Role of the osaA Gene in Aspergillus fumigatus Development, Secondary Metabolism and Virulence

Aspergillus fumigatus is the leading cause of aspergillosis, associated with high mortality rates, particularly in immunocompromised individuals. In search of novel genetic targets against aspergillosis, we studied the WOPR transcription factor OsaA. The deletion of the osaA gene resulted in colony growth reduction. Conidiation is also influenced by osaA; both osaA deletion and overexpression resulted in a decrease in spore production. Wild-type expression levels of osaA are necessary for the expression of the conidiation regulatory genes brlA, abaA, and wetA. In addition, osaA is necessary for normal cell wall integrity. Furthermore, the deletion of osaA resulted in a reduction in the ability of A. fumigatus to adhere to surfaces, decreased thermotolerance, as well as increased sensitivity to oxidative stress. Metabolomics analysis indicated that osaA deletion or overexpression led to alterations in the production of multiple secondary metabolites, including gliotoxin. This was accompanied by changes in the expression of genes in the corresponding secondary metabolite gene clusters. These effects could be, at least in part, due to the observed reduction in the expression levels of the veA and laeA global regulators when the osaA locus was altered. Importantly, our study shows that osaA is indispensable for virulence in both neutropenic and corticosteroid-immunosuppressed mouse models.


Introduction
The ubiquitous fungus Aspergillus fumigatus is one of the most common fungal pathogens of humans worldwide [1], particularly affecting immunosuppressed patients, including those with hematological malignancies (such as leukemia), solid-organ and hematopoietic stem cell transplant patients, individuals with prolonged corticosteroid therapy, patients with genetic immunodeficiencies such as chronic granulomatous disease, cancer patients undergoing chemotherapy, and individuals infected with HIV [2][3][4][5][6][7].The main reason for the increase in systemic infections lies in the constant increase in the number of immunocompromised individuals [8], such as a higher number of transplants for end-organ disease, the development of immunosuppressive and myeloablative therapies for autoimmune and neoplastic diseases, the HIV pandemic [2,[8][9][10], and the COVID-19 pandemic [11][12][13][14].Mortality rates range from 40% to 90% [8,9,15,16].The World Health Organization recently ranked A. fumigatus third in the critical priority group of fungal pathogens [17].The leap from being an organism on the watch list in 2019 to a globally critical fungal pathogen in three years can be traced back to unmet health problems that are associated with aspergillosis [17,18].
For most patients, the main site of entry and infection for A. fumigatus is the respiratory tract.In immunosuppressed patients, A. fumigatus infection can cause aspergilloma and invasive aspergillosis (IA).Diagnostic methods are still limited, as well as the possibilities of therapeutic treatments, leading to high mortality rates [19][20][21].A. fumigatus causes approximately 90% of systemic Aspergillus infections [22].Fungal asexual spores (conidia) are only 2.5-3.0 µm in diameter, which allows them to reach the lung alveoli [23,24].In healthy individuals, conidia that are not removed via mucociliary clearance encounter epithelial cells or alveolar macrophages, which are responsible for the phagocytosis and killing of conidia, as well as for initiating a pro-inflammatory response that recruits neutrophils (PMN type).IA is primarily a consequence of a dysfunction in these defenses.The most common host immunodeficiencies responsible for an increased risk of IA are neutropenia and corticosteroid-induced immunosuppression [2,[25][26][27][28].In neutropenic patients or in a murine model of chemotherapy-induced neutropenia, IA can cause thrombosis and hemorrhage due to rapid hyphal angioinvasion and systemic dissemination to other organs [9,29].Corticosteroid-induced immunosuppressed patients usually present tissue necrosis and excessive inflammation.Corticosteroid treatments impair the function of phagocytes [30], thus rendering them unable to kill both conidia and hyphae [31][32][33][34][35][36].In most cases, consequent excessive inflammation leading to tissue damage is considered the cause of death.
Common treatments with azole drugs present side effects such as skin rashes and photopsia, especially in the early stages of treatment [50].Studies have also revealed the presence of resistant isolates of A. fumigatus to azole treatment [51].Prolonged azole therapy could result in the development of azole-resistant A. fumigatus strains during infection [52].The widespread application of azole fungicides in crop fields is also a driving force for azole-resistant infections worldwide [53,54].It is paramount to find new strategies against fungal infections such as aspergillosis, including novel genetic targets for the treatment or prevention of A. fumigatus infection.In the present study, we investigated the role of OsaA, a WOPR-domain transcription factor, in A. fumigatus.
The name WOPR evolved from closely related proteins, Wor1 (in Candida albicans), Pac2 (in Schizosaccharomyce pombe), and Ryp1 (in Histoplasma capsulatum), where these proteins play a major role in morphological development and pathogenesis [55][56][57].Wor1 regulates white-opaque switching in C. albicans [55], affecting mating frequency and tissue specificity during infection [58].Pac2 may mediate signaling between nutritional conditions and the activation of the transcription factor gene ste11, which is necessary for sexual development in S. pombe [56].Ryp1, from the human pathogen H. capsulatum, is a regulator of the yeast-mycelia transition and affects the transcription of hundreds of genes [57].In Fusarium graminearum, the WOPR protein Fgp1 is a key regulator of reproduction, toxin biosynthesis, and pathogenesis [59], as well as Sge1 in Fusarium oxysporum [60].In Aspergillus nidulans, osaA was shown to be epistatic to the veA regulatory gene in maintaining a balance between sexual and asexual development [61].
Our present work revealed that osaA is necessary for proper colony growth and conidiation in A. fumigatus.In addition, our analysis indicated that secondary metabolite production was also influenced by osaA.Furthermore, osaA is indispensable for virulence in both the neutropenic and corticosteroid-immunosuppressed mouse models.Other virulence factors, such as cell wall stability, thermotolerance, adhesion capacity, and sensi-tivity to oxidative stress, were also decreased in the absence of osaA in this opportunistic human pathogen.

Strains and Culture Conditions
The strains of A. fumigatus used in this study are listed in Table 1.All the strains were grown in glucose minimal medium (GMM) [62] plus the appropriate supplements for the corresponding auxotrophic markers [62] unless otherwise indicated.A solid medium was prepared by adding 1% agar.Strains were stored as 30% glycerol stocks at −80 • C.

Generation of the osaA Deletion Strain
To construct the osaA deletion strain (∆osaA) in A. fumigatus, a deletion of the DNA cassette was generated to replace the osaA gene with the Aspergillus parasiticus pyrG selectable marker [63].First, the 5 ′ UTR and 3 ′ UTR were PCR-amplified from A. fumigatus genomic DNA using the primers osaA_F1, osaA_R2, and osaA_F3, osaA_R4, respectively (the primers used in this study are included in Table S1).The pyrG marker gene was amplified from A. parasiticus genomic DNA using osaA_F5 and osaA_R6 primers.The three fragments were fused using the nested primers, osaA_F7, and osaA_R8, resulting in a 5157 bp PCR product.The obtained deletion fusion cassette was transformed in A. fumigatus CEA17 via polythene glycol-mediated fungal transformation.Transformants were selected on a stabilized minimal medium (SMM) [64] containing 1 M sucrose and no supplemental uracil.The ∆osaA strain was confirmed using a diagnostic PCR with primers osaA_F1 and A para pyrG_R and designated as TAD1.1.

Generation of the osaA Complementation Strain
To generate the osaA complementation strain (Com), a recombinant plasmid was constructed with pBC-hygro as the backbone vector.The osaA coding region, along with 1.5 kb of 5 ′ and 3 ′ UTR were PCR-amplified using the primers osaA_com1 and osaA_com2.The forward primer contained an engineered HindIII site, whereas the reverse primer contained a NotI site.The amplified fragment was digested with the respective restriction enzymes and ligated to the pBC-hygro vector, previously digested with the same enzymes.The pBC-hygro vector includes the selection marker Hyg.The recombinant plasmid was named pAD1.1 and was transformed in the ∆osaA strain, resulting in the complementation strain (TAD2.1).The strain was confirmed using a diagnostic PCR with primers osaA_OE1 and osaA_OE2.

Construction of the osaA Overexpression Strain
A strain overexpressing osaA (OE) was conducted as follows: the coding region of osaA was PCR-amplified from A. fumigatus genomic DNA using the primers osaA_OE1 and osaA_OE2 with AscI and NotI engineered restriction sites, respectively.The 1443 bp PCR product was then digested using the corresponding enzymes and ligated into the pSSP1.1 plasmid, previously digested with the same enzymes.The pSSP1.1 vector includes the A. nidulans gpdA promoter and trpC terminator, along with A. fumigatus pyrG.The resulting recombinant plasmid (pAD3.1)was transformed into the CEA17 strain.Transformants were confirmed using a diagnostic PCR with the primers osaA_OE1 and A fum pyrG_R.

Morphological Analysis
To examine the role of osaA in colony growth, spores of wild-type, ∆osaA, Com, and OE strains were point-inoculated on GMM and incubated at 37 • C in the dark.Colony growth was estimated as the colony diameter, which was measured every two days.The experiment was carried out with four replicates.
For an assessment of the effect of osaA on conidiation, the same A. fumigatus strains were top-agar-inoculated on GMM (5 × 10 6 spores/plate) and incubated at 37 • C in the dark.Cores were harvested from each plate at 24 h, 48 h, and 72 h after inoculation and homogenized in water.Conidia were quantified using a haemocytometer (Hausser Scientific, Horsham, PA, USA) and a Nikon Eclipse E-400 bright field microscope (Nikon, Inc., Melville, NY, USA).The experiment was performed in triplicates.Also, the spore size was measured under the Nikon E-400 microscope.Measurements of spore diameter (n = 40) were taken.
To evaluate whether osaA affects the spore germination rate in A. fumigatus, approximately 10 6 spores/mL of each of the strains were grown in liquid-shaken cultures containing 50 mL of GMM at 250 rpm.After 4 h post-inoculation, the cultures were observed under a bright field microscope, using a haemocytometer, every 2 h.This experiment was carried out in triplicates.

Environmental Stress Tests
To test sensitivity to a range of temperatures, wild-type, ∆osaA, Com and OE strains were point-inoculated on GMM and incubated in the dark at 25 • C, 30 • C, 37 • C, and 42 • C for 5 days when photographs and colony diameter measurements were taken.This experiment included three replicates.
To evaluate the importance of osaA in the resistance to oxidative stress, the strains were point-inoculated on solid GMM supplemented with 0, 15, 20, 25, or 30 µM menadione and incubated at 37 • C. Photographs were taken after an incubation period of 5 days.The experiment was performed with three replicates.

Cell Wall Tests
To elucidate possible alterations of cell wall integrity due to changes in the osaA locus, the wild-type, and ∆osaA, Com, and OE strains were point-inoculated in a 24-well plate on solid GMM supplemented with different concentrations (0, 0.005, 0.01, 0.015, and 0.02%) of sodium dodecyl sulfate (SDS) [65], and incubated at 37 • C for 72 h.In a separate experiment, the strains were inoculated on GMM with Congo red at concentrations of 0, 5, 10, 15, 50, or 100 µg/mL and incubated at 37 • C. Photographs were also taken at 72 h after inoculation.The experiments were carried out with three replicates.
Whether osmotic stabilization allowed the recovery of the wild-type phenotype in strains with an altered osaA locus and possible cell wall defects was assessed as follows.The conidia (10 6 spores/mL) of each strain were point-inoculated on GMM supplemented with either 0.6 M of KCl, 1 M of sucrose, 0.7 M of NaCl, or 1.2 M of sorbitol, and incubated at 37 • C for 72 h when photographs were taken, and colony diameters were measured.This experiment was performed in triplicate.

Adhesion Assay
To assess the role of osaA in adhesion capacity, each strain was inoculated in 50 mL of GMM (10 6 spores/mL).Aliquots (130 µL) from these suspensions were then added to each well of a 96-well microtiter plate.Cultures were incubated at 37 • C for a period of 24 h and 48 h.The mycelial mat and supernatant were removed from the wells.Biomass adhered to the well walls and was washed three times with water and then stained with 130 µL of 0.01% Crystal Violet (in water) for 20 min at room temperature.The stained wells were washed again three times with water, allowed to dry, and then de-stained using 130 µL of 30% acetic acid.The absorbance was measured at 560 nm on an Epoch spectrophotometer (Biotek, Winooski, VT, USA).

