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1.  Human Platelets Attenuate Aspergillus Species via Granule-Dependent Mechanisms 
The Journal of infectious diseases  2008;198(8):1243-1246.
Using laser scanning microscopy, we investigated whether platelets are capable of internalizing Aspergillus conidia and examined Aspergillus-platelet adherence. The influence of platelets on fungal growth was evaluated by assessing galactomannan (GM) release, hyphal elongation, and colony size. A secretion assay with [3H]-serotonin (5-hydroxytryptamine [5-HT]) was performed. Exposure to platelets resulted in significantly decreased GM release (P<.05), hyphal elongation (P<.001), colony size, pig-mentation, and 5-HT release (P<.05). A lack of antifungal effects was observed with the microfilament inhibitor cytochalasin D. Platelets attenuate the virulence of Aspergillus species in vitro on the basis of granule-dependent effects.
doi:10.1086/591458
PMCID: PMC2980866  PMID: 18752432
2.  Cross-species discovery of syncretic drug combinations that potentiate the antifungal fluconazole 
The authors screen for compounds that show synergistic antifungal activity when combined with the widely-used fungistatic drug fluconazole. Chemogenomic profiling explains the mode of action of synergistic drugs and allows the prediction of additional drug synergies.
The authors screen for compounds that show synergistic antifungal activity when combined with the widely-used fungistatic drug fluconazole. Chemogenomic profiling explains the mode of action of synergistic drugs and allows the prediction of additional drug synergies.
Chemical screens with a library enriched for known drugs identified a diverse set of 148 compounds that potentiated the action of the antifungal drug fluconazole against the fungal pathogens Cryptococcus neoformans, Cryptococcus gattii and Candida albicans, and the model yeast Saccharomyces cerevisiae, often in a species-specific manner.Chemogenomic profiles of six confirmed hits in S. cerevisiae revealed different modes of action and enabled the prediction of additional synergistic combinations; three-way synergistic interactions exhibited even stronger synergies at low doses of fluconazole.The synergistic combination of fluconazole and the antidepressant sertraline was active against fluconazole-resistant clinical fungal isolates and in an in vivo model of Cryptococcal infection.
Rising fungal infection rates, especially among immune-suppressed individuals, represent a serious clinical challenge (Gullo, 2009). Cancer, organ transplant and HIV patients, for example, often succumb to opportunistic fungal pathogens. The limited repertoire of approved antifungal agents and emerging drug resistance in the clinic further complicate the effective treatment of systemic fungal infections. At the molecular level, the paucity of fungal-specific essential targets arises from the conserved nature of cellular functions from yeast to humans, as well as from the fact that many essential yeast genes can confer viability at a fraction of wild-type dosage (Yan et al, 2009). Although only ∼1100 of the ∼6000 genes in yeast are essential, almost all genes become essential in specific genetic backgrounds in which another non-essential gene has been deleted or otherwise attenuated, an effect termed synthetic lethality (Tong et al, 2001). Genome-scale surveys suggest that over 200 000 binary synthetic lethal gene combinations dominate the yeast genetic landscape (Costanzo et al, 2010). The genetic buffering phenomenon is also manifest as a plethora of differential chemical–genetic interactions in the presence of sublethal doses of bioactive compounds (Hillenmeyer et al, 2008). These observations frame the difficulty of interdicting network functions in eukaryotic pathogens with single agent therapeutics. At the same time, however, this genetic network organization suggests that judicious combinations of small molecule inhibitors of both essential and non-essential targets may elicit additive or synergistic effects on cell growth (Sharom et al, 2004; Lehar et al, 2008). Unbiased screens for drugs that synergistically enhance a specific bioactive effect, but which are not themselves individually active—termed a syncretic combination—are one means to substantially elaborate chemical space (Keith et al, 2005). Indeed, compounds that enhance the activity of known agents in model yeast and cancer cell line systems have been identified both by focused small molecule library screens and by computational methods (Borisy et al, 2003; Lehar et al, 2007; Nelander et al, 2008; Jansen et al, 2009; Zinner et al, 2009).
To extend the stratagem of chemical synthetic lethality to clinically relevant fungal pathogens, we screened a bioactive library of known drugs for synergistic enhancers of the widely used fungistatic drug fluconazole against the clinically relevant pathogens C. albicans, C. neoformans and C. gattii, as well as the genetically tractable budding yeast S. cerevisiae. Fluconazole is an azole drug that inhibits lanosterol 14α-demethylase, the gene product of ERG11, an essential cytochrome P450 enzyme in the ergosterol biosynthetic pathway (Groll et al, 1998). We identified 148 drugs that potentiate the antifungal action of fluconazole against the four species. These syncretic compounds had not been previously recognized in the clinic as antifungal agents, and many acted in a species-specific manner, often in a potent fungicidal manner.
To understand the mechanisms of synergism, we interrogated six syncretic drugs—trifluoperazine, tamoxifen, clomiphene, sertraline, suloctidil and L-cycloserine—in genome-wide chemogenomic profiles of the S. cerevisiae deletion strain collection (Giaever et al, 1999). These profiles revealed that membrane, vesicle trafficking and lipid biosynthesis pathways are targeted by five of the synergizers, whereas the sphingolipid biosynthesis pathway is targeted by L-cycloserine. Cell biological assays confirmed the predicted membrane disruption effects of the former group of compounds, which may perturb ergosterol metabolism, impair fluconazole export by drug efflux pumps and/or affect active import of fluconazole (Kuo et al, 2010; Mansfield et al, 2010). Based on the integration of chemical–genetic and genetic interaction space, a signature set of deletion strains that are sensitive to the membrane active synergizers correctly predicted additional drug synergies with fluconazole. Similarly, the L-cycloserine chemogenomic profile correctly predicted a synergistic interaction between fluconazole and myriocin, another inhibitor of sphingolipid biosynthesis. The structure of genetic networks suggests that it should be possible to devise higher order drug combinations with even greater selectivity and potency (Sharom et al, 2004). In an initial test of this concept, we found that the combination of a non-synergistic pair drawn from the membrane active and sphingolipid target classes exhibited potent three-way synergism with a low dose of fluconazole. Finally, the combination of sertraline and fluconazole was active in a G. mellonella model of Cryptococcal infection, and was also efficacious against fluconazole-resistant clinical isolates of C. albicans and C. glabrata.
Collectively, these results demonstrate that the combinatorial redeployment of known drugs defines a powerful antifungal strategy and establish a number of potential lead combinations for future clinical assessment.
Resistance to widely used fungistatic drugs, particularly to the ergosterol biosynthesis inhibitor fluconazole, threatens millions of immunocompromised patients susceptible to invasive fungal infections. The dense network structure of synthetic lethal genetic interactions in yeast suggests that combinatorial network inhibition may afford increased drug efficacy and specificity. We carried out systematic screens with a bioactive library enriched for off-patent drugs to identify compