1. Introduction
Invasive aspergillosis (IA) has emerged as one of the most common life-threatening fungal diseases affecting immunocompromised individuals, with mortality rates as high as 90% [
1]. IA is predominantly caused by
Aspergillus fumigatus, a worldwide environmental and airborne fungal pathogen in humans [
2].
Aspergillus infection is on the rise due to the use of antibiotics and immunosuppressants, causing great concern in the medical community [
3]. Currently available antifungal drugs to treat IA are limited because of increasing drug resistance and side effects/toxicities [
4]. Recent developments in
A. fumigatus have heightened the need for examining functional genes that contribute to its pathogenesis and may provide novel drug targets.
Fungal vacuoles are acidic compartments that play numerous roles, including amino acid storage and phosphate, pH and ion homeostasis maintenance [
5,
6]. Vacuole-type H
+-ATPase (V-ATPase) is found in almost every eukaryotic cell and is responsible for supplying energy to various internal organelles and membrane assemblies [
7]. V-ATPase produces proton motive force by hydrolyzing ATP. The energy provided by V-ATPase promotes the acidification of intracellular compartments in eukaryotic cells and plays a crucial role in receptor-mediated endocytosis, protein degradation and intracellular trafficking processes [
8,
9]. V-ATPase is a multisubunit protein consisting of two distinct functional domains, V
1 and V
0. The V
1 domain consists of eight subunits, designated A–H, which are encoded by the yeast
vma1,
vma2,
vma5,
vma8,
vma4,
vma7,
vma10 and
vma13 genes, respectively. The V
0 domain is composed of six different subunits (a, d, e, c, c′, c′′), which are encoded by the
vph1 (or
stv1),
vma3,
vma11,
vma16 and
vma6 genes [
10]. The V
0 domain also contains a conserved acidic residue required for proton translocation and a hydrophobic carboxyl domain that is part of the proton pore [
9,
11]. The V
1 sector is required for ATP hydrolysis, and the V
0 sector participates in proton transport [
9,
12].
V-ATPase is a central player in pH regulation and calcium homeostasis. A previous study demonstrated that loss of V-ATPase function in yeast caused pH-dependent conditional lethality, with mutant strains unable to grow at pH 7 or above but able to grow at pH 5–5.5 [
13]. Other fungi, including
Candida albicans,
Neurospora crassa, and
Schizosaccharomyces pombe, also exhibit a pH-dependent growth phenotype when V-ATPase subunits are disrupted [
14,
15,
16]. It has been proposed that
vma mutants grow at a lower extracellular pH because they can acidify the vacuole via ingestion of acidic extracellular fluid [
17]. Furthermore, yeast
vma mutants are deficient in many aspects of metal ion homeostasis, among which the calcium sensitivity of
vma mutants is best understood. For example, Kane et al. discovered that
vma mutants were both sensitive to high levels of extracellular calcium and unable to grow on nonfermentable carbon sources [
18]. Elevated calcium caused morphological defects in
vma4-1ts mutants at the nonpermissive temperature [
19]. It has also been reported that V-ATPase subunit C is required for the maintenance of calcium homeostasis in
S. cerevisiae and
C. albicans [
20,
21].
Although the combination of pH-dependent growth and calcium sensitivity is a typical result of loss of V-ATPase activity, it should be highlighted that these are far from being the only physiological defects seen in V-ATPase mutants. The fungal cell wall is a crucial organelle that provides a structural barrier, enables biofilm formation, and participates in host-pathogen interactions [
22,
23]. In
S. cerevisiae, the main regulatory pathway responsible for maintaining cell wall biosynthesis and responding to cell wall stress is the cell wall integrity (CWI) pathway [
24]. Fungi exposed to sublethal concentrations of cell- wall-targeted reagents, such as calcofluor white, Congo Red or caspofungin, exhibit disturbed cell wall biosynthesis and activation of the CWI pathway [
25,
26]. It has been reported that disruption of
vma6 or
vph2 not only leads to weakened hyphal development but also affects cell wall composition and stress resistance in
C. albicans [
27]. In
A. niger, cell-wall-related genes, including
agsA,
fksA and
phiA, were upregulated in
vma mutants [
28], indicating that the differential expression of these transcription factors may affect the cell wall components of V-ATPase. Taken together, these findings suggest that V-ATPase plays a key role in regulating the CWI pathway.