Metabolomics Analysis
For these analyses, the wild-type, ∆osaA, Com, and OE strains were top-agar-inoculated on GMM (5 × 10 6 spores/plate) and cultured at 37 • C for 72 h.Three cores (16 mm diameter) were harvested from each plate and extracted with chloroform.The strain set was also grown in a liquid yeast extract-glucose-supplements (YES) medium [66] and cultured at 37 • C and 250 rpm for 7 days.The filtrate (30 mL) was collected and extracted with chloroform.Extracts were analyzed using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS).Samples were dissolved in 500 µL of an acetonitrile/water mixture (50:50, v/v) and shaken vigorously for 30 s, and then treated with a sonicator (Bransonic 221 Ultrasonic bath, Emerson Electric, St. Louis, MO, USA) for 2 h.A volume of 250 µL of pure acetonitrile was added to each sample, and vigorously shaken for 30 s and centrifuged (pulse).LC-MS analyses were performed using Acquity HPLC (Waters, Saint-Quentin-en-Yvelines, France) linked to an LTQ Orbitrap XL high-resolution mass spectrometer (Thermo Fisher Scientific, Les Ulis, France).A reversed-phase Luna ® C18 column (150 mm × 2.0 mm, 5 µm) (Phenomenex, Torrance, CA, USA) was utilized with water acidified with 0.1% formic acid and 100% acetonitrile as the mobile phases.The elution gradient was as follows: 0-30 min, B: 20-50%; 30-35 min, B: 50-90%; 35-45 min, B: 90%; 45-50 min, B: 90-20%; and 50-60 min, B: 20%.The flow rate was 0.2 mL min −1 , the column temperature was 30 • C, and a volume of 10 µL was injected.HRMS acquisitions were carried out with electrospray ionization (ESI) in positive and negative modes, as previously reported [67].MS/MS spectra were obtained with the CID mode at low resolution and a collision energy of 35%.

Gene Expression Analysis
Total RNA was obtained from lyophilized mycelial samples using TRIsure TM (Meridian Bioscience, Bioline, Cincinnati, OH, USA), following the manufacturer's instructions.Gene expression was assessed using qRT-PCR.Ten micrograms of total RNA was treated with the Ambion Turbo DNA-free Kit (ThermoFisher Scientific, Waltham, MA, USA).cDNA was synthesized with Moloney murine leukemia virus (MMLV) reverse transcriptase (Promega, Madison, WI, USA).qRT-PCR was performed with the BioRad CFX96 Real-Time PCR System or Applied Biosystems 7000, using Q SYBR green Supermix (Bio-Rad, Hercules, CA, USA) or SYBR green Jumpstart Taq Ready mix (Sigma, St. Louis, MO, USA) for fluorescence detection.The primer pairs used for qRT-PCR are listed in Table S1.The gene expression data for each gene were normalized to that of the A. fumigatus histone H4 unless specified otherwise.Relative expression levels were calculated using the 2 −∆∆CT method [68].

Virulence Assay
The inoculum was generated as follows: all strains were inoculated on stabilized minimal medium (GMM + 1.2 M sorbitol) and grown at 37 • C for 5 days.Conidia were collected by flooding the plates with 0.01% Tween-80 and scraping them with sterile cotton tip applicators.The conidia were filtered through Miracloth, washed twice with sterile water, and resuspended in sterile phosphate-buffered saline (PBS, ThermoFisher Scientific, Waltham, MA, USA).
Outbred female CD-1 mice (Charles River), six to eight weeks old, weighing 18-24 g, were used for the virulence experiments.For a neutropenic mouse model, immunosuppression was carried out via intraperitoneal injection with 175 mg/kg cyclophosphamide (Baxter, Deerfield, IL, USA) two days before inoculation [69], followed by a subcutaneous injection 40 mg/kg of triamcinolone acetonide (Kenalog-10 from Bristol-Myers Squibb, Lawrenceville, NJ, USA) one day before inoculation with A. fumigatus spores.Immunosuppressed mice were briefly anesthetized using a small rodent anesthesia machine and inoculated with A. fumigatus wild-type, ∆osaA, osaA Com, or osaA OE in the concentration of 2 × 10 6 spores in 40 µL of PBS.A control group of five mice were immunosuppressed and injected with PBS.All the mice were monitored three times daily from days 1 to 7 and once daily from days 8 to 14. Injections with cyclophosphamide and Kenalog were repeated on days +3 and +6, respectively.A statistical comparison of the associated Kaplan-Meier curves was conducted with the log-rank test using GraphPad Prism (version 8.0).
For the corticosteroid-induced immune suppression mouse model, mice were immunosuppressed with a single 40 mg/kg injection of Kenalog-10 one day before infection with A. fumigatus conidia.Mice were monitored three times daily from days 1 to 7 and once daily from days 8-14.A statistical comparison of the associated Kaplan-Meier curves was also conducted with the log-rank test using GraphPad Prism (version 8.0).
This study was carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Research Council.The protocol was approved by the Institutional Animal Care and Use Committee of Clemson University (Permit #AUP2022-0236).All efforts were made to minimize suffering.Humane euthanasia via CO 2 inhalation was performed when mice met the criteria indicating a moribund state; these endpoints include behaviors of unresponsiveness to tactile stimuli, inactivity, lethargy, staggering, anorexia, and/or clinical signs of bleeding from the nose or mouth, labored breathing, agonal respirations, a purulent exudate from the eyes or nose, abnormally ruffled fur, or greater than 20% weight loss.The method of euthanasia via CO 2 inhalation is consistent with recommendations of the Panel on Euthanasia of the American Veterinary Medical Association.

Statistical Analysis
Statistical analysis was carried out using analysis of variance (ANOVA) in conjunction with Tukey's multiple test comparison test, unless stated otherwise.

