Deletion of Aspergillus nidulans GDP-mannose transporters affects hyphal morphometry, cell wall architecture, spore surface character, cell adhesion, and biofilm formation

Deletion of Aspergillus nidulans GDP-mannose transporters affects hyphal morphometry, cell wall... Abstract Systemic human fungal infections are increasingly common. Aspergillus species cause most of the airborne fungal infections. Life-threatening invasive aspergillosis was formerly found only in immune-suppressed patients, but recently some strains of A. fumigatus have become primary pathogens. Many fungal cell wall components are absent from mammalian systems, so they are potential drug targets. Cell-wall-targeting drugs such as echinocandins are used clinically, although echinocandin-resistant strains were discovered shortly after their introduction. Currently there are no fully effective anti-fungal drugs. Fungal cell wall glycoconjugates modulate human immune responses, as well as fungal cell adhesion, biofilm formation, and drug resistance. Guanosine diphosphate (GDP) mannose transporters (GMTs) transfer GDP-mannose from the cytosol to the Golgi lumen prior to mannosylation. Aspergillus nidulans GMTs are encoded by gmtA and gmtB. Here we elucidate the roles of A. nidulans GMTs. Strains engineered to lack either or both GMTs were assessed for hyphal and colonial morphology, cell wall ultrastructure, antifungal susceptibility, spore hydrophobicity, adherence and biofilm formation. The gmt-deleted strains had smaller colonies with reduced sporulation and with thicker hyphal walls. The gmtA deficient spores had reduced hydrophobicity and were less adherent and less able to form biofilms in vitro. Thus, gmtA not only participates in maintaining the cell wall integrity but also plays an important role in biofilm establishment and adherence of A. nidulans. These findings suggested that GMTs have roles in A. nidulans growth and cell-cell interaction and could be a potential target for new antifungals that target virulence determinants. GDP-mannose transporters, Aspergillus nidulans, spore hydrophobicity, adherence, biofilm ABBREVIATIONS ABBREVIATIONS CR Congo red CV crystal violet GDP Guanosine diphosphate GMT GDP mannose transporter RMPI Roswell Park Memorial Institute SDA Sabourand dextrose agar SEM Scanning electron microscope TEM transmission electron microscope Introduction Fungi cause superficial, subcutaneous, and systemic human infections, the latter especially in immune-compromised patients.1,2 Fungal infections kill more than 1.3 million people annually, comparable to drug-resistant Mycobacterium tuberculosis.3Aspergillus fumigatus is the most common airborne fungal pathogen, and now some A. fumigatus strains even cause invasive pulmonary disease in immune-competent people.4Aspergillus nidulans is a genetically tractable congeneric of A. fumigatus that has overall similar cell wall sugar and protein composition.5,6 The cell wall is essential for fungal survival and virulence. Many wall components are not found in humans, so the proteins synthesizing the cell wall components are potential targets for antifungal drug development.7 Echinocandin-class antifungals targets wall β-glucan synthesis by inhibiting fksA8 and are fungicidal in Candida spp. and fungistatic in Aspergillus spp. However, clinically-relevant echinocandin-resistant strains were identified shortly after wide-spread therapeutic use began.9 Due to the elevated problem of antifungal resistance, there is a new paradigm to target virulence determinants rather than targeting essential processes to reduce evolution of resistance.10 Glycosylation is a post-translational modification of some membrane and wall components that is important for growth, virulence, and immunogenicity of many pathogens including Aspergillus.11–15 Mannosylation is one of the glycosylation processes, in which mannan is added to proteins or other carbohydrates to form different glycoconjugates. Mannan is an essential component in many fungal species. In Aspergillus, mannan is not present as a homogeneous mannose polymer but as galactomannan (GM) or mannoproteins.16 GM is covalently bound to the glucan–chitin core of the cell wall, found in both the inner and outer layers of the cell wall and can be used to diagnose invasive aspergillosis.17 In addition, GM also bound to the plasma membrane by a lipid anchor of glycosylphosphatidylinositol (GPI).18 The guanosine diphosphate (GDP)-mannose transporter (GMT) is required for mannosylation of galactomannan, mannoproteins and the GPI anchor.19 Mammalian cells lack GMTs, making them excellent new targets for antifungal drug development.20 In A. nidulans, GMT is encoded by gmtA and gmtB, 21 both of which co-localize with the Golgi equivalent.22 GMT was reported to be essential in Saccharomyces, Candida, and A. niger.23–25 Although gmtA is not essential for viability of A. fumigatus, its deletion leads to severe growth defects.26 GMT is also required for virulence in Cryptococcus neoformans and the protist Leishmania donovani.27,28 the roles of GMTs in Aspergillus nidulans is not fully understood. Here, we explore the in vitro roles of GMTs in A. nidulans strains that were deleted for gmtA and/or gmtB. Growth morphometry, cell wall ultrastructure, and sensitivity to antifungal drugs were studied. In addition spore hydrophobicity and its effect on adherence and biofilm formation; as two important virulence factors; were assessed in comparison to the wild-type parent strain. Methods Strains and culture conditions Strains used in this study are listed in Table 1.29Aspergillus nidulans strains were maintained on Roswell Park Memorial Institute (RPMI) 1640 agar medium (Sigma Aldrich, St Louis, MO, USA) supplemented with uridine and uracil (Sigma Aldrich, St Louis, MO, USA) for pyrG-deficient strains. Sabourand dextrose agar (SDA) was used as media for the double deletion strain, while SDA plus uridine and uracil was used as media for gmtBΔ strain. Strains were stored in 50% glycerol at –20°C.30 Most procedures were carried out in Thermoscientific class II microbiological safety cabinet. Table 1. Strains included in this study. Aspergillus nidulans strains  Strain genetic characters  1A1148  pyrG89; pyroA4; riboB2; nkuB:: A. fumigatus riboB; nkuA:: argB  2gmtAΔ  pyrG89; pyroA4; riboB2; nkuB:: A. fumigatus riboB; nkuA::argB; AN8848:: A. fumigatus pyrG  2gmtBΔ  wA3; pyroA4; argB2; nkuA::argB; AN9298:: A. fumigatus pyroA.  2gmtAΔ- gmtBΔ  pyrG89; pyroA4; riboB2; nkuB::A. fumigatus riboB; nkuA::argB; AN8848::A. fumigatus pyrG; AN9298:: A. fumigatus pyroA  Aspergillus nidulans strains  Strain genetic characters  1A1148  pyrG89; pyroA4; riboB2; nkuB:: A. fumigatus riboB; nkuA:: argB  2gmtAΔ  pyrG89; pyroA4; riboB2; nkuB:: A. fumigatus riboB; nkuA::argB; AN8848:: A. fumigatus pyrG  2gmtBΔ  wA3; pyroA4; argB2; nkuA::argB; AN9298:: A. fumigatus pyroA.  2gmtAΔ- gmtBΔ  pyrG89; pyroA4; riboB2; nkuB::A. fumigatus riboB; nkuA::argB; AN8848::A. fumigatus pyrG; AN9298:: A. fumigatus pyroA  1: Fungal genetics stock center (FGSC). 2: El-Ganiny et al. 2015. View Large Table 1. Strains included in this study. Aspergillus nidulans strains  Strain genetic characters  1A1148  pyrG89; pyroA4; riboB2; nkuB:: A. fumigatus riboB; nkuA:: argB  2gmtAΔ  pyrG89; pyroA4; riboB2; nkuB:: A. fumigatus riboB; nkuA::argB; AN8848:: A. fumigatus pyrG  2gmtBΔ  wA3; pyroA4; argB2; nkuA::argB; AN9298:: A. fumigatus pyroA.  2gmtAΔ- gmtBΔ  pyrG89; pyroA4; riboB2; nkuB::A. fumigatus riboB; nkuA::argB; AN8848::A. fumigatus pyrG; AN9298:: A. fumigatus pyroA  Aspergillus nidulans strains  Strain genetic characters  1A1148  pyrG89; pyroA4; riboB2; nkuB:: A. fumigatus riboB; nkuA:: argB  2gmtAΔ  pyrG89; pyroA4; riboB2; nkuB:: A. fumigatus riboB; nkuA::argB; AN8848:: A. fumigatus pyrG  2gmtBΔ  wA3; pyroA4; argB2; nkuA::argB; AN9298:: A. fumigatus pyroA.  2gmtAΔ- gmtBΔ  pyrG89; pyroA4; riboB2; nkuB::A. fumigatus riboB; nkuA::argB; AN8848::A. fumigatus pyrG; AN9298:: A. fumigatus pyroA  1: Fungal genetics stock center (FGSC). 2: El-Ganiny et al. 2015. View Large Colony growth and hyphal morphometry The colony diameter and number of spores per colony were measured for wild-type and deleted strains as described previously.31 Briefly, strains were streaked on RPMI media and incubated for 3 d at 28°C to give isolated colonies. The diameter of 10 colonies/strain was measured to the nearest millimeter. The number of spores per colony was counted for four colonies/strain. Isolated colonies were vortexed in a microfuge tube containing 1 ml water, then samples of spores were counted with a hemocytometer. For hyphal morphometry, hyphae were grown at 37°C for 16 h and examined using Leica light microscopy (DM500). Imaging used a Leica ICC50 HD camera and Leica DM500 image analysis software. Hyphal width was measured at septa and basal cell length was measured between adjacent septa (50/strain) as previously described.32 Electron microscopy Scanning electron microscopy (SEM) followed El-Ganiny et al.31 Briefly, strains were grown on dialysis tubing overlaying RPMI agar for 3 d at 28°C. Isolated colonies were vapor-fixed at 100% relative humidity over 4% aqueous osmium tetroxide (OsO4) for 2 h, frozen to −80°C, then lyophilized overnight. Samples were mounted on SEM stubs, gold sputter-coated for 6 min, and examined with a Quanta FEG250 (FE-SEM) in Central Metallurgical Research Institute (Tebbin, Helwan, Egypt). The accelerating voltage was 20 kV. The beam current for sample examination was 1.5 nA, and for image acquisition it was 50 pA. Transmission electron microscopy (TEM) samples were prepared and imaged as described previously.28 Strains were grown submerged in liquid medium for 16 h at 28°C. Cells were fixed for 1 h in 1% glutaraldehyde in 50 mM phosphate buffer, pH 7, then post-fixed for 1 h in 1% OsO4 in phosphate buffer. Samples were dehydrated in a gradient ethanol series, then transferred to 100% anhydrous acetone, and embedded in epoxy resin. Sections of 70–100 nm thickness were cut with a Leica Ultracut UCT microtome and then stained with uranyl acetate and lead citrate as described previously.30 Stained sections were examined using a JEOL JEM-1400 at the Faculty of Agriculture, Cairo University Research Park (FA-CURP). Images were captured by CCD camera model AMT. Transverse TEM sections of wild-type and deleted strains hyphae (with well resolved cell membrane) were used to compare hyphal diameter and wall thickness. Antifungal susceptibility testing Drug sensitivity of wild-type, gmtAΔ, gmtBΔ and gmtAΔ-gmtBΔ strains was compared using disk diffusion method.33 Briefly; strains were grown on RPMI agar for 4 d. Spores were collected, and the suspension was filtered using sterile filter paper to remove any hyphae or conidiophores and counted by hemocytometer. Then 1 × 107 freshly harvested spores were mixed with 20 ml of molten RPMI agar and poured into Petri plates. After the medium had hardened, 6 mm sterile blank paper disks (BBL, Becton Dickinson, Cockeysville, MD, USA) were placed on agar surface. Tested commercial antifungals were from four drug classes: polyenes (amphotericin B; Bristol-Meyers Squibb, France), allylamines (terbinafine; Mash Premier, Egypt), azoles (voriconazole; Pfizer, Ireland), and echinocandins (micafungin; Astellas Toyama, Japan). Stock solutions were prepared using sterile water, except terbinafine in 50% ethanol (Alnasr chemicals, Egypt) in the following concentrations: amphotericin B and micafungin (20 mg/ml), voriconazole and terbinafine (1.6 mg/ml). Then 2 μl of each antifungal drug stock solution was pipetted onto individual disks; control solvent disc was also included. Also, Congo red (CR) was tested as it interferes with chitin crystallization in the cell wall and hence has antifungal effects.34 CR stock solution contain 10 mg/ml in water; 10 μl was added to blank disc surface.33 The plates were incubated at 37 °C for 48 h. The radius of the zone of inhibition (showing no fungal growth) was calculated as [inhibition diameter – 6 mm]/2. Five replicates were measured for each strain. Spores hydrophobicity assay Hydrophobicity assay was performed as described previously.35 Briefly, spores were collected from 4 day-old colonies, and the number of spores was counted using a hemocytometer. Equal number of spores were resuspended in 3 ml mineral oil-water mix (1:1 v/v) and shaken vigorously. After settlement, the number of spores remaining in the water was counted and the percent portioning in oily phase was calculated as: (1- N/N0) × 100, where N0 and N were the initial number and final number of spores in the aqueous phase after partitioning, respectively, the hydrophobicity of Gmt-deleted strains was calculated relative to that of wild-type.36 The results are the average of three independent experiments. Adherence of A. nidulans spores to human epithelial cells For epithelial cells collection, a swab with a rayon tip and plastic applicator (167KS01; Copan Italia S.P.A., Brescia, Italy) was used to obtain a sample of the posterior oropharyngeal mucosal membrane from healthy volunteers.37 Collected swabs were soaked in phosphate-buffered saline (PBS), vortexed, and then centrifuged at 350 rpm for 10 min. The sediment was washed twice in PBS and resuspended in RPMI medium. The number of cells was estimated with a hemocytometer and standardized to 105 cells/ml. Also, A. nidulans spores were collected from 4 day-old colonies, and the number of spores was counted using hemocytometer and adjusted to 106 spore/ml. A. nidulans spores were tested for their adherence ability to epithelial cells in 96 well microtiter plates as described previously with some modifications.38 Briefly, the microtiter plates were inoculated with 100 μl of previously prepared epithelial cells. After 24 h incubation at 37°C, the unattached epithelial cells were washed once with PBS. Each well containing attached epithelial cells were inoculated with 100 μl of A. nidulans spore suspension, and the mixture was incubated at 37°C for 60 min. The unattached spores were washed out with PBS three times, and the remaining attached spores were fixed with 3.7% formaldehyde for 15 min. Adherence of spores was estimated by staining with 150 μl of 0.5% (w/v) Crystal Violet (CV) solution for 15 min. Excess stain was gently removed under running water then the wells were destained by adding 100 μl of glacial acetic acid. The adherence of spores was estimated by determining the absorbance of the destaining solution at 590 nm using Synergy HT ELISA reader and Gen5 1.11 Biotek software. Absorbance values of negative control of liquid media was used as blank. Assays were performed in 3 independent wells on at least three separate days. Adherence and biofilm formation of A. nidulans spores on polystyrene Biofilm study was based on methods described previously.39,40 Briefly, 96-well microtiter plates were inoculated with 100 μl of 105 spores per well in RPMI media, and media-only blanks were also set up. Plates were incubated at 37°C, and analysis was made at 4 and 24 h representing adhesion and biofilm formation, respectively. The medium was removed from each well and the adherent hyphae were washed three times with PBS and then fixed with 3.7% formaldehyde. Adherence and biofilm formation were estimated as described in adherence of spores to human epithelial cells (see the section “Adherence of A. nidulans spores to human epithelial cells”). RESULTS Effect of deleting gmtA, gmtB, and gmtA gmtB on colony growth and sporulation The effect of gmtA deletion on colony morphology was clear, colonies of gmtAΔ are paler in colour than parent wild-type strain (A1148) due to the severely reduced sporulation, but the appearance of hyphal margin was generally similar to the parent strain. Colonies lacking GmtB and the double deletion strain (lacked GmtA and GmtB) had both smaller colonies and fewer spores (Fig. 1A). Compared to the wild-type, sporulation was reduced in the GmtAΔ, GmtBΔ, and [GmtAΔ, GmtBΔ] strains, respectively by about 90-fold, 170-fold, and 230-fold (Table 2). Figure 1. View largeDownload slide Morphology of wild-type and gmt-deleted strains. A: colony morphology, B: SEM showing colony surface, scale bar 100 μm, C: SEM showing conidiophores, scale bar 10 μm, and D: SEM showing spore surface character, scale bar 1 μm. This Figure is reproduced in color in the online version of Medical Mycology. Figure 1. View largeDownload slide Morphology of wild-type and gmt-deleted strains. A: colony morphology, B: SEM showing colony surface, scale bar 100 μm, C: SEM showing conidiophores, scale bar 10 μm, and D: SEM showing spore surface character, scale bar 1 μm. This Figure is reproduced in color in the online version of Medical Mycology. Table 2. Morphometric comparison of wild-type and GMT-deletion strains.   wild-type  gmtAΔ  gmtBΔ  gmtAΔ-gmtBΔ  Colony diameter (mm)  16 ± 0.5  4.2a ± 0.2  3.5a ± 0.4  1.2a ± 0.3  Spores/colony × 106  138 ± 2  1.5a ± 0.5  0.8a ± 0.4  0.6a ± 0.3  Hyphal width (μm)  2.5± 0.3  3.1a ± 0.8  3.7a ± 0.9  4.4a ± 0.5  Basal cell length (μm)  25 ± 0.3  14.7a ± 0.6  15.9a ± 0.1  14.0 a ± 0 .4  Cell wall thickness (nm)  77.5 ± 4.2  117a ± 7.5  124.8a± 10.5  133a ± 8.9    wild-type  gmtAΔ  gmtBΔ  gmtAΔ-gmtBΔ  Colony diameter (mm)  16 ± 0.5  4.2a ± 0.2  3.5a ± 0.4  1.2a ± 0.3  Spores/colony × 106  138 ± 2  1.5a ± 0.5  0.8a ± 0.4  0.6a ± 0.3  Hyphal width (μm)  2.5± 0.3  3.1a ± 0.8  3.7a ± 0.9  4.4a ± 0.5  Basal cell length (μm)  25 ± 0.3  14.7a ± 0.6  15.9a ± 0.1  14.0 a ± 0 .4  Cell wall thickness (nm)  77.5 ± 4.2  117a ± 7.5  124.8a± 10.5  133a ± 8.9  Values are presented as mean ± standard error. a:Values are significantly different from wild-type by Graphpad Prism 6.0 using one-way ANOVA at P < .05. View Large Table 2. Morphometric comparison of wild-type and GMT-deletion strains.   wild-type  gmtAΔ  gmtBΔ  gmtAΔ-gmtBΔ  Colony diameter (mm)  16 ± 0.5  4.2a ± 0.2  3.5a ± 0.4  1.2a ± 0.3  Spores/colony × 106  138 ± 2  1.5a ± 0.5  0.8a ± 0.4  0.6a ± 0.3  Hyphal width (μm)  2.5± 0.3  3.1a ± 0.8  3.7a ± 0.9  4.4a ± 0.5  Basal cell length (μm)  25 ± 0.3  14.7a ± 0.6  15.9a ± 0.1  14.0 a ± 0 .4  Cell wall thickness (nm)  77.5 ± 4.2  117a ± 7.5  124.8a± 10.5  133a ± 8.9    wild-type  gmtAΔ  gmtBΔ  gmtAΔ-gmtBΔ  Colony diameter (mm)  16 ± 0.5  4.2a ± 0.2  3.5a ± 0.4  1.2a ± 0.3  Spores/colony × 106  138 ± 2  1.5a ± 0.5  0.8a ± 0.4  0.6a ± 0.3  Hyphal width (μm)  2.5± 0.3  3.1a ± 0.8  3.7a ± 0.9  4.4a ± 0.5  Basal cell length (μm)  25 ± 0.3  14.7a ± 0.6  15.9a ± 0.1  14.0 a ± 0 .4  Cell wall thickness (nm)  77.5 ± 4.2  117a ± 7.5  124.8a± 10.5  133a ± 8.9  Values are presented as mean ± standard error. a:Values are significantly different from wild-type by Graphpad Prism 6.0 using one-way ANOVA at P < .05. View Large The parental strain had abundant conidiophores with wild-type morphology (Fig. 1B), whereas the GmtAΔ strain had fewer conidiophores and, of those, fewer with normal spore chains (Fig. 1B). The GmtBΔ strain had aerial hyphae, and few mature conidiophores, since many appeared to have stalled at the vesicle stage (Fig. 1B) or produced short spore chains. The [GmtAΔ, GmtBΔ] strain had abundant aerial hyphae, and lower sporulation than single deletion strains (Fig. 1B, C). Figure 1D shows the spore surface for these strains. The spores of wild-type had few ornaments that partially cover the rodlet layer, the spores of gmtAΔ had a rougher surface with more ornaments than the wild-type. But spores of gmtBΔ and gmtAΔ-gmtBΔ lost the ornamented outer layer of wild-type, which cause exposure of the underlying rodlet layer. Hyphal morphometry Hyphal width increased markedly in the gene-deleted strains compared to the wild-type parental strain: wild-type ≪ gmtAΔ < gmtBΔ < gmtAΔgmtBΔ. Basal cell length decreased as hyphal width increased. Morphometry of parental and deletion strains is shown in Table 2 and Figure 2A. Figure 2. View largeDownload slide Hyphae of wild-type and gmt-deleted strains as shown by light microscopy and TEM. A: light microscopy, scale bar 10 μm; B: TEM images, scale bar = 100 nm Figure 2. View largeDownload slide Hyphae of wild-type and gmt-deleted strains as shown by light microscopy and TEM. A: light microscopy, scale bar 10 μm; B: TEM images, scale bar = 100 nm Cell wall ultrastructure TEM images of 16-h old hyphae grown in liquid media demonstrate the effect of gmtA and gmtB deletion on the cell wall ultrastructure. Hyphal wall thickness was increased in the gmt-deleted strains compared to wild-type parental, so that parental ≪ gmtAΔ < gmtBΔ <gmtAΔ-gmtBΔ (Table 2). The wild-type and gmtAΔ strains (grown submerged in liquid cultures) accumulated large amount of exogenous materials that not observed for the gmtBΔ strains (Fig. 2B). The hyphal walls of the wild-type and gmtAΔ strains had two distinct layers while the walls of gmtBΔ and gmtAΔ-gmtBΔ strains appear as one diffused layer (Fig. 2B). Susceptibility to antifungal drugs Micafungin susceptibility (target β-1, 3-glucan synthesis) was compared with drugs that target ergosterol (amphotericin B) or ergosterol biosynthesis (voriconazole and terbinafine). There were no significant changes in the sensitivity to amphotericin B, voriconazole or terbinafine. In contrast, reduced sensitivity to micafungin was noticed in gmtAΔ, gmtBΔ, and gmtAΔ-gmtBΔ compared to wild-type. Regarding CR, there was a slight increase in the sensitivity of the gmtAΔ, gmtBΔ, and gmtAΔ-gmtBΔ strains compared with the wild-type strain (Table 3, Suppl. Fig. 1). Table 3. Sensitivity of wild-type and deletion strains to antifungal drugs.   Radius of inhibition zone in mm    Strains  Amphotericin B  Micafungin  Terbinafine  Voriconazole  Congo Red  wild-type  14 ± 0.2  2 ± 0.4  26 ± 0.3  25 ± 0.3  12.3 ± 0.7  gmtAΔ  16 ± 0.3  1a ± 0.2  26 ± 0.1  27 ± 0.2  12.8 ± 0.4  gmtBΔ  17 ± 0.1  0.5a ± 0.1  26 ± 0.3  26 ± 0.3  13.2 ± 0.4  gmtAΔ-gmtBΔ  16 ± 0.2  0.5a ± 0.1  27 ± 0.2  27 ± 0.1  14.8 ± 0.3    Radius of inhibition zone in mm    Strains  Amphotericin B  Micafungin  Terbinafine  Voriconazole  Congo Red  wild-type  14 ± 0.2  2 ± 0.4  26 ± 0.3  25 ± 0.3  12.3 ± 0.7  gmtAΔ  16 ± 0.3  1a ± 0.2  26 ± 0.1  27 ± 0.2  12.8 ± 0.4  gmtBΔ  17 ± 0.1  0.5a ± 0.1  26 ± 0.3  26 ± 0.3  13.2 ± 0.4  gmtAΔ-gmtBΔ  16 ± 0.2  0.5a ± 0.1  27 ± 0.2  27 ± 0.1  14.8 ± 0.3  Values are presented as mean ± standard error. a:Values are significantly different from wild-type by Graphpad Prism 6.0 using one-way ANOVA at P < .05. View Large Table 3. Sensitivity of wild-type and deletion strains to antifungal drugs.   Radius of inhibition zone in mm    Strains  Amphotericin B  Micafungin  Terbinafine  Voriconazole  Congo Red  wild-type  14 ± 0.2  2 ± 0.4  26 ± 0.3  25 ± 0.3  12.3 ± 0.7  gmtAΔ  16 ± 0.3  1a ± 0.2  26 ± 0.1  27 ± 0.2  12.8 ± 0.4  gmtBΔ  17 ± 0.1  0.5a ± 0.1  26 ± 0.3  26 ± 0.3  13.2 ± 0.4  gmtAΔ-gmtBΔ  16 ± 0.2  0.5a ± 0.1  27 ± 0.2  27 ± 0.1  14.8 ± 0.3    Radius of inhibition zone in mm    Strains  Amphotericin B  Micafungin  Terbinafine  Voriconazole  Congo Red  wild-type  14 ± 0.2  2 ± 0.4  26 ± 0.3  25 ± 0.3  12.3 ± 0.7  gmtAΔ  16 ± 0.3  1a ± 0.2  26 ± 0.1  27 ± 0.2  12.8 ± 0.4  gmtBΔ  17 ± 0.1  0.5a ± 0.1  26 ± 0.3  26 ± 0.3  13.2 ± 0.4  gmtAΔ-gmtBΔ  16 ± 0.2  0.5a ± 0.1  27 ± 0.2  27 ± 0.1  14.8 ± 0.3  Values are presented as mean ± standard error. a:Values are significantly different from wild-type by Graphpad Prism 6.0 using one-way ANOVA at P < .05. View Large Hydrophobicity of spores Spores from wild-type and gmt-deleted strains were shaken in water/mineral oil mixture to determine spore hydrophobicity (Fig. 3A). For each strain, number of spores in aqueous layer were counted and used to calculate the % partitioning in mineral oil and the relative hydrophobicity of Gmt-deleted strains relative to wild-type (Fig. 3B). The gmtAΔ strain shows decreased hydrophobicity while gmtB-deleted strains show increased hydrophobicity. Figure 3. View largeDownload slide Hydrophobicity of gmt-deleted strains relative to wild-type strain. Figure 3. View largeDownload slide Hydrophobicity of gmt-deleted strains relative to wild-type strain. Assessment of spore adhesion to human epithelial cells The adherence to human epithelial cells was assessed for wild-type and gmt-deleted strains by staining with 0.5% CV. There was a significant decrease in absorbance of gmtAΔ strain and a slight increase for gmtBΔ and gmtA-gmtBΔ compared to wild-type (Table 4). Table 4. Adherence on epithelial cells and polystyrene plate and biofilm formation of wild-type and gmt-deleted strains.   % absorbance measured at 590 nm (A590) after  Strain  Adherence to epithelial cells  Adherence to polystyrene plates  In vitro biofilm formation  wild-type  72.33 ± 3.0  13.8 ± 3.7  72.1 ± 5.0  gmtAΔ  52.96a ± 5.0  10.3a ± 1.3  60.7a ± 5.0  gmtBΔ  77.14 ± 3.0  12.2 ± 8.0  93.4b ± 5.0  gmtAΔ-gmtBΔ  76.83 ± 1.0  12.4 ± 2.9  93.3b ± 6.0    % absorbance measured at 590 nm (A590) after  Strain  Adherence to epithelial cells  Adherence to polystyrene plates  In vitro biofilm formation  wild-type  72.33 ± 3.0  13.8 ± 3.7  72.1 ± 5.0  gmtAΔ  52.96a ± 5.0  10.3a ± 1.3  60.7a ± 5.0  gmtBΔ  77.14 ± 3.0  12.2 ± 8.0  93.4b ± 5.0  gmtAΔ-gmtBΔ  76.83 ± 1.0  12.4 ± 2.9  93.3b ± 6.0  a, b: values are significantly different from wild-type and other stains. Statistical analysis was done using Graphpad Prism 6.0 (One Way ANOVA at P ≤ 0.05). View Large Table 4. Adherence on epithelial cells and polystyrene plate and biofilm formation of wild-type and gmt-deleted strains.   % absorbance measured at 590 nm (A590) after  Strain  Adherence to epithelial cells  Adherence to polystyrene plates  In vitro biofilm formation  wild-type  72.33 ± 3.0  13.8 ± 3.7  72.1 ± 5.0  gmtAΔ  52.96a ± 5.0  10.3a ± 1.3  60.7a ± 5.0  gmtBΔ  77.14 ± 3.0  12.2 ± 8.0  93.4b ± 5.0  gmtAΔ-gmtBΔ  76.83 ± 1.0  12.4 ± 2.9  93.3b ± 6.0    % absorbance measured at 590 nm (A590) after  Strain  Adherence to epithelial cells  Adherence to polystyrene plates  In vitro biofilm formation  wild-type  72.33 ± 3.0  13.8 ± 3.7  72.1 ± 5.0  gmtAΔ  52.96a ± 5.0  10.3a ± 1.3  60.7a ± 5.0  gmtBΔ  77.14 ± 3.0  12.2 ± 8.0  93.4b ± 5.0  gmtAΔ-gmtBΔ  76.83 ± 1.0  12.4 ± 2.9  93.3b ± 6.0  a, b: values are significantly different from wild-type and other stains. Statistical analysis was done using Graphpad Prism 6.0 (One Way ANOVA at P ≤ 0.05). View Large Adherence of A. nidulans spores to polystyrene plate and biofilm formation The adherence of A. nidulans spores to polystyrene plate was assessed. The absorbance values were directly proportional to the degree of adherence. After 4 h, adherence of the gmtAΔ was reduced significantly, while adherence of the gmtBΔ, and gmtAΔ-gmtBΔ was comparable to the wild-type (Table 4). The formation of biofilm on polystyrene plates by A. nidulans hyphae was assessed after 24 h. The results showed that absorbance was significantly increased in the double deletion strain, gmtAΔ-gmtBΔ and gmtBΔ indicating increased biofilm formation, while gmtAΔ had the least absorbance value indicating that the biofilm was significantly reduced than wild-type (Table 4). Discussion In Aspergillus, GDP-mannose is the precursor of all the mannosylation processes.26,42 GDP-mannose is synthesized in the cytoplasm then trans-located into the Golgi lumen by action of GDP mannose transporters (GMTs).21 GMTs could be potential drug targets for new antifungals, as disruption of any step of GDP-mannose biosynthesis can affect viability, growth or virulence of fungi. For example, in A. fumigatus repression of GDP-mannose pyrophosphorylase (GMPP), which catalyses the synthesis of GDP-mannose produces a lethal phenotype including hyphal lysis, defective cell wall, and impaired polarity maintenance.41 In this work we studied A. nidulans strains that were deleted for either or both of the GMT genes (gmtA and gmtB). When gmtA was deleted from A1148 parent strain, the gmtAΔ strain was viable, but its colonies were paler in color than the parent strain. It has been reported previously that melanin layer (responsible for spore's surface coloration) is attached to the underlying layers by α-glucan.42 Reduction in cell wall α-glucan content was observed in an A. fumigatus ΔgmtA strain, 26 which might explain the paling of A. nidulans gmtAΔ colony color in our study. A C. neoformans double deletion strain was viable,27 similar to our [gmtAΔ-gmtBΔ] strain. This may be explained based on a previous report about nucleotide sugar transporters (NST) redundancy, which can be considered as evolutionary backup mechanism in case of any NST impairment, deletion or mutation.43 In this investigation, we studied the roles of GMTs in colony and hyphal morphogenesis. The morphological defects occurred in Gmt-deleted strains included: smaller colony size, reduced sporulation, increased hyphal width and decreased basal cell length. These defective phenotypes generally happen due to disturbances during cell wall biosynthesis. For examples, A. nidulans ugmAΔ and ugeAΔ strains (defective in GM biosynthesis) showed similar phenotype.31,44 Also partial repression of GMPP in A. fumigatus led to defective cell wall and reduced sporulation. 41 Furthermore, deletion of A. fumigatus mannosyl transferases led to the reduction in mannan content of the conidial cell wall but not the hyphal wall. This was associated with a partial disorganization of the cell wall leading to defects in conidial survival.45 TEM showed an increase in the cell wall thickness of the deletion strains. Similarly, hyphal walls of strains defective in UDP-galactofuranose transporter (ugtAΔ) were at least threefold thicker than wild-type strains and were thickly coated with exogenous materials.33 Cell wall ultrastructure in wild-type had a bilayered appearance, which was not obvious in the GmtBΔ strains. Similarly, hyphal cell wall in Aspergillus nidulans mnpA mutant (defective in mannosylation) had an electron-dense irregular and disrupted outer layer, along with a broad electron-translucent inner layer.46 Although the GmtB deletion phenotype was subtle, this gene function appears to include cell wall integrity, especially in conidial and hyphal walls. Antifungal susceptibility testing revealed reduction in sensitivity of deleted strains to micafungin (inhibitor of β-glucan synthesis) and increased in sensitivity to the chitin binding agent CR. Strains that have defects in cell wall are usually hypersensitive to CR.47 In a similar manner, mutation of A. nidulans GmtA increased the sensitivity to the cell wall targeting agent Calcofluor White (CFW).21 Increased chitin synthesis has been known as an important compensatory response to cell wall stress in fungi.48 Oka and colleagues reported that the N-acetyl glucosamine (GlcNAc) content of the A. nidulans pmtA disrupted strain was one third higher than wild-type, and pmtAΔ strain was also hypersensitive to CR.34 Furthermore, Engel and colleagues analyzed the cell wall monosaccharides of A. fumigatus ΔgmtA and found that no mannose was detected, and the glucosamine (precursor for chitin) was twice as abundant as wild-type, whereas the glucose content was reduced by 65%.26 Previous findings strongly suggest alterations in β-glucan and chitin content and might explain the increased sensitivity to CR and reduced towards echinocandin in current investigation. In this study, we studied the effect of gmtA and gmtB deletion on spore hydrophobicity. The results showed that the gmtAΔ spores were less hydrophobic than wild-type, whereas the gmtBΔ and gmtAΔ-gmtBΔ spores were more hydrophobic. This agrees with the spore surface character reveled by SEM. A previous study in A. fumigatus reported that the Δpes1 strain (defective in peptide synthetase gene) had a conidial surface with a lower degree of ornamentation and was 51% more hydrophobic than the wild-type.49 We expect that GmtB is responsible for formation of the external ornamentation layer of spore surface; thus its deletion causes disappearance of this layer and exposure of the underlying rodlet layer. Rodlets are composed mainly of hydrophobins,50 Adherence to human epithelial cells was in the following pattern: gmtAΔ < wild-type < gmtBΔ < gmtAΔ-gmtBΔ. This might be attributed to the spore hydrophobicity as the more hydrophobic the spores the more they will be adhesive. It has been reported that the hydrophobin RodA is one of the proteins that mediate spore adherence in A. fumigatus.51 Biofilm formation follows the same pattern of adherence, it is known that adherence to a surface is the first step of biofilm formation.39 The rough spore surface of gmtAΔ strain may play a role in biofilm reduction. As it was previously reported in A. fumigatus that A ΔcspA strain (defective in GPI- anchored cell wall protein) had a rougher surface with more ornaments and showed reduction in biofilm formation.52 Previous studies in other Aspergillus species found a relation between spore surface character and the ability of spores to form biofilm. For example, it has been reported that A. fumigatus ΔmedA strain was less hydropbobic and showed reduction in biofilm formation.35 Also in A. niger, the Δalb1 spores has a smooth surface and were more adhesive to polystyrene plates.53 To our knowledge, double deletion mutant in both Gmt(s) has not been reported in Aspergillus nidulans before. Our double deletion strain was impaired but still viable. Similarly, the cryptococcal double mutant strain was also viable but clearly demonstrates the critical role of mannose glycosylation in C. neoformans, as this strain was sensitive to stress and temperature, was acapsular, and avirulent in a mouse model of infection.27 In conclusion, the altered conidial surface characters, decreased adherence and biofilm formation of gmtAΔ mutant, coupled with the fact that humans do not have GDP-mannose transporters, suggests GmtA can be a potential target for new antifungal that targets virulence determinants. Future work will further investigate the role of GMTs in virulence in animal model. Supplementary material Supplementary data are available at MMYCOL online. Acknowledgements The authors would like to thank Prof Elham Khalifa (Faculty of Agriculture, Cairo University, Cairo, Egypt) for helping with TEM. Funding No specific funding has been received. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. REFERENCES 1. Person AK, Kontoyianni DP, Alexander BD. Fungal infections in transplant and oncology patients. Infect Dis Clin North Am . 2010; 24: 439– 459. Google Scholar CrossRef Search ADS PubMed  2. Moran GP, Coleman DC, Sullivan DJ. Comparative genomics and the evolution of pathogenicity in human pathogenic fungi. Eukaryot Cell . 2011; 10: 34– 42. Google Scholar CrossRef Search ADS PubMed  3. Calderone R, Sun N, Gay-Andrieu F et al.   Antifungal drug discovery: the process and outcomes. 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Deletion of Aspergillus nidulans GDP-mannose transporters affects hyphal morphometry, cell wall architecture, spore surface character, cell adhesion, and biofilm formation

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© The Author(s) 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.
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Abstract

Abstract Systemic human fungal infections are increasingly common. Aspergillus species cause most of the airborne fungal infections. Life-threatening invasive aspergillosis was formerly found only in immune-suppressed patients, but recently some strains of A. fumigatus have become primary pathogens. Many fungal cell wall components are absent from mammalian systems, so they are potential drug targets. Cell-wall-targeting drugs such as echinocandins are used clinically, although echinocandin-resistant strains were discovered shortly after their introduction. Currently there are no fully effective anti-fungal drugs. Fungal cell wall glycoconjugates modulate human immune responses, as well as fungal cell adhesion, biofilm formation, and drug resistance. Guanosine diphosphate (GDP) mannose transporters (GMTs) transfer GDP-mannose from the cytosol to the Golgi lumen prior to mannosylation. Aspergillus nidulans GMTs are encoded by gmtA and gmtB. Here we elucidate the roles of A. nidulans GMTs. Strains engineered to lack either or both GMTs were assessed for hyphal and colonial morphology, cell wall ultrastructure, antifungal susceptibility, spore hydrophobicity, adherence and biofilm formation. The gmt-deleted strains had smaller colonies with reduced sporulation and with thicker hyphal walls. The gmtA deficient spores had reduced hydrophobicity and were less adherent and less able to form biofilms in vitro. Thus, gmtA not only participates in maintaining the cell wall integrity but also plays an important role in biofilm establishment and adherence of A. nidulans. These findings suggested that GMTs have roles in A. nidulans growth and cell-cell interaction and could be a potential target for new antifungals that target virulence determinants. GDP-mannose transporters, Aspergillus nidulans, spore hydrophobicity, adherence, biofilm ABBREVIATIONS ABBREVIATIONS CR Congo red CV crystal violet GDP Guanosine diphosphate GMT GDP mannose transporter RMPI Roswell Park Memorial Institute SDA Sabourand dextrose agar SEM Scanning electron microscope TEM transmission electron microscope Introduction Fungi cause superficial, subcutaneous, and systemic human infections, the latter especially in immune-compromised patients.1,2 Fungal infections kill more than 1.3 million people annually, comparable to drug-resistant Mycobacterium tuberculosis.3Aspergillus fumigatus is the most common airborne fungal pathogen, and now some A. fumigatus strains even cause invasive pulmonary disease in immune-competent people.4Aspergillus nidulans is a genetically tractable congeneric of A. fumigatus that has overall similar cell wall sugar and protein composition.5,6 The cell wall is essential for fungal survival and virulence. Many wall components are not found in humans, so the proteins synthesizing the cell wall components are potential targets for antifungal drug development.7 Echinocandin-class antifungals targets wall β-glucan synthesis by inhibiting fksA8 and are fungicidal in Candida spp. and fungistatic in Aspergillus spp. However, clinically-relevant echinocandin-resistant strains were identified shortly after wide-spread therapeutic use began.9 Due to the elevated problem of antifungal resistance, there is a new paradigm to target virulence determinants rather than targeting essential processes to reduce evolution of resistance.10 Glycosylation is a post-translational modification of some membrane and wall components that is important for growth, virulence, and immunogenicity of many pathogens including Aspergillus.11–15 Mannosylation is one of the glycosylation processes, in which mannan is added to proteins or other carbohydrates to form different glycoconjugates. Mannan is an essential component in many fungal species. In Aspergillus, mannan is not present as a homogeneous mannose polymer but as galactomannan (GM) or mannoproteins.16 GM is covalently bound to the glucan–chitin core of the cell wall, found in both the inner and outer layers of the cell wall and can be used to diagnose invasive aspergillosis.17 In addition, GM also bound to the plasma membrane by a lipid anchor of glycosylphosphatidylinositol (GPI).18 The guanosine diphosphate (GDP)-mannose transporter (GMT) is required for mannosylation of galactomannan, mannoproteins and the GPI anchor.19 Mammalian cells lack GMTs, making them excellent new targets for antifungal drug development.20 In A. nidulans, GMT is encoded by gmtA and gmtB, 21 both of which co-localize with the Golgi equivalent.22 GMT was reported to be essential in Saccharomyces, Candida, and A. niger.23–25 Although gmtA is not essential for viability of A. fumigatus, its deletion leads to severe growth defects.26 GMT is also required for virulence in Cryptococcus neoformans and the protist Leishmania donovani.27,28 the roles of GMTs in Aspergillus nidulans is not fully understood. Here, we explore the in vitro roles of GMTs in A. nidulans strains that were deleted for gmtA and/or gmtB. Growth morphometry, cell wall ultrastructure, and sensitivity to antifungal drugs were studied. In addition spore hydrophobicity and its effect on adherence and biofilm formation; as two important virulence factors; were assessed in comparison to the wild-type parent strain. Methods Strains and culture conditions Strains used in this study are listed in Table 1.29Aspergillus nidulans strains were maintained on Roswell Park Memorial Institute (RPMI) 1640 agar medium (Sigma Aldrich, St Louis, MO, USA) supplemented with uridine and uracil (Sigma Aldrich, St Louis, MO, USA) for pyrG-deficient strains. Sabourand dextrose agar (SDA) was used as media for the double deletion strain, while SDA plus uridine and uracil was used as media for gmtBΔ strain. Strains were stored in 50% glycerol at –20°C.30 Most procedures were carried out in Thermoscientific class II microbiological safety cabinet. Table 1. Strains included in this study. Aspergillus nidulans strains  Strain genetic characters  1A1148  pyrG89; pyroA4; riboB2; nkuB:: A. fumigatus riboB; nkuA:: argB  2gmtAΔ  pyrG89; pyroA4; riboB2; nkuB:: A. fumigatus riboB; nkuA::argB; AN8848:: A. fumigatus pyrG  2gmtBΔ  wA3; pyroA4; argB2; nkuA::argB; AN9298:: A. fumigatus pyroA.  2gmtAΔ- gmtBΔ  pyrG89; pyroA4; riboB2; nkuB::A. fumigatus riboB; nkuA::argB; AN8848::A. fumigatus pyrG; AN9298:: A. fumigatus pyroA  Aspergillus nidulans strains  Strain genetic characters  1A1148  pyrG89; pyroA4; riboB2; nkuB:: A. fumigatus riboB; nkuA:: argB  2gmtAΔ  pyrG89; pyroA4; riboB2; nkuB:: A. fumigatus riboB; nkuA::argB; AN8848:: A. fumigatus pyrG  2gmtBΔ  wA3; pyroA4; argB2; nkuA::argB; AN9298:: A. fumigatus pyroA.  2gmtAΔ- gmtBΔ  pyrG89; pyroA4; riboB2; nkuB::A. fumigatus riboB; nkuA::argB; AN8848::A. fumigatus pyrG; AN9298:: A. fumigatus pyroA  1: Fungal genetics stock center (FGSC). 2: El-Ganiny et al. 2015. View Large Table 1. Strains included in this study. Aspergillus nidulans strains  Strain genetic characters  1A1148  pyrG89; pyroA4; riboB2; nkuB:: A. fumigatus riboB; nkuA:: argB  2gmtAΔ  pyrG89; pyroA4; riboB2; nkuB:: A. fumigatus riboB; nkuA::argB; AN8848:: A. fumigatus pyrG  2gmtBΔ  wA3; pyroA4; argB2; nkuA::argB; AN9298:: A. fumigatus pyroA.  2gmtAΔ- gmtBΔ  pyrG89; pyroA4; riboB2; nkuB::A. fumigatus riboB; nkuA::argB; AN8848::A. fumigatus pyrG; AN9298:: A. fumigatus pyroA  Aspergillus nidulans strains  Strain genetic characters  1A1148  pyrG89; pyroA4; riboB2; nkuB:: A. fumigatus riboB; nkuA:: argB  2gmtAΔ  pyrG89; pyroA4; riboB2; nkuB:: A. fumigatus riboB; nkuA::argB; AN8848:: A. fumigatus pyrG  2gmtBΔ  wA3; pyroA4; argB2; nkuA::argB; AN9298:: A. fumigatus pyroA.  2gmtAΔ- gmtBΔ  pyrG89; pyroA4; riboB2; nkuB::A. fumigatus riboB; nkuA::argB; AN8848::A. fumigatus pyrG; AN9298:: A. fumigatus pyroA  1: Fungal genetics stock center (FGSC). 2: El-Ganiny et al. 2015. View Large Colony growth and hyphal morphometry The colony diameter and number of spores per colony were measured for wild-type and deleted strains as described previously.31 Briefly, strains were streaked on RPMI media and incubated for 3 d at 28°C to give isolated colonies. The diameter of 10 colonies/strain was measured to the nearest millimeter. The number of spores per colony was counted for four colonies/strain. Isolated colonies were vortexed in a microfuge tube containing 1 ml water, then samples of spores were counted with a hemocytometer. For hyphal morphometry, hyphae were grown at 37°C for 16 h and examined using Leica light microscopy (DM500). Imaging used a Leica ICC50 HD camera and Leica DM500 image analysis software. Hyphal width was measured at septa and basal cell length was measured between adjacent septa (50/strain) as previously described.32 Electron microscopy Scanning electron microscopy (SEM) followed El-Ganiny et al.31 Briefly, strains were grown on dialysis tubing overlaying RPMI agar for 3 d at 28°C. Isolated colonies were vapor-fixed at 100% relative humidity over 4% aqueous osmium tetroxide (OsO4) for 2 h, frozen to −80°C, then lyophilized overnight. Samples were mounted on SEM stubs, gold sputter-coated for 6 min, and examined with a Quanta FEG250 (FE-SEM) in Central Metallurgical Research Institute (Tebbin, Helwan, Egypt). The accelerating voltage was 20 kV. The beam current for sample examination was 1.5 nA, and for image acquisition it was 50 pA. Transmission electron microscopy (TEM) samples were prepared and imaged as described previously.28 Strains were grown submerged in liquid medium for 16 h at 28°C. Cells were fixed for 1 h in 1% glutaraldehyde in 50 mM phosphate buffer, pH 7, then post-fixed for 1 h in 1% OsO4 in phosphate buffer. Samples were dehydrated in a gradient ethanol series, then transferred to 100% anhydrous acetone, and embedded in epoxy resin. Sections of 70–100 nm thickness were cut with a Leica Ultracut UCT microtome and then stained with uranyl acetate and lead citrate as described previously.30 Stained sections were examined using a JEOL JEM-1400 at the Faculty of Agriculture, Cairo University Research Park (FA-CURP). Images were captured by CCD camera model AMT. Transverse TEM sections of wild-type and deleted strains hyphae (with well resolved cell membrane) were used to compare hyphal diameter and wall thickness. Antifungal susceptibility testing Drug sensitivity of wild-type, gmtAΔ, gmtBΔ and gmtAΔ-gmtBΔ strains was compared using disk diffusion method.33 Briefly; strains were grown on RPMI agar for 4 d. Spores were collected, and the suspension was filtered using sterile filter paper to remove any hyphae or conidiophores and counted by hemocytometer. Then 1 × 107 freshly harvested spores were mixed with 20 ml of molten RPMI agar and poured into Petri plates. After the medium had hardened, 6 mm sterile blank paper disks (BBL, Becton Dickinson, Cockeysville, MD, USA) were placed on agar surface. Tested commercial antifungals were from four drug classes: polyenes (amphotericin B; Bristol-Meyers Squibb, France), allylamines (terbinafine; Mash Premier, Egypt), azoles (voriconazole; Pfizer, Ireland), and echinocandins (micafungin; Astellas Toyama, Japan). Stock solutions were prepared using sterile water, except terbinafine in 50% ethanol (Alnasr chemicals, Egypt) in the following concentrations: amphotericin B and micafungin (20 mg/ml), voriconazole and terbinafine (1.6 mg/ml). Then 2 μl of each antifungal drug stock solution was pipetted onto individual disks; control solvent disc was also included. Also, Congo red (CR) was tested as it interferes with chitin crystallization in the cell wall and hence has antifungal effects.34 CR stock solution contain 10 mg/ml in water; 10 μl was added to blank disc surface.33 The plates were incubated at 37 °C for 48 h. The radius of the zone of inhibition (showing no fungal growth) was calculated as [inhibition diameter – 6 mm]/2. Five replicates were measured for each strain. Spores hydrophobicity assay Hydrophobicity assay was performed as described previously.35 Briefly, spores were collected from 4 day-old colonies, and the number of spores was counted using a hemocytometer. Equal number of spores were resuspended in 3 ml mineral oil-water mix (1:1 v/v) and shaken vigorously. After settlement, the number of spores remaining in the water was counted and the percent portioning in oily phase was calculated as: (1- N/N0) × 100, where N0 and N were the initial number and final number of spores in the aqueous phase after partitioning, respectively, the hydrophobicity of Gmt-deleted strains was calculated relative to that of wild-type.36 The results are the average of three independent experiments. Adherence of A. nidulans spores to human epithelial cells For epithelial cells collection, a swab with a rayon tip and plastic applicator (167KS01; Copan Italia S.P.A., Brescia, Italy) was used to obtain a sample of the posterior oropharyngeal mucosal membrane from healthy volunteers.37 Collected swabs were soaked in phosphate-buffered saline (PBS), vortexed, and then centrifuged at 350 rpm for 10 min. The sediment was washed twice in PBS and resuspended in RPMI medium. The number of cells was estimated with a hemocytometer and standardized to 105 cells/ml. Also, A. nidulans spores were collected from 4 day-old colonies, and the number of spores was counted using hemocytometer and adjusted to 106 spore/ml. A. nidulans spores were tested for their adherence ability to epithelial cells in 96 well microtiter plates as described previously with some modifications.38 Briefly, the microtiter plates were inoculated with 100 μl of previously prepared epithelial cells. After 24 h incubation at 37°C, the unattached epithelial cells were washed once with PBS. Each well containing attached epithelial cells were inoculated with 100 μl of A. nidulans spore suspension, and the mixture was incubated at 37°C for 60 min. The unattached spores were washed out with PBS three times, and the remaining attached spores were fixed with 3.7% formaldehyde for 15 min. Adherence of spores was estimated by staining with 150 μl of 0.5% (w/v) Crystal Violet (CV) solution for 15 min. Excess stain was gently removed under running water then the wells were destained by adding 100 μl of glacial acetic acid. The adherence of spores was estimated by determining the absorbance of the destaining solution at 590 nm using Synergy HT ELISA reader and Gen5 1.11 Biotek software. Absorbance values of negative control of liquid media was used as blank. Assays were performed in 3 independent wells on at least three separate days. Adherence and biofilm formation of A. nidulans spores on polystyrene Biofilm study was based on methods described previously.39,40 Briefly, 96-well microtiter plates were inoculated with 100 μl of 105 spores per well in RPMI media, and media-only blanks were also set up. Plates were incubated at 37°C, and analysis was made at 4 and 24 h representing adhesion and biofilm formation, respectively. The medium was removed from each well and the adherent hyphae were washed three times with PBS and then fixed with 3.7% formaldehyde. Adherence and biofilm formation were estimated as described in adherence of spores to human epithelial cells (see the section “Adherence of A. nidulans spores to human epithelial cells”). RESULTS Effect of deleting gmtA, gmtB, and gmtA gmtB on colony growth and sporulation The effect of gmtA deletion on colony morphology was clear, colonies of gmtAΔ are paler in colour than parent wild-type strain (A1148) due to the severely reduced sporulation, but the appearance of hyphal margin was generally similar to the parent strain. Colonies lacking GmtB and the double deletion strain (lacked GmtA and GmtB) had both smaller colonies and fewer spores (Fig. 1A). Compared to the wild-type, sporulation was reduced in the GmtAΔ, GmtBΔ, and [GmtAΔ, GmtBΔ] strains, respectively by about 90-fold, 170-fold, and 230-fold (Table 2). Figure 1. View largeDownload slide Morphology of wild-type and gmt-deleted strains. A: colony morphology, B: SEM showing colony surface, scale bar 100 μm, C: SEM showing conidiophores, scale bar 10 μm, and D: SEM showing spore surface character, scale bar 1 μm. This Figure is reproduced in color in the online version of Medical Mycology. Figure 1. View largeDownload slide Morphology of wild-type and gmt-deleted strains. A: colony morphology, B: SEM showing colony surface, scale bar 100 μm, C: SEM showing conidiophores, scale bar 10 μm, and D: SEM showing spore surface character, scale bar 1 μm. This Figure is reproduced in color in the online version of Medical Mycology. Table 2. Morphometric comparison of wild-type and GMT-deletion strains.   wild-type  gmtAΔ  gmtBΔ  gmtAΔ-gmtBΔ  Colony diameter (mm)  16 ± 0.5  4.2a ± 0.2  3.5a ± 0.4  1.2a ± 0.3  Spores/colony × 106  138 ± 2  1.5a ± 0.5  0.8a ± 0.4  0.6a ± 0.3  Hyphal width (μm)  2.5± 0.3  3.1a ± 0.8  3.7a ± 0.9  4.4a ± 0.5  Basal cell length (μm)  25 ± 0.3  14.7a ± 0.6  15.9a ± 0.1  14.0 a ± 0 .4  Cell wall thickness (nm)  77.5 ± 4.2  117a ± 7.5  124.8a± 10.5  133a ± 8.9    wild-type  gmtAΔ  gmtBΔ  gmtAΔ-gmtBΔ  Colony diameter (mm)  16 ± 0.5  4.2a ± 0.2  3.5a ± 0.4  1.2a ± 0.3  Spores/colony × 106  138 ± 2  1.5a ± 0.5  0.8a ± 0.4  0.6a ± 0.3  Hyphal width (μm)  2.5± 0.3  3.1a ± 0.8  3.7a ± 0.9  4.4a ± 0.5  Basal cell length (μm)  25 ± 0.3  14.7a ± 0.6  15.9a ± 0.1  14.0 a ± 0 .4  Cell wall thickness (nm)  77.5 ± 4.2  117a ± 7.5  124.8a± 10.5  133a ± 8.9  Values are presented as mean ± standard error. a:Values are significantly different from wild-type by Graphpad Prism 6.0 using one-way ANOVA at P < .05. View Large Table 2. Morphometric comparison of wild-type and GMT-deletion strains.   