Among the subunits of V-ATPase,
vma5 encodes subunit C of the V
1 domain of V-ATPase and is responsible for V
1 domain assembly in the vacuolar membrane in
S. cerevisiae [
12]. Disruption of
vma5 in
S. cerevisiae generates a characteristic
vma deletion phenotype characterized by the inability to grow at high pH and high calcium concentrations [
29]. In
C. albicans, the loss of
vma5 significantly affects filamentous development, vacuole function and calcium homeostasis [
21]. Furthermore, in
N. crassa, the
vma5 mutant shows defects in calcium mobilization [
30]. These data indicate that subunit C is essential for the V-ATPase function. However, the functions of yeast VmaC homolog in
A. fumigatus-VmaC (
AfVmaC) have never been explored.
In this study, VmaC was characterized and investigated via sequence alignment and functional analysis in A. fumigatus. VmaC, putatively encoding subunit C of V-ATPase, was further deleted to uncover its functions during the growth and development of A. fumigatus. Our results demonstrated the importance of AfvmaC in vacuolar function, maintenance of pH and calcium homeostasis, and regulation of the CWI pathway.
2. Materials and Methods
2.1. Strains, Media, and Cultural Conditions
All strains used in this study are listed in
Table S1 in the supplemental material. Strains were grown in minimal medium (MM) containing 1% glucose, 2% agar, 20× salt solution, and trace elements. MM was supplemented with 5 mM uracil and 10 mM uridine for uracil and uridine auxotroph strains. The liquid glucose MM recipe is identical to that for MM, except without agar. To induce the expression of
vmaC in the
tet-vmaC mutant, the medium was supplemented with 1 μg/mL doxycycline.
NiiA-vmaC strains were grown on MM with 70 mM NaNO
3 as a nitrogen source, and the
alcA-vmaC mutant was induced with glycerin as a carbon source. All strains were cultured at 37 °C.
2.2. Construction of Strains
Deletions of vmaC: the selective marker pyr4 was amplified from the pAL5 plasmid using the primer pair Pyr4-F/Pyr4-R. Approximately 1 kb of the upstream and downstream flanking sequences of the vmaC ORF was amplified with the primer pairs VmaC-P1/P3 and VmaC-P4/P6, respectively. These three PCR products were used as the template to generate the vmaC knockout cassette with the primers VmaC-P2/P5. The resulting fusion products were cloned into pEASY-Blunt Zero using a cloning kit (TransGen Biotech, Beijing, China) and used to transform the recipient strain A1160. The transformants were grown on MM and verified by diagnostic PCR using the primers VmaC-diagnostic F/R, VmaC-P1/HPH-R, and VmaC-P6/HPH-F. A similar strategy was used to construct the ΔvmaA mutant.
For the construction of complementary vmaC, the selected marker hph from pAN7-1 was amplified via PCR using the primers HPH-F and HPH-R and then cloned into the pEASY-Blunt vector (TransGen Biotech) to generate the plasmid p-zero-hph. The primers vmaC-NotI-F and vmaC-NotI-R were used to generate a fragment that included the promoter sequence, the complete ORF, and the 3′UTR of vmaC. This fragment was then cloned into the NotI site of the plasmid p-zero-hph and used to transform the ΔvmaC deletion strain. For verification of transformants, diagnostic PCR was carried out with the primers vmaC-com-up and vmaC-com-down.