Identification of OsaA in A. fumigatus
The OsaA protein (corresponding to AFUB_093810) of A. fumigatus is conserved with respect to homologs in other species of the genus Aspergillus (Figures 1 and 2, and Table 2).Our BLASTP analysis demonstrated that A. fumigatus OsaA presents the highest similarity to Aspergillus fischeri (98.96% percent identity, accession number: XP_001257654.1),followed by the homolog in Aspergillus flavus (82.32% identity, accession number: AFLA_004600).The OsaA in A. nidulans (AN6578) showed a 64.24% identity with OsaA in A. fumigatus (Table 2).Our BLASTP analysis also revealed the high conservation of OsaA with other species within Ascomycota, beyond Aspergillus.The sequence alignment of A. fumigatus OsaA with other fungal homologs showed the conservation of the WOPR domain (Figure 1).The WOPR domain in A. fumigatus OsaA was divided into two regions (9-94 and 158-222 residues), connected to each other by an interlinked sequence.Conserved domain analysis revealed the presence of a Gti1/Pac2 domain (Accession pfam09729) in the N-terminal region of the protein.Members of this family are orthologs of Saccharomyces pombe, such as the Pac2 WOPR fragment [56].
OsaA with other species within Ascomycota, beyond Aspergillus.The sequence alignm of A. fumigatus OsaA with other fungal homologs showed the conservation of the WO domain (Figure 1).The WOPR domain in A. fumigatus OsaA was divided into two regi (9-94 and 158-222 residues), connected to each other by an interlinked sequence.C served domain analysis revealed the presence of a Gti1/Pac2 domain (Access pfam09729) in the N-terminal region of the protein.Members of this family are orthol of Saccharomyces pombe, such as the Pac2 WOPR fragment [56].To determine the function of osaA in A. fumigatus, an osaA deletion, ∆osaA, as well as complementation and overexpression strains were generated (Figure S1).A diagnostic PCR confirmed the gene replacement of osaA with the pyrG marker and obtained the expected 2.64 kb product.PCR was also used to verify the integration of the wild-type allele in the ∆osaA strain in the Com strain, amplifying a 1.4 kb product (Figure S1).In addition, osaA overexpression strains were confirmed via a PCR amplifying the expected 1.97 kb product.
An evaluation of the colony growth revealed a significant reduction in colony diameter in the osaA deletion mutant compared to the wild-type (Figure 3).The back of the mutant colonies showed dark pigmentation, which was not present in the wild-type colonies.The complementation of ∆osaA with the osaA wild-type allele recovered the wild-type phenotype.The constitutive overexpression of osaA also reduced colony growth but to a lesser extent.The back of the osaA overexpression strain colony exhibited a yellowish pigmentation and distinct furrowing when compared to the wild-type colony.to a lesser extent.The back of the osaA overexpression strain colony exhibited a yellow pigmentation and distinct furrowing when compared to the wild-type colony.Conidia are the inoculum for A. fumigatus infections.Previous studies in A. nidu showed that the production of these asexual air-borne spores was affected by osaA in model fungus [61].In the present study, the role of osaA in conidiation was assessed i fumigatus.The ΔosaA and OE strains showed a significant reduction in conidial produc compared to the wild-type and Com strain, particularly in the deletion strain (Figur Our experiments revealed that both the deletion and overexpression of osaA resulte alterations in expression levels of the transcription factor genes brlA, abaA, and wetA of central regulatory pathway leading to conidiation [70], particularly in the absence of o resulting in a significant reduction (Figure 4).Interestingly, micrographs of ΔosaA sp indicated a significant enlargement of conidial size (57%) compared to spores produ Conidia are the inoculum for A. fumigatus infections.Previous studies in A. nidulans showed that the production of these asexual air-borne spores was affected by osaA in the model fungus [61].In the present study, the role of osaA in conidiation was assessed in A. fumigatus.The ∆osaA and OE strains showed a significant reduction in conidial production compared to the wild-type and Com strain, particularly in the deletion strain (Figure 4).Our experiments revealed that both the deletion and overexpression of osaA resulted in alterations in expression levels of the transcription factor genes brlA, abaA, and wetA of the central regulatory pathway leading to conidiation [70], particularly in the absence of osaA, resulting in a significant reduction (Figure 4).Interestingly, micrographs of ∆osaA spores indicated a significant enlargement of conidial size (57%) compared to spores produced by the wild-type.Furthermore, a reduction in germination rate was observed in the absence of osaA, while the overexpression of osaA resulted in precocious germination (Figure 4).compared to the wild-type and Com strain, particularly in the deletion strain (Figure Our experiments revealed that both the deletion and overexpression of osaA resulted alterations in expression levels of the transcription factor genes brlA, abaA, and wetA of central regulatory pathway leading to conidiation [70], particularly in the absence of os resulting in a significant reduction (Figure 4).Interestingly, micrographs of ΔosaA spo indicated a significant enlargement of conidial size (57%) compared to spores produ by the wild-type.Furthermore, a reduction in germination rate was observed in the sence of osaA, while the overexpression of osaA resulted in precocious germination (Fig 4 ).

osaA Influences A. fumigatus Temperature and Oxidative Stress Resistance
Our results revealed that the absence of osaA alters A. fumigatus thermotolerance (Figure 5).The osaA gene is required for growth at 42 • C.This is relevant since the pathogenic nature of A. fumigatus is attributed to its thermotolerance, allowing this fungus to survive in a warmer environment and to combat human inflammatory responses.The colony growth defect of this mutant is slightly remediated at temperatures lower than 37 • C. We also noted that the knockout mutant showed increased pigmentation compared to the wild-type, particularly when grown at 25 • C and 30 • C. ure 5).The osaA gene is required for growth at 42 °C.This is relevant since the pathogenic nature of A. fumigatus is attributed to its thermotolerance, allowing this fungus to survive in a warmer environment and to combat human inflammatory responses.The colony growth defect of this mutant is slightly remediated at temperatures lower than 37 °C.We also noted that the knockout mutant showed increased pigmentation compared to the wild-type, particularly when grown at 25 °C and 30 °C.An important component in the host's defense against A. fumigatus infections includes the formation of an oxidative stress environment [16,71].In our study, either the absence or constitutive overexpression of osaA both resulted in increased sensitivity to oxidative stress.Although at 10 µM of menadione, the deletion strain colony grew slightly healthier, both deletion and overexpression osaA strain colonies were unable to grow in the presence of 30 µM of menadione, a condition that still allowed for growth in wild-type and Com colonies (Figure 6).An important component in the host's defense against A. fumigatus infections includes the formation of an oxidative stress environment [16,71].In our study, either the absence or constitutive overexpression of osaA both resulted in increased sensitivity to oxidative stress.Although at 10 µM of menadione, the deletion strain colony grew slightly healthier, both deletion and overexpression osaA strain colonies were unable to grow in the presence of 30 µM of menadione, a condition that still allowed for growth in wild-type and Com colonies (Figure 6). Figure 6.osaA is necessary for resistance to oxidative stress.Plates of solid GMM and GMM supplemented with increasing concentrations of menadione (from 0 to 30 µM) were point-inoculated with A. fumigatus wild-type (WT), ΔosaA, complementation (Com) and overexpression (OE) strains and incubated at 37 °C for 5 days.The experiment was conducted with three replicates.

