasic response in the vacuole area was seen, which we explored in detail. We observe rapid mitochondrial fragmentation upon manogepix exposure, a known precursor for fungal cell death (
62). Our data suggest that vacuoles rapidly enlarge and fuse in the first 2 h of exposure, and then diminish in size, possibly due to releasing their contents. However, the precise mechanisms driving these observations remain to be further investigated.
In summary, our results open new opportunities to advance fluorescence imaging in A. fumigatus. Our identification of successful FPs in A. fumigatus will provide valuable tools for not only molecular assays but also drug discovery and efficacy studies. We describe new approaches to evaluate and quantify the mechanisms behind antifungal treatments and reiterate the need for in vivo assays to validate FP usability in the species of choice as described in a previous work on FPs in other fungal species.