For the construction of the conditional
tet-vmaC mutant, the endogenous promoter of
vmaC was replaced with a conditional doxycycline-inducible Tet-On promoter [
31,
32].The pyrithiamine resistance cassette and the Tet system from pCH008 were amplified with the primer pair Tet-F/Tet-R. Approximately 1 kb of the upstream and downstream flanking sequences of the
vmaC promoter regions at positions 802 and +1 were amplified with the primer pairs
vmaC-conditional-P1/
vmaC-tet-P3 and
vmaC-tet-P4/
vmaC-conditional-P6, respectively. The three purified PCR products were then used as a template to generate the
tet-vmaC cassette with the primers
vmaC-conditional-P2/
vmaC-conditional-P5. The resulting fusion product was cloned into pEASY-Blunt Zero using a cloning kit (TransGen Biotech) and used to transform the WT recipient strain. Transformants were grown on medium supplemented with 0.1 g/mL pyrithiamine (Sigma, St. Louis, MO, USA) and verified by diagnostic PCR using the primer pairs
vmaC-conditional-P1/tet-ptrA-down and
vmaC-conditional-P6/tet-ptrA-up.
Tet-vmaCc was constructed by transforming the
tet-vmaC mutant with the p-zero-hph-
vmaC plasmid.
For the generation of the NiiA-vmaC conditional strain, the NiiA fragment was first amplified with the primer pair NiiA-fusion-up/NiiA-down. The primer pairs vmaC-conditional-P1/vmaC-NiiA-P3 and vmaC-NiiA-P4/vmaC-conditional-P6 were used to generate the upstream and downstream flanking sequences, respectively. The above-purified PCR products were then used as a template to generate the NiiA-vmaC cassette with the primers vmaC-conditional-P2/vmaC-conditional-P5. Then, the three fusion fragments were cloned into pEASY-Blunt Zero using a cloning kit (TransGen Biotech) and used to transform the WT recipient strain. Diagnostic PCR was performed to verify the transformants using the primer pairs vmaC-conditional-P1/NiiA-down and vmaC-conditional-P6/NiiA-fusion-up.
For the construction of the conditional alc-vmaC mutant, the alc fragment was first amplified with the primer pair PyrG+alc-up/PyrG+alc-down. Next, the upstream and downstream flanking sequences of the vmaC promoter regions were amplified with the primer pairs vmaC-conditional-P1/vmaC-alc-P3 and vmaC-alc-P4/vmaC-conditional-P6, respectively. The three purified PCR products were then used as a template to generate the alc-vmaC cassette with the primers vmaC-conditional-P2/vmaC-conditional-P5. The resulting fusion product was then cloned into pEASY-Blunt Zero using a cloning kit (TransGen Biotech) and used to transform the WT recipient strain. Transformants were verified by diagnostic PCR using the primer pairs vmaC-conditional-P1/PyrG+alc-down and vmaC-conditional-P6/PyrG+alc-up.
VmaC-GFP was generated as follows: GFP was amplified with the primers GFP+PyrgF and GFP+PyrgR. The upstream and downstream flanking sequences of the vmaC promoter regions were amplified with the primer pairs vmaC-GFP-P1/vmaC-GFP-P3 and vmaC-GFP-P4/vmaC-GFP-P6, respectively. The three purified PCR products were then used as a template to generate the vmaC cassette with the primers vmaC-GFP-P2/vmaC-GFP-P5. Transformants were verified by diagnostic PCR using the primer pairs GFP-P1/GFP+PyrgR or GFP+PyrgF/vmaC-GFP-P6. A similar strategy was used to construct the CccA-RFP strain.
To measure the calcium level in vacuoles, the strain gpd-cpy-Aeq-tet-vmaC was generated as follows: PCR was performed using the primers ClaI-cpyA-R and ClaI-cpyA-F to generate a cpyA’ ORF fragment. The fragment was subcloned into the ClaI site of pBARGPE, which has a PgpdA promoter in front of its ClaI site, to generate pBARGPE-PgpdA-cpyA. The PgpdA-cpyA fragment was amplified from pBARGPE-PgpdA-cpyA with Ama1-BamHI-gpd-F and CpyA-linker-R primers and then fused with the Aeq-TrpC fragment generated using Linker-Aeq-F and Ama1-BamHI-trpC-R to yield the fusion fragment PgpdA-cpyA-Aeq. The pAMA1-PgpdA-CpyA-Aeq plasmid was generated by ligating PgpdA-cpyA-Aeq into prg3-AMAI-NotI, and then the plasmid was used to transform the recipient strain Tet-vmaC to generate the gpd-cpy-Aeq-tet-vmaC mutant. A similar strategy was used to construct the gpd-cpy-Aeq-A1160 strain.
To study the ortholog complementation of A. fumigatus, the Tet-vmaCc-an and Tet-vmaCc-sc strains were generated using a similar strategy. In brief, the vmaC promoter was amplified using the primers Af-vmaC-promoter-p1 and Af-vmaC-promoter-p3, and the coding sequence was amplified using the primers An-vmaC-up, An-vmaC-down, Sc-vmaC-up and Sc-vmaC-up. Finally, the above fusion PCR products and the selective marker pyr4 were cloned into pEASY-Blunt Zero using a cloning kit (TransGen Biotech) and used to transform the recipient strain Tet-vmaC.
2.3. Plate Assays
To test the sensitivity of the WT and Tet-vmaC strains to cell-wall-perturbing agents, minimal medium was supplemented with 20 μg/mL CFW or 5 μg/mL CR. Then, 2 μL portions of conidial suspensions (1 × 107, 1 × 106, or 1 × 105 conidia/mL) of the indicated strains were spotted on the relevant media plates with or without doxycycline and grown at 37 °C for 48 h for observation and imaging.
2.4. RNA Isolation and RT–qPCR
To analyze the relative expression levels of
vmaC under normal growth conditions, the WT,
Tet-vmaC,
NiiA-vmaC, and
alcA-vmaC strains were incubated in MM for 48 h at 37 °C. To analyze the relative expression of cell wall synthesis genes, WT and the
Tet-vmaC mutant were incubated in MM for 48 h at 37 °C. To induce the expression of
vmaC in the
Tet-vmaC mutant, the medium was supplemented with 1 μg/mL doxycycline. Samples were collected and subsequently frozen using liquid nitrogen. Total RNA was isolated using a UNIQ-10 column total RNA purification kit (Shanghai Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions. For gDNA digestion and cDNA synthesis, a HiScriptII Q RT SuperMix for qPCR (gDNA wiper) kit (Vazyme) was used according to the manufacturer’s instructions. To analyze the relative expression of the genes of interest, the resulting cDNAs were used for quantitative PCR, performed with an ABI one-step fast thermocycler (Applied Biosystems) and AceQ qPCR SYBR green master mix (Vazyme). The results were then normalized to
tubA expression, and expression levels were calculated using the 2
−ΔΔCT method [
33].
2.5. Western Blotting Analysis
To extract proteins from A. fumigatus mycelia, conidia from related strains were incubated in liquid-inducing medium and then shaken at 220 rpm on a rotary shaker at 37 °C for 48 h. The mycelium was ground in liquid nitrogen with a mortar and pestle and suspended in an ice-cold extraction buffer (50 mM HEPES pH 7.4, 137 mM KCl, 10% glycerol, 1 mM EDTA, 1 μg/mL pepstatin A, 1 μg/mL leupeptin, 1 mM PMSF). Equal amounts of proteins (40 μg) per lane were subjected to 10% SDS–PAGE and transferred to PVDF membranes (Immobilon-P, Millipore) in 384 mM glycine, 50 mM Tris (pH 8.4), and 20% methanol at 250 mA for 1.5 h, and the membranes were then blocked with phosphate-buffered saline (PBS), 5% milk, and 0.1% Tween 20. Next, the membrane was probed sequentially with 1:3000 dilutions of an anti-GFP primary antibody (Sigma) and goat anti-rabbit IgG-horseradish peroxidase secondary antibody (Abclonal Co., AS014, Woburn, MA, USA) diluted in PBS, 5% milk, and 0.1% Tween 20. Blots were developed using Clarity ECL western blotting detection reagents (Bio-Rad, Hercules, CA, USA), and images were acquired with a Tanon 4200 Chemiluminescence Imaging System (Tanon, St Andrews, Scotland).
2.6. Fluorescence Microscopy
The size of the cover glasses was about 18 × 18 mm (Sangon Biotech, F518211-0001). The medium (1.0 mL) was added gently into the culture dish having the cover glass. For microscopic observation of germlings, fresh conidia of strains in 1.0 mL of liquid MM were grown on sterile glass at 37 °C for 48 h. The resulting hyphae were gently washed with PBS buffer three times and then fixed with 4% paraformaldehyde (Polysciences, Warrington, PA, USA) for 1 h. Then, the cover glasses adhered with germlings were placed upside down in slides for observation and imaging by microscopy. To assess the localization of VmaC-GFP, the mycelia were washed with PBS and then fixed with 4% paraformaldehyde for 40 min at room temperature in the dark. FM4–64 (Sigma–Aldrich, St. Louis, MO, USA) staining was conducted on ice following the manufacturer’s protocol. For CFW staining, hyphae were washed with PBS and stained with 20 μg/mL CFW for ~2 min. All images of the cells were collected with a Zeiss Axio Imager A1 microscope (Zeiss, Jena, Germany).
2.7. Transmission Electron Microscopy Analysis of the Cell Wall
The cell walls of the WT and
Tet-vmaC (Off) strains were examined via TEM as previously described [
34]. After the indicated incubation period, the mycelia were fixed overnight in 0.1 M sodium phosphate buffer containing 2.5% glutaraldehyde at 4 °C. The samples were embedded in 1% (wt/vol) agar, fixed in 0.1 M sodium phosphate buffer containing 1% OsO4 for 2 h, and sequentially washed three times in 0.1 M sodium phosphate buffer (15 min each). Next, the samples were dehydrated in 50, 70, 90 and 100% ethanol and 100% acetone for 15 min each. Samples were embedded in 812 epoxy resin monomers (SPI), sliced into 60- to 80-nm ultrathin sections using an ultrathin microtome (Leica UC7), stained with uranyl acetate and lead citrate, and imaged at 80 kV using a transmission electron microscope (Hitachi HT7700, Tokyo, Japan).
2.8. Cell Wall Polysaccharide Analysis
WT and Δ
vmaC strains were incubated in MM for 48 h at 37 °C. After incubation, the mycelia were washed with distilled water and then lyophilized. Fungal cell wall polysaccharides were extracted and quantitatively determined as previously described [
35].
2.9. Measurement of Intracellular [Ca2+]c and Vacuolar Calcium [Ca2+]v
The strains expressing Aeq/cyp-Aeq were cultured for 2 days at 37 °C to form fresh spores. Fresh spores were filtered through nylon cloth and washed 10 times in distilled deionized water. One million (106) spores in 100 mL liquid MM with/without 2 mM CaCl2 were inoculated into each well of a 96-well microtiter plate (Thermo Fisher, Waltham, MA, USA) and incubated at 37 °C for 48 h. The medium was then removed, and the cells in each well were washed twice with PGM (20 mM PIPES pH 6.7, 50 mM glucose, 1 mM MgCl2). Aequorin was reconstituted by incubating mycelia in 100 μL PGM containing 2.5 μM coelenterazine f (Sigma–Aldrich, St. Louis, MO, USA) for 4 h at 4 °C in the dark. After the aequorin constitution, mycelia were washed twice with 1 mL PGM and allowed to recover to room temperature for 1 h. Luminescence was measured with an LB 960 Microplate Luminometer (Berthold Technologies, Bad Wildbad, Germany), controlled by a dedicated computer running MikroWin 2000 software. At the 20-s time point of luminescence reading, 0.1 M CaCl2 was applied as a stimulant. At the end of each experiment, the active aequorin was completely discharged by permeabilizing the cells with 20% (v/v) ethanol in the presence of an excess of calcium (3 M CaCl2) to determine the total aequorin luminescence in each culture. The conversion of luminescence (relative light units [RLU]) into [Ca2+] was performed using Excel 2019 software (Microsoft). Input data were converted using the following empirically derived calibration formula: pCa = 0.332588 (−log k) + 5.5593, where k is luminescence (in RLU) s−1/total luminescence (in RLU).
2.10. Coimmunoprecipitation and Mass Spectrometry Assay
A GFP antibody (Roche, Basel, Switzerland, 11814460001) was used to pull down VmaC-interacting proteins. A nonlabeled strain under similar conditions was used as a negative and nonspecific VmaC binding control. HPLC was performed at BGI Genomics as a commercial service. In brief, proteins were digested with trypsin (Promega) and labelled using a TMT kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. The labelled tryptic peptides were fractionated via HPLC using a Thermo Betasil C18 column (5 μM particles, 10 mm diameter, 250 mm length). After fractioning, the tryptic peptides were analyzed with an LC-MS/MS system. MS/MS data processing was performed using the MaxQuant search engine (v.1.5.2.8).
2.11. Data Analysis
Data are given as the means ± SDs. The SD was obtained from at least three biological replicates. Statistical significance was estimated with Student’s t-test using GraphPad Prism 7 software. p values less than 0.05 were considered statistically significant.
4. Discussion
V-ATPase is a multisubunit complex that is involved in a variety of cellular processes [
6]. In this study, we functionally characterized the roles of an evolutionarily conserved subunit of the V-ATPase complex, VmaC, in
A. fumigatus. According to the sequence alignments and functional analysis, we identified
AfvmaC as a subunit of the V-ATPase complex. Both the
vmaC deletion and the
vmaC-turned-off mutants resulted in phenotypic defects in
A. fumigatus hyphal growth and conidiation, which highlights the importance of VmaC in these biological processes. Complementation experiments between different fungal species verified that the homologous VmaC of
S. cerevisiae could rescue the sick phenotype of the
tet-vmaC (off) conditional strain (
Figure 1E), indicating that VmaC functions as a subunit of V-ATPase and its functions are relatively conserved. In yeast, cells appear to be able to tolerate the loss of V-ATPase function but with conditional defective growth and morphological phenotypes [
37]. However, in
A. fumigatus, deletion of
AfvmaC or turn-off of
AfvmaC expression was not lethal but led to a very sick and tiny colony phenotype in all tested media, which is different from the conditional defects caused by deletion of
vmaC in yeast. This suggests that although VmaC functions are conserved among various fungal species, the roles of important functional domains may differ. In addition, disruption of the C-terminal hydrophobic domain of VmaC in
A. fumigatus not only resulted in a colony phenotype similar to that of the
vmaC deletion mutant but also led to mislocalization of VmaC, indicating that the C-terminal hydrophobic domain of VmaC was needed for the function and proper distribution of VmaC. As a storage organelle, vacuoles are associated with many aspects of cellular homeostasis [
5]; thus, vacuole-localized VmaC in
A. fumigatus may be involved in the normal functions of vacuoles. Defects in VmaC-involved V-ATPase activity may result in an inability to store or exchange nutrients between vacuoles and the cytosol during germination.