The Effect of osaA on A. fumigatus Cell Wall and Adhesion Capacity
The fungal cell wall is the first point of contact with the host.To elucidate whether osaA influences cell wall integrity, we exposed the deletion and overexpression of osaA strains, as well as their controls, to cell wall stressors.When grown on a medium supplemented with SDS, the growth of the osaA deletion mutant was compromised (Figure S2).

The Effect of osaA on A. fumigatus Cell Wall and Adhesion Capacity
The fungal cell wall is the first point of contact with the host.To elucidate whether osaA influences cell wall integrity, we exposed the deletion and overexpression of osaA strains, as well as their controls, to cell wall stressors.When grown on a medium supplemented with SDS, the growth of the osaA deletion mutant was compromised (Figure S2).In addition, when Congo red was supplemented to the medium, the growth of ∆osaA was severely affected at a concentration of 5 µg/mL and was unable to grow at 10 µg/mL (Figure 7).The growth of the OE strain was also dramatically affected at 25 µg/mL of Congo red. Figure 6.osaA is necessary for resistance to oxidative stress.Plates of solid GMM and GMM supplemented with increasing concentrations of menadione (from 0 to 30 µM) were point-inoculated with A. fumigatus wild-type (WT), ΔosaA, complementation (Com) and overexpression (OE) strains and incubated at 37 °C for 5 days.The experiment was conducted with three replicates.

The Effect of osaA on A. fumigatus Cell Wall and Adhesion Capacity
The fungal cell wall is the first point of contact with the host.To elucidate whether osaA influences cell wall integrity, we exposed the deletion and overexpression of osaA strains, as well as their controls, to cell wall stressors.When grown on a medium supplemented with SDS, the growth of the osaA deletion mutant was compromised (Figure S2).In addition, when Congo red was supplemented to the medium, the growth of ΔosaA was severely affected at a concentration of 5 µg/mL and was unable to grow at 10 µg/mL (Figure 7).The growth of the OE strain was also dramatically affected at 25 µg/mL of Congo red.The presence of osmotic stabilizers (0.6 M KCl, 1 M sucrose, 0.7 M NaCl, or 1.2 M sorbitol) in the medium allowed for the healthier growth of the deletion osaA mutant than in their absence, although the colony growth was still significantly smaller than that of the wild-type growing under the same experimental conditions (Figure S3).
Biofilm formation in the host is relevant in A. fumigatus resistance to anti-fungal drugs and to evade the host immune system.Importantly, adhesion to surfaces is required for the formation of biofilm [60].Our analysis indicated a delay in the capacity to bind to abiotic surfaces in the absence of osaA at 24 h after inoculation (Figure S4).At 48 h, the adhesion capacity in ∆osaA increased over time; however, it was still significantly lower with respect to that of the wild-type.Interestingly, the overexpression of osaA resulted in a significant loss of adhesion capacity after 48 h.

osaA Regulates Secondary Metabolism in A. fumigatus
Aspergillus fumigatus produces numerous bioactive compounds that serve multiple functions, including those involved in pathogenicity.Our metabolomics analysis revealed that osaA positively regulated secondary metabolism, including the production of fumiquinazoline C, helvolic acid, pyripyropene A, and fumagillin when growing on GMM (Figure 8).In addition, the deletion of osaA completely abolished the production of pseurotin A (Figure S5) when growing on the same medium.In the case of the OE strain, the production of pseurotin A was significantly overproduced compared to the wild-type under these culture conditions (Figure S5).When the YES medium was used (Table 3), the osaA deletion mutant presented a reduction or was unable to produce the above-mentioned metabolites, along with helvolinic acid, 1,2 dihydrohelvolic acid and gliotoxin, which were absent in the deletion mutant.The overexpression of osaA in YES medium leads to a significant decrease or lack of production of fumiquinazoline C, helvolinic acid, 1,2dihydrohelvolic acid, fumagillin, and gliotoxin detected in the wild-type strain under the same experimental conditions.fumiquinazoline C, helvolic acid, pyripyropene A, and fumagillin when growing on GMM (Figure 8).In addition, the deletion of osaA completely abolished the production of pseurotin A (Figure S5) when growing on the same medium.In the case of the OE strain, the production of pseurotin A was significantly overproduced compared to the wild-type under these culture conditions (Figure S5).When the YES medium was used (Table 3), the osaA deletion mutant presented a reduction or was unable to produce the above-mentioned metabolites, along with helvolinic acid, 1,2 dihydrohelvolic acid and gliotoxin, which were absent in the deletion mutant.The overexpression of osaA in YES medium leads to a significant decrease or lack of production of fumiquinazoline C, helvolinic acid, 1,2-dihydrohelvolic acid, fumagillin, and gliotoxin detected in the wild-type strain under the same experimental conditions.

Gene Expression Analysis of Secondary Metabolite Genes
Genes from gene clusters associated with the secondary metabolites whose synthesis was found to be osaA-dependent were selected as indicators of cluster activation.Their expression was analyzed in the wild-type, ∆osaA, Com, and OE strains using qRT-PCR.Our results indicated that osaA is necessary for the normal transcription of the fmqD gene, encoding a FAD-dependent oxidoreductase in the fumiquinazoline C biosynthetic gene cluster [72].The transcription of the pdsA cyclase gene, involved in the synthesis of helvolic acid [73], and pypC, necessary for the synthesis of pyripyropene A [74], were significantly downregulated in the deletion strain.In addition, the transcription factor gene, fumR [75], involved in the regulation of other genes in the fumagillin gene cluster, displayed a considerable loss of expression when osaA was deleted (Figure 8).Furthermore, our study revealed that osaA was required for the normal transcription of psoA, encoding a polyketide synthase, in the pseurotin A gene cluster [65,76] (Figure S5).Since gliotoxin production was reduced in the ∆osaA strain, we also examined whether the expression of key genes in the gliotoxin gene cluster was osaA-dependent.Our results indicated a drastic reduction in the expression of the Zn(II)2Cys6 binuclear transcription factor gliZ [77] in the absence of osaA with respect to the wild-type control (Figure S6).In addition, the expression of gliP, encoding a nonribosomal peptide synthase [78], was also downregulated in the osaA deletion strain.Interestingly, our results also showed that the expression of the global regulatory genes veA and laeA is osaA-dependent (Figure 9).
Our results indicated that osaA is necessary for the normal transcription of the fmqD gene, encoding a FAD-dependent oxidoreductase in the fumiquinazoline C biosynthetic gene cluster [72].The transcription of the pdsA cyclase gene, involved in the synthesis of helvolic acid [73], and pypC, necessary for the synthesis of pyripyropene A [74], were significantly downregulated in the deletion strain.In addition, the transcription factor gene, fumR [75], involved in the regulation of other genes in the fumagillin gene cluster, displayed a considerable loss of expression when osaA was deleted (Figure 8).Furthermore, our study revealed that osaA was required for the normal transcription of psoA, encoding a polyketide synthase, in the pseurotin A gene cluster [65,76] (Figure S5).
Since gliotoxin production was reduced in the ΔosaA strain, we also examined whether the expression of key genes in the gliotoxin gene cluster was osaA-dependent.Our results indicated a drastic reduction in the expression of the Zn(II)2Cys6 binuclear transcription factor gliZ [77] in the absence of osaA with respect to the wild-type control (Figure S6).In addition, the expression of gliP, encoding a nonribosomal peptide synthase [78], was also downregulated in the osaA deletion strain.Interestingly, our results also showed that the expression of the global regulatory genes veA and laeA is osaA-dependent (Figure 9).

osaA Is Indispensable for Normal A. fumigatus Virulence in Both Neutropenic and Corticosteroid-Immunodepressed Murine Models
Since A. fumigatus osaA affects multiple cellular processes in this fungus, including those influencing pathogenicity, we tested whether osaA is involved in virulence (Figure 10).Importantly, the groups infected with spores from the deletion osaA mutant showed higher survival rates when compared to the groups infected with wild-type spores, indicating that osaA is indispensable for virulence in either neutropenic or corticosteroid immunerepressed environments.The complementation strain presented a similar virulence pattern to that of the wild-type strain in both experiments, indicating the recovery of the wild-type phenotype when the osaA wild-type allele was reintroduced in the deletion mutant.The overexpression of osaA resulted in a slight reduction in the mortality rate in the neutropenic model, while, in the corticosteroid model, OE presented a similar mortality rate to that of the wild-type.

osaA Is Indispensable for Normal A. fumigatus Virulence in Both Neutropenic and Corticosteroid-Immunodepressed Murine Models
Since A. fumigatus osaA affects multiple cellular processes in this fungus, including those influencing pathogenicity, we tested whether osaA is involved in virulence (Figure 10).Importantly, the groups infected with spores from the deletion osaA mutant showed higher survival rates when compared to the groups infected with wild-type spores, indicating that osaA is indispensable for virulence in either neutropenic or corticosteroid immune-repressed environments.The complementation strain presented a similar virulence pattern to that of the wild-type strain in both experiments, indicating the recovery of the wild-type phenotype when the osaA wild-type allele was reintroduced in the deletion mutant.The overexpression of osaA resulted in a slight reduction in the mortality rate in the neutropenic model, while, in the corticosteroid model, OE presented a similar mortality rate to that of the wild-type.CD-1 mice.(A) For the neutropenic model, mice received an intraperitoneal injection of cyclophosphamide and Kenalog, as detailed in the Section 2. Uninfected controls were treated with cyclophosphamide and Kenalog but not fungal spores.Post-infection, mice were observed three times daily for the first 7 days and once daily from days 8 to 14.Each group included 10 mice.Statistical analysis was performed utilizing GraphPad PRISM and the Mantel-Cox test.Different letters indicate that values are statistically different.Survival was significantly different between ∆osaA and WT (p < 0.0001) and OE and WT (p < 0.0001).(B) For the non-neutropenic model, mice received only Kenalog one day prior to the infection.Uninfected controls were treated with Kenalog but not fungal spores.Mice were monitored for survival for 14 days.Statistical analysis was performed using GraphPad PRISM statistics.This was significantly different between ∆osaA and WT (p < 0.0001) and between OE and WT (p ≤ 0.001).