wild-type  gmtAΔ  gmtBΔ  gmtAΔ-gmtBΔ  Colony diameter (mm)  16 ± 0.5  4.2a ± 0.2  3.5a ± 0.4  1.2a ± 0.3  Spores/colony × 106  138 ± 2  1.5a ± 0.5  0.8a ± 0.4  0.6a ± 0.3  Hyphal width (μm)  2.5± 0.3  3.1a ± 0.8  3.7a ± 0.9  4.4a ± 0.5  Basal cell length (μm)  25 ± 0.3  14.7a ± 0.6  15.9a ± 0.1  14.0 a ± 0 .4  Cell wall thickness (nm)  77.5 ± 4.2  117a ± 7.5  124.8a± 10.5  133a ± 8.9    wild-type  gmtAΔ  gmtBΔ  gmtAΔ-gmtBΔ  Colony diameter (mm)  16 ± 0.5  4.2a ± 0.2  3.5a ± 0.4  1.2a ± 0.3  Spores/colony × 106  138 ± 2  1.5a ± 0.5  0.8a ± 0.4  0.6a ± 0.3  Hyphal width (μm)  2.5± 0.3  3.1a ± 0.8  3.7a ± 0.9  4.4a ± 0.5  Basal cell length (μm)  25 ± 0.3  14.7a ± 0.6  15.9a ± 0.1  14.0 a ± 0 .4  Cell wall thickness (nm)  77.5 ± 4.2  117a ± 7.5  124.8a± 10.5  133a ± 8.9  Values are presented as mean ± standard error. a:Values are significantly different from wild-type by Graphpad Prism 6.0 using one-way ANOVA at P < .05. View Large The parental strain had abundant conidiophores with wild-type morphology (Fig. 1B), whereas the GmtAΔ strain had fewer conidiophores and, of those, fewer with normal spore chains (Fig. 1B). The GmtBΔ strain had aerial hyphae, and few mature conidiophores, since many appeared to have stalled at the vesicle stage (Fig. 1B) or produced short spore chains. The [GmtAΔ, GmtBΔ] strain had abundant aerial hyphae, and lower sporulation than single deletion strains (Fig. 1B, C). Figure 1D shows the spore surface for these strains. The spores of wild-type had few ornaments that partially cover the rodlet layer, the spores of gmtAΔ had a rougher surface with more ornaments than the wild-type. But spores of gmtBΔ and gmtAΔ-gmtBΔ lost the ornamented outer layer of wild-type, which cause exposure of the underlying rodlet layer. Hyphal morphometry Hyphal width increased markedly in the gene-deleted strains compared to the wild-type parental strain: wild-type ≪ gmtAΔ < gmtBΔ < gmtAΔgmtBΔ. Basal cell length decreased as hyphal width increased. Morphometry of parental and deletion strains is shown in Table 2 and Figure 2A. Figure 2. View largeDownload slide Hyphae of wild-type and gmt-deleted strains as shown by light microscopy and TEM. A: light microscopy, scale bar 10 μm; B: TEM images, scale bar = 100 nm Figure 2. View largeDownload slide Hyphae of wild-type and gmt-deleted strains as shown by light microscopy and TEM. A: light microscopy, scale bar 10 μm; B: TEM images, scale bar = 100 nm Cell wall ultrastructure TEM images of 16-h old hyphae grown in liquid media demonstrate the effect of gmtA and gmtB deletion on the cell wall ultrastructure. Hyphal wall thickness was increased in the gmt-deleted strains compared to wild-type parental, so that parental ≪ gmtAΔ < gmtBΔ <gmtAΔ-gmtBΔ (Table 2). The wild-type and gmtAΔ strains (grown submerged in liquid cultures) accumulated large amount of exogenous materials that not observed for the gmtBΔ strains (Fig. 2B). The hyphal walls of the wild-type and gmtAΔ strains had two distinct layers while the walls of gmtBΔ and gmtAΔ-gmtBΔ strains appear as one diffused layer (Fig. 2B). Susceptibility to antifungal drugs Micafungin susceptibility (target β-1, 3-glucan synthesis) was compared with drugs that target ergosterol (amphotericin B) or ergosterol biosynthesis (voriconazole and terbinafine). There were no significant changes in the sensitivity to amphotericin B, voriconazole or terbinafine. In contrast, reduced sensitivity to micafungin was noticed in gmtAΔ, gmtBΔ, and gmtAΔ-gmtBΔ compared to wild-type. Regarding CR, there was a slight increase in the sensitivity of the gmtAΔ, gmtBΔ, and gmtAΔ-gmtBΔ strains compared with the wild-type strain (Table 3, Suppl. Fig. 1). Table 3. Sensitivity of wild-type and deletion strains to antifungal drugs.   Radius of inhibition zone in mm    Strains  Amphotericin B  Micafungin  Terbinafine  Voriconazole  Congo Red  wild-type  14 ± 0.2  2 ± 0.4  26 ± 0.3  25 ± 0.3  12.3 ± 0.7  gmtAΔ  16 ± 0.3  1a ± 0.2  26 ± 0.1  27 ± 0.2  12.8 ± 0.4  gmtBΔ  17 ± 0.1  0.5a ± 0.1  26 ± 0.3  26 ± 0.3  13.2 ± 0.4  gmtAΔ-gmtBΔ  16 ± 0.2  0.5a ± 0.1  27 ± 0.2  27 ± 0.1  14.8 ± 0.3    Radius of inhibition zone in mm    Strains  Amphotericin B  Micafungin  Terbinafine  Voriconazole  Congo Red  wild-type  14 ± 0.2  2 ± 0.4  26 ± 0.3  25 ± 0.3  12.3 ± 0.7  gmtAΔ  16 ± 0.3  1a ± 0.2  26 ± 0.1  27 ± 0.2  12.8 ± 0.4  gmtBΔ  17 ± 0.1  0.5a ± 0.1  26 ± 0.3  26 ± 0.3  13.2 ± 0.4  gmtAΔ-gmtBΔ  16 ± 0.2  0.5a ± 0.1  27 ± 0.2  27 ± 0.1  14.8 ± 0.3  Values are presented as mean ± standard error. a:Values are significantly different from wild-type by Graphpad Prism 6.0 using one-way ANOVA at P < .05. View Large Table 3. Sensitivity of wild-type and deletion strains to antifungal drugs.   Radius of inhibition zone in mm    Strains  Amphotericin B  Micafungin  Terbinafine  Voriconazole  Congo Red  wild-type  14 ± 0.2  2 ± 0.4  26 ± 0.3  25 ± 0.3  12.3 ± 0.7  gmtAΔ  16 ± 0.3  1a ± 0.2  26 ± 0.1  27 ± 0.2  12.8 ± 0.4  gmtBΔ  17 ± 0.1  0.5a ± 0.1  26 ± 0.3  26 ± 0.3  13.2 ± 0.4  gmtAΔ-gmtBΔ  16 ± 0.2  0.5a ± 0.1  27 ± 0.2  27 ± 0.1  14.8 ± 0.3    Radius of inhibition zone in mm    Strains  Amphotericin B  Micafungin  Terbinafine  Voriconazole  Congo Red  wild-type  14 ± 0.2  2 ± 0.4  26 ± 0.3  25 ± 0.3  12.3 ± 0.7  gmtAΔ  16 ± 0.3  1a ± 0.2  26 ± 0.1  27 ± 0.2  12.8 ± 0.4  gmtBΔ  17 ± 0.1  0.5a ± 0.1  26 ± 0.3  26 ± 0.3  13.2 ± 0.4  gmtAΔ-gmtBΔ  16 ± 0.2  0.5a ± 0.1  27 ± 0.2  27 ± 0.1  14.8 ± 0.3  Values are presented as mean ± standard error. a:Values are significantly different from wild-type by Graphpad Prism 6.0 using one-way ANOVA at P < .05. View Large Hydrophobicity of spores Spores from wild-type and gmt-deleted strains were shaken in water/mineral oil mixture to determine spore hydrophobicity (Fig. 3A). For each strain, number of spores in aqueous layer were counted and used to calculate the % partitioning in mineral oil and the relative hydrophobicity of Gmt-deleted strains relative to wild-type (Fig. 3B). The gmtAΔ strain shows decreased hydrophobicity while gmtB-deleted strains show increased hydrophobicity. Figure 3. View largeDownload slide Hydrophobicity of gmt-deleted strains relative to wild-type strain. Figure 3. View largeDownload slide Hydrophobicity of gmt-deleted strains relative to wild-type strain. Assessment of spore adhesion to human epithelial cells The adherence to human epithelial cells was assessed for wild-type and gmt-deleted strains by staining with 0.5% CV. There was a significant decrease in absorbance of gmtAΔ strain and a slight increase for gmtBΔ and gmtA-gmtBΔ compared to wild-type (Table 4). Table 4. Adherence on epithelial cells and polystyrene plate and biofilm formation of wild-type and gmt-deleted strains.   % absorbance measured at 590 nm (A590) after  Strain  Adherence to epithelial cells  Adherence to polystyrene plates  In vitro biofilm formation  wild-type  72.33 ± 3.0  13.8 ± 3.7  72.1 ± 5.0  gmtAΔ  52.96a ± 5.0  10.3a ± 1.3  60.7a ± 5.0  gmtBΔ  77.14 ± 3.0  12.2 ± 8.0  93.4b ± 5.0  gmtAΔ-gmtBΔ  76.83 ± 1.0  12.4 ± 2.9  93.3b ± 6.0    % absorbance measured at 590 nm (A590) after  Strain  Adherence to epithelial cells  Adherence to polystyrene plates  In vitro biofilm formation  wild-type  72.33 ± 3.0  13.8 ± 3.7  72.1 ± 5.0  gmtAΔ  52.96a ± 5.0  10.3a ± 1.3  60.7a ± 5.0  gmtBΔ  77.14 ± 3.0  12.2 ± 8.0  93.4b ± 5.0  gmtAΔ-gmtBΔ  76.83 ± 1.0  12.4 ± 2.9  93.3b ± 6.0  a, b: values are significantly different from wild-type and other stains. Statistical analysis was done using Graphpad Prism 6.0 (One Way ANOVA at P ≤ 0.05). View Large Table 4. Adherence on epithelial cells and polystyrene plate and biofilm formation of wild-type and gmt-deleted strains.   % absorbance measured at 590 nm (A590) after  Strain  Adherence to epithelial cells  Adherence to polystyrene plates  In vitro biofilm formation  wild-type  72.33 ± 3.0  13.8 ± 3.7  72.1 ± 5.0  gmtAΔ  52.96a ± 5.0  10.3a ± 1.3  60.7a ± 5.0  gmtBΔ  77.14 ± 3.0  12.2 ± 8.0  93.4b ± 5.0  gmtAΔ-gmtBΔ  76.83 ± 1.0  12.4 ± 2.9  93.3b ± 6.0    % absorbance measured at 590 nm (A590) after  Strain  Adherence to epithelial cells  Adherence to polystyrene plates  In vitro biofilm formation  wild-type  72.33 ± 3.0  13.8 ± 3.7  72.1 ± 5.0  gmtAΔ  52.96a ± 5.0  10.3a ± 1.3  60.7a ± 5.0  gmtBΔ  77.14 ± 3.0  12.2 ± 8.0  93.4b ± 5.0  gmtAΔ-gmtBΔ  76.83 ± 1.0  12.4 ± 2.9  93.3b ± 6.0  a, b: values are significantly different from wild-type and other stains. Statistical analysis was done using Graphpad Prism 6.0 (One Way ANOVA at P ≤ 0.05). View Large Adherence of A. nidulans spores to polystyrene plate and biofilm formation The adherence of A. nidulans spores to polystyrene plate was assessed. The absorbance values were directly proportional to the degree of adherence. After 4 h, adherence of the gmtAΔ was reduced significantly, while adherence of the gmtBΔ, and gmtAΔ-gmtBΔ was comparable to the wild-type (Table 4). The formation of biofilm on polystyrene plates by A. nidulans hyphae was assessed after 24 h. The results showed that absorbance was significantly increased in the double deletion strain, gmtAΔ-gmtBΔ and gmtBΔ indicating increased biofilm formation, while gmtAΔ had the least absorbance value indicating that the biofilm was significantly reduced than wild-type (Table 4). Discussion In Aspergillus, GDP-mannose is the precursor of all the mannosylation processes.26,42 GDP-mannose is synthesized in the cytoplasm then trans-located into the Golgi lumen by action of GDP mannose transporters (GMTs).21 GMTs could be potential drug targets for new antifungals, as disruption of any step of GDP-mannose biosynthesis can affect viability, growth or virulence of fungi. For example, in A. fumigatus repression of GDP-mannose pyrophosphorylase (GMPP), which catalyses the synthesis of GDP-mannose produces a lethal phenotype including hyphal lysis, defective cell wall, and impaired polarity maintenance.41 In this work we studied A. nidulans strains that were deleted for either or both of the GMT genes (gmtA and gmtB). When gmtA was deleted from A1148 parent strain, the gmtAΔ strain was viable, but its colonies were paler in color than the parent strain. It has been reported previously that melanin layer (responsible for spore's surface coloration) is attached to the underlying layers by α-glucan.42 Reduction in cell wall α-glucan content was observed in an A. fumigatus ΔgmtA strain, 26 which might explain the paling of A. nidulans gmtAΔ colony color in our study. A C. neoformans double deletion strain was viable,27 similar to our [gmtAΔ-gmtBΔ] strain. This may be explained based on a previous report about nucleotide sugar transporters (NST) redundancy, which can be considered as evolutionary backup mechanism in case of any NST impairment, deletion or mutation.43 In this investigation, we studied the roles of GMTs in colony and hyphal morphogenesis. The morphological defects occurred in Gmt-deleted strains included: smaller colony size, reduced sporulation, increased hyphal width and decreased basal cell length. These defective phenotypes generally happen due to disturbances during cell wall biosynthesis. For examples, A. nidulans ugmAΔ and ugeAΔ strains (defective in GM biosynthesis) showed similar phenotype.31,44 Also partial repression of GMPP in A. fumigatus led to defective cell wall and reduced sporulation. 41 Furthermore, deletion of A. fumigatus mannosyl transferases led to the reduction in mannan content of the conidial cell wall but not the hyphal wall. This was associated with a partial disorganization of the cell wall leading to defects in conidial survival.45 TEM showed an increase in the cell wall thickness of the deletion strains. Similarly, hyphal walls of strains defective in UDP-galactofuranose transporter (ugtAΔ) were at least threefold thicker than wild-type strains and were thickly coated with exogenous materials.33 Cell wall ultrastructure in wild-type had a bilayered appearance, which was not obvious in the GmtBΔ strains. Similarly, hyphal cell wall in Aspergillus nidulans mnpA mutant (defective in mannosylation) had an electron-dense irregular and disrupted outer layer, along with a broad electron-translucent inner layer.46 Although the GmtB deletion phenotype was subtle, this gene function appears to include cell wall integrity, especially in conidial and hyphal walls. Antifungal susceptibility testing revealed reduction in sensitivity of deleted strains to micafungin (inhibitor of β-glucan synthesis) and increased in sensitivity to the chitin binding agent CR. Strains that have defects in cell wall are usually hypersensitive to CR.47 In a similar manner, mutation of A. nidulans GmtA increased the sensitivity to the cell wall targeting agent Calcofluor White (CFW).21 Increased chitin synthesis has been known as an important compensatory response to cell wall stress in fungi.48 Oka and colleagues reported that the N-acetyl glucosamine (GlcNAc) content of the A. nidulans pmtA disrupted strain was one third higher than wild-type, and pmtAΔ strain was also hypersensitive to CR.34 Furthermore, Engel and colleagues analyzed the cell wall monosaccharides of A. fumigatus ΔgmtA and found that no mannose was detected, and the glucosamine (precursor for chitin) was twice as abundant as wild-type, whereas the glucose content was reduced by 65%.26 Previous findings strongly suggest alterations in β-glucan and chitin content and might explain the increased sensitivity to CR and reduced towards echinocandin in current investigation. In this study, we studied the effect of gmtA and gmtB deletion on spore hydrophobicity. The results showed that the gmtAΔ spores were less hydrophobic than wild-type, whereas the gmtBΔ and gmtAΔ-gmtBΔ spores were more hydrophobic. This agrees with the spore surface character reveled by SEM. A previous study in A. fumigatus reported that the Δpes1 strain (defective in peptide synthetase gene) had a conidial surface with a lower degree of ornamentation and was 51% more hydrophobic than the wild-type.49 We expect that GmtB is responsible for formation of the external ornamentation layer of spore surface; thus its deletion causes disappearance of this layer and exposure of the underlying rodlet layer. Rodlets are composed mainly of hydrophobins,50 Adherence to human epithelial cells was in the following pattern: gmtAΔ < wild-type < gmtBΔ < gmtAΔ-gmtBΔ. This might be attributed to the spore hydrophobicity as the more hydrophobic the spores the more they will be adhesive. It has been reported that the hydrophobin RodA is one of the proteins that mediate spore adherence in A. fumigatus.51 Biofilm formation follows the same pattern of adherence, it is known that adherence to a surface is the first step of biofilm formation.39 The rough spore surface of gmtAΔ strain may play a role in biofilm reduction. As it was previously reported in A. fumigatus that A ΔcspA strain (defective in GPI- anchored cell wall protein) had a rougher surface with more ornaments and showed reduction in biofilm formation.52 Previous studies in other Aspergillus species found a relation between spore surface character and the ability of spores to form biofilm. For example, it has been reported that A. fumigatus ΔmedA strain was less hydropbobic and showed reduction in biofilm formation.35 Also in A. niger, the Δalb1 spores has a smooth surface and were more adhesive to polystyrene plates.53 To our knowledge, double deletion mutant in both Gmt(s) has not been reported in Aspergillus nidulans before. Our double deletion strain was impaired but still viable. Similarly, the cryptococcal double mutant strain was also viable but clearly demonstrates the critical role of mannose glycosylation in C. neoformans, as this strain was sensitive to stress and temperature, was acapsular, and avirulent in a mouse model of infection.27 In conclusion, the altered conidial surface characters, decreased adherence and biofilm formation of gmtAΔ mutant, coupled with the fact that humans do not have GDP-mannose transporters, suggests GmtA can be a potential target for new antifungal that targets virulence determinants. Future work will further investigate the role of GMTs in virulence in animal model. Supplementary material Supplementary data are available at MMYCOL online. Acknowledgements The authors would like to thank Prof Elham Khalifa (Faculty of Agriculture, Cairo University, Cairo, Egypt) for helping with TEM. Funding No specific funding has been received. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. REFERENCES 1. Person AK, Kontoyianni DP, Alexander BD. Fungal infections in transplant and oncology patients. Infect Dis Clin North Am . 2010; 24: 439– 459. Google Scholar CrossRef Search ADS PubMed  2. Moran GP, Coleman DC, Sullivan DJ. Comparative genomics and the evolution of pathogenicity in human pathogenic fungi. Eukaryot Cell . 2011; 10: 34– 42. Google Scholar CrossRef Search ADS PubMed  3. Calderone R, Sun N, Gay-Andrieu F et al.   Antifungal drug discovery: the process and outcomes. 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Medical MycologyOxford University Press

Published: Oct 9, 2017

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