Calcium is vital for cells to translate diverse developmental cues and environmental stresses into specific cellular compartment developmental responses, and V-ATPase plays an important role in the maintenance of ion homeostasis in eukaryotes [
38]. It has been reported that cytosolic calcium homeostasis is a constitutive function of the V-ATPase in
S. cerevisiae [
39]. However, due to an inability to examine the dynamic vacuolar calcium concentration, there was no direct evidence showing a relationship between VmaC and calcium homeostasis before this study. Here, through an intracellular calcium dynamic monitoring strategy, we discovered that VmaC dysfunction disturbed calcium homeostasis in both the cytosol and vacuole, resulting in significant cell growth inhibition in the presence of a calcium stimulus. Typically, the resting [Ca
2+]
c in
A. fumigatus ranges from 0.1 to 1.1 μM. In the
vmaC-turned-off mutant, the cytosolic resting calcium level increased by 4.5-fold compared to that in the WT strain before the calcium stimulus. However, when cells were challenged with a high calcium concentration, the Δ
vmaC mutant displayed a 16% decrease in the [Ca
2+]
c amplitude compared to the WT strain. The sustained increase in [Ca
2+]
c led us to consider whether calcium homeostasis in the vacuole would also be affected by a lack of VmaC. Surprisingly, we found that the basal resting [Ca
2+]
v and the transient [Ca
2+]
v in the
vmaC-turned-off mutant were significantly increased in the presence of an extracellular calcium stimulus. Collectively, these data demonstrated that VmaC not only regulates resting cytosolic calcium but also affects the vacuolar calcium transient response in
A. fumigatus. Because of the evolutionarily conserved role of VmaC, we suggest that VmaC may also participate in maintaining cytosolic calcium homeostasis in other fungal species. In addition to disordered intracellular calcium homeostasis, the Δ
vmaC mutant exhibited hypersensitivity to high pH, which is in accord with the Δ
vmaA mutant. Since V-ATPase is involved in the maintenance of intracellular pH homeostasis through its role in pumping excess protons into the vacuole [
6], we speculate that VmaC is involved in the reciprocal transport of Ca
2+ and H
+ in
A. fumigatus. Defects in pH and/or calcium homeostasis could potentially account for both the cytoskeletal defects and the morphological changes in the Δ
vmaC mutant [
40,
41,
42].
Furthermore, the conditional
vmaC-turned-off mutants were tolerant of the cell wall stress reagent CR, and GFP-trap data further revealed that VmaC may interact with cell wall biosynthesis-related proteins (
Figure 2B), indicating that VmaC may play a central role in the cell wall biosynthesis pathway. Since proper V-ATPase function affects multiple cellular processes, several scenarios can be envisioned to explain this phenomenon. One scenario is that the loss of VmaC affects the transport of secretory vesicles to the tip of the fungal cell (
Figure 5B). These vesicles contain the enzymes that are needed for proper cell wall biosynthesis, and it is conceivable that less efficient secretion leads to improper cell wall biosynthesis. Another possibility is that the
vmaC mutant triggered the CWI pathway via regulation of related transcription factors (
Figure 5E). Accordingly, we found that the cell wall of the conditional
vmaC-turned-off mutant was significantly thicker than that of the wild-type strain. In addition, qRT–PCR analysis showed that the transcription factor
fksA was significantly upregulated in the Δ
vmaC mutant, in accord with findings in
A. niger [
28], while other transcription factors (e.g.,
rlmA,
adsA,
chsC,
chsD, and
mnn1) were downregulated, suggesting that VmaC plays a central role in regulating cell wall components. We demonstrated that the chitin and mannose components of cell walls were decreased in the Δ
vmaC mutant, and galactose and β-glucan were increased compared to levels in the parental WT, further indicating that the functions of VmaC are related to the CWI pathway. In addition, azoles function by inhibiting ergosterol synthesis, while ergosterol is a critical requirement in V-ATPase function. The mutants defective in ergosterol biosynthesis exhibited most of these characteristic
vma deletion phenotypes [
43]. Notably, deletion of
vmaC leads to disorders in vacuolar localization and function [
43]. In both
S. cerevisiae and
C. albicans, fluconazole impaired vacuolar acidification, whereas concomitant ergosterol feeding restored V-ATPase function and cell growth [
43]. These findings suggest that the critical requirement for ergosterol in V-ATPase function may underlie the antifungal activity of azoles. Although there is no direct evidence showing that
vma mutants are related to drug resistance in
A. fumigatus, with the high conservation of VmaC, the role of VmaC in drug resistance may exist in
A. fumigatus as well. This is an interesting research area which can be addressed in the near future.
Taken together, to the best of our knowledge, this is the first report in which the function of the VmaC subunit of the V-ATPase complex has been identified in A. fumigatus. Our study reveals that VmaC is essential for hyphal growth and conidiation in A. fumigatus. Notably, deletion of vmaC leads to disorders in vacuolar localization and function, probably by affecting calcium homeostasis. Furthermore, vmaC deletion increases the tolerance to cell-wall-stress reagents and regulates the cell wall pathway in A. fumigatus. Therefore, our findings enrich the understanding of V-ATPase and may provide a potential antifungal target.