Discussion
Transcriptional regulators with a WOPR domain are found across different fungal genera [79] and have been known to act as master regulators governing morphological development, sporogenesis, and pathogenesis [55][56][57][58][59][60].Our work shows that OsaA in A. fumigatus includes a WOPR domain in the N-terminal region.This domain is present in two sub-regions and interconnected with a short linker region, as is the case in the same conserved domain in the C. albicans Wor1 protein [55,79,80].
In closely related dimorphic fungi, H. capsulatum and Coccidioides spp., the WOPR protein Ryp1 is involved in regulating host responses to temperature that result in a switch between two phenotypic forms necessary for the organism's commensalism [81,82].Specifically, in the absence of Ryp1, the mycelial form of H. capsulatum is genetically dominant at 37 • C [57], whereas, in Coccidioides, it maintains genetic development in the environmental hyphae form as well as the pathogenic spherulated form [81].In A. nidulans, the role of osaA was identified to be a crucial factor in development.Our analysis also indicated that although A. fumigatus osaA presents commonalities regarding its function in other species, including A. nidulans, it also presents some distinct roles.For example, as in A. nidulans [61], osaA is important for proper conidiation in A. fumigatus; however, osaA also has a role in germination and vegetative growth in this opportunistic pathogen.
Importantly, in this study, we demonstrated that osaA is indispensable for virulence in both neutropenic and corticosteroid-induced murine models.The pathogenicity of A. fumigatus is associated with multiple virulent factors; some of these factors are affected by osaA.Our microscopic observation indicated that the spore size of the deletion osaA strain is larger than that of the wild-type strain.The small spore size of A. fumigatus allows them to easily enter the respiratory tract of humans and other animals.Spore enlargement in the absence of osaA could reduce the capability to reach the lung alveoli, reducing systemic infection [37,83,84].In addition, decreased thermotolerance in a strain with an osaA-altered locus could negatively impact its ability to grow inside mammalian lungs [85,86].Recent studies have also indicated that thermotolerance regulatory genes play a vital role in the A. fumigatus cell wall integrity pathway [86].Our results revealed that both the deletion and overexpression of osaA (particularly the former) are more susceptible to cell wall stressors, suggesting a loss of integrity.The addition of osmotic stabilizers to the deletion of osaA cultures partially restored colony growth, further supporting the role of osaA in the cellwall function.Importantly, osaA affects the capacity of the fungus to adhere to surfaces, a condition necessary for the formation of biofilm, which is crucial for fungal pathogenicity, protecting the fungus from external stresses, including antifungal agents [40,87,88].
During A. fumigatus infection, the host immune system elicits an elevated production of the macrophage and neutrophil-mediated killing of the conidia, one of which is through the generation of reactive oxygen species (ROS) [16].Here, we also explored whether osaA is necessary for resistance to oxidative stress in A. fumigatus.Our results showed an increase in sensitivity to high levels of menadione in the deletion and overexpression of osaA strains compared to the wild-type.The addition of menadione in the medium simulates the production of ROS inside the cell [89,90], thus creating an oxidative stressful condition.This higher sensitivity to ROS in A. fumigatus could influence the survival of the infected host.
In addition, A. fumigatus is known to produce numerous secondary metabolites that impact host defenses at different stages [91,92].These compounds are known to either suppress or avoid immune sentinel cells [93].Our metabolomics analysis revealed a profound effect of osaA in A. fumigatus secondary metabolism.For example, pseurotin A and fumagillin, both associated with the same biosynthetic gene cluster [57,94], are not produced in the osaA mutant.This is relevant, as fumagillin alters neutrophil activity [95], and pseurotin A is an antioxidant [96].It is possible that the lack of pseurotin A in the osaA mutant could contribute to the observed higher sensitivity to ROS in this strain.Other compounds not produced in the osaA deletion strain and produced at significantly lower levels when osaA is overexpressed are helvolic acid, a cilioinhibitor, and the derivatives helvolinic acid and 1,2-dihydrohelvolic acid, which presents antibacterial properties against Streptococcus aureus [97], fumiquinazoline, which presents cytotoxic activity, and pyripyropenes, an acyl-CoA/cholesterol acyltransferase inhibitor [98][99][100][101].Furthermore, the production of gliotoxin was severely compromised when osaA was differentially expressed in A. fumigatus.Gliotoxin's role in A. fumigatus infection has been previously studied in depth [8,16,93,102], where it was shown to affect host macrophage phagocytosis and to inhibit the activity of other immune cells [93].
The osaA gene was required for the expression of numerous genes in biosynthetic gene clusters associated with the production of secondary metabolites; for instance, gliZ and gliP [77,78], involved in the production of gliotoxin, and fmqD, pdsA, pypC, and fumR, involved in the synthesis of fumiquinazoline C, helvolic acid, pyripyropene, and fumagillin, respectively [65,72,73,75,76,103] were downregulated in the osaA deletion mutant with respect to the wild-type.Based on our results, the effect of osaA on the expression of these gene clusters could be, at least in part, mediated by osaA's influence on the expression of the global regulatory genes veA and laeA.VeA and LaeA are components of the velvet protein complex [16,48], which governs several biological processes in numerous fungi, including secondary metabolism and development [48,74,[104][105][106][107][108][109].In A. nidulans, osaA has been shown to regulate the developmental balance between conidiation and sexual reproduction in a veA-dependent manner [61].In A. fumigatus, we observed the downregulation of the expression of the developmental genes brlA, abaA, and wetA in the absence of osaA, which was associated with a decrease in conidiation.Based on our expression analysis results, it is likely that the effect of osaA on veA and laeA in A. fumigatus could result in multiple outputs, affecting secondary metabolism, development, and sensitivity to oxidative stress, among other biological processes [105].

Conclusions
In conclusion, A. fumigatus osaA is indispensable for virulence in neutropenic and corticosteroid-induced immune-repressed environments.The osaA gene influences growth, development, and sensitivity to environmental stressors, such as oxidative and temperature stresses, which could affect pathogenicity in this fungus.Furthermore, osaA was found to positively regulate the capacity of adhesion to surfaces, which is an important characteristic for the formation of biofilm.Importantly, this WOPR domain transcription factor gene is also necessary for the normal expression of secondary metabolite gene clusters and the concomitant synthesis of natural products, many of which are known to play a role during infection, including gliotoxin.In addition, osaA regulates the expression of veA and laeA genes, known global regulators governing development and secondary metabolism in A. fumigatus and other fungi.This study suggests that osaA or its product are promising targets with the potential for use in a control strategy against A. fumigatus infections.

Figure 3 .
Figure 3. osaA influences growth and development in A. fumigatus.(A) The wild-type (WT), Δo complementation strain (Com), and overexpression strain (OE) were point-inoculated on GMM grown in the dark at 37 °C.Photographs were taken after 10 days.(B) Colony diameter was m ured every 2 days.The error bars represent standard errors.Different letters on the columns ind values that are statistically different (p < 0.05), as determined using one-way ANOVA with Tuk test comparison.

Figure 3 .
Figure 3. osaA influences growth and development in A. fumigatus.(A) The wild-type (WT), ∆osaA, complementation strain (Com), and overexpression strain (OE) were point-inoculated on GMM and grown in the dark at 37 • C. Photographs were taken after 10 days.(B) Colony diameter was measured every 2 days.The error bars represent standard errors.Different letters on the columns indicate values that are statistically different (p < 0.05), as determined using one-way ANOVA with Tukey's test comparison.

Figure 4 .
Figure 4. osaA regulates conidial production and spore size.(A) Conidial quantification of top-ag inoculated plates grown at 37 °C for 24 h, 48 h, and 72 h.Conidia were observed under a bright fi microscope and counted with a hemocytometer.(B) Germination rate assessment.Liquid GM cultures were grown at 37 °C under shaking conditions.Germination was evaluated every 2 h un the microscope using a hemocytometer.(C) Measurements of spore diameter (n = 40 for each stra

Figure 4 .
Figure 4. osaA regulates conidial production and spore size.(A) Conidial quantification of top-agarinoculated plates grown at 37 • C for 24 h, 48 h, and 72 h.Conidia were observed under a bright field microscope and counted with a hemocytometer.(B) Germination rate assessment.Liquid GMM cultures were grown at 37 • C under shaking conditions.Germination was evaluated every 2 h under the microscope using a hemocytometer.(C) Measurements of spore diameter (n = 40 for each strain).Error bars represent the standard error.Different letters on the columns indicate values that are statistically different (p < 0.05).(D) Micrographs of A. fumigatus wild-type (WT), ∆osaA, osaA complementation (Com), and osaA overexpression (OE) conidia.The scale bar represents 10 µm.(E-G) Gene expression analysis of developmental genes brlA (E), abaA (F), and wetA (G).WT, ∆osaA, Com, and OE strains were grown in GMM liquid stationary cultures at 37 • C for 48 h.Expression was calculated using the 2 −∆∆CT method [68].Error bars represent the standard error, and different letters on the bar represent significantly different values (p < 0.05).Color key in (B) also applies to (A-C) and (E-G).

Figure 5 .
Figure 5. Thermotolerance is affected by osaA in A. fumigatus.A. fumigatus wild-type (WT), ΔosaA, complementation (Com), and overexpression (OE) strains were point-inoculated on GMM and incubated in the dark at 25 °C, 30 °C, 37 °C, or 42 °C.Photographs were taken 5 days after inoculation.The experiment was carried out with three replicates.

Figure 5 .
Figure 5. Thermotolerance is affected by osaA in A. fumigatus.A. fumigatus wild-type (WT), ∆osaA, complementation (Com), and overexpression (OE) strains were point-inoculated on GMM and incubated in the dark at 25 • C, 30 • C, 37 • C, or 42 • C. Photographs were taken 5 days after inoculation.The experiment was carried out with three replicates.

Figure 6 .
Figure 6.osaA is necessary for resistance to oxidative stress.Plates of solid GMM and GMM supplemented with increasing concentrations of menadione (from 0 to 30 µM) were point-inoculated with A. fumigatus wild-type (WT), ∆osaA, complementation (Com) and overexpression (OE) strains and incubated at 37 • C for 5 days.The experiment was conducted with three replicates.

Figure 7 .
Figure 7. Effect of osaA in response to cell wall stress.A. fumigatus wild-type (WT), ΔosaA, complementation (Com), and overexpression (OE) strains were point-inoculated on GMM and GMM supplemented with different concentrations of Congo red.Plates were incubated at 37 °C for 72 h.The experiment was carried out in triplicates.

Figure 7 .
Figure 7. Effect of osaA in response to cell wall stress.A. fumigatus wild-type (WT), ∆osaA, complementation (Com), and overexpression (OE) strains were point-inoculated on GMM and GMM supplemented with different concentrations of Congo red.Plates were incubated at 37 • C for 72 h.The experiment was carried out in triplicates.

Figure 8 .Figure 8 .
Figure 8. osaA regulates the production of (A) fumiquinazoline C, (B) helvolic acid, (C) pyripyropene A and (D) fumagillin in A. fumigatus.The A. fumigatus wild-type (WT), ΔosaA, complementation (Com), and overexpression (OE) strains were top-agar-inoculated on GMM and incubated at 37 °C for 72 h.Extracts were analyzed using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS).On the right in each panel, the expression of key genes in the corresponding secondary metabolite genes clusters, fmqD, pdsA, pypC, and fumR, respectively, were selected as indicators of cluster activation.Samples were obtained from GMM liquid stationary cultures incubated for 48 h.Expression was calculated following the 2 −ΔΔCT method [68].Error bars represent the standard error.Different letters on the columns indicate values that are statistically different (p < Figure 8. osaA regulates the production of (A) fumiquinazoline C, (B) helvolic acid, (C) pyripyropene A and (D) fumagillin in A. fumigatus.The A. fumigatus wild-type (WT), ∆osaA, complementation (Com), and overexpression (OE) strains were top-agar-inoculated on GMM and incubated at 37 • C for 72 h.Extracts were analyzed using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS).On the right in each panel, the expression of key genes in the corresponding secondary metabolite genes clusters, fmqD, pdsA, pypC, and fumR, respectively, were selected as indicators of cluster activation.Samples were obtained from GMM liquid stationary cultures incubated for 48 h.Expression was calculated following the 2 −∆∆CT method [68].Error bars represent the standard error.Different letters on the columns indicate values that are statistically different (p < 0.05), as determined using one-way ANOVA with Tukey's test comparison.All the experiments were performed in triplicates.

Figure 9 .
Figure 9. osaA affects the gene expression of the global regulators veA and laeA in A. fumigatus.A. fumigatus wild-type (WT), ΔosaA, complementation (Com) and overexpression (OE) strains were grown on GMM stationary cultures for 48 h.The relative gene expression of (A) veA and (B) laeA was calculated using the 2 −ΔΔCT method [68].Error bars represent the standard error.Different letters on the columns indicate values that are statistically different (p < 0.05).

Figure 9 .
Figure 9. osaA affects the gene expression of the global regulators veA and laeA in A. fumigatus.A. fumigatus wild-type (WT), ∆osaA, complementation (Com) and overexpression (OE) strains were grown on GMM stationary cultures for 48 h.The relative gene expression of (A) veA and (B) laeA was calculated using the 2 −∆∆CT method [68].Error bars represent the standard error.Different letters on the columns indicate values that are statistically different (p < 0.05).

Figure 10 .
Figure 10.osaA is indispensable for A. fumigatus virulence in both neutropenic and corticosteroidinduced immune-repressed murine models.The A. fumigatus wild-type (WT), ΔosaA, complementation (Com), and overexpression (OE) strains (2 × 10 6 spores/40 µL PBS) were used to infect female outbred CD-1 mice.(A) For the neutropenic model, mice received an intraperitoneal injection of cyclophosphamide and Kenalog, as detailed in the Materials and Methods section.Uninfected controls were treated with cyclophosphamide and Kenalog but not fungal spores.Post-infection, mice were observed three times daily for the first 7 days and once daily from days 8 to 14.Each group included 10 mice.Statistical analysis was performed utilizing GraphPad PRISM and the Mantel-Cox test.Different letters indicate that values are statistically different.Survival was significantly different between ΔosaA and WT (p < 0.0001) and OE and WT (p < 0.0001).(B) For the non-neutropenic model, mice received only Kenalog one day prior to the infection.Uninfected controls were treated with Kenalog but not fungal spores.Mice were monitored for survival for 14 days.Statistical

Table 1 .
Strains used in this study.

Table 2 .
Percentage of identity and similarity of A. fumigatus OsaA with homologous proteins in species within the Ascomycota phylum.
Figure 2. Phylogenetic tree of OsaA homologs from different fungal species.Construction of a phylogenetic tree was carried out using MEGA v11.0.The tree was generated with the maximum likelihood model with a bootstrap value of 1000 (http://megasoftware.net/, 11 June 2023).

Table 2 .
Percentage of identity and similarity of A. fumigatus OsaA with homologous proteins in species within the Ascomycota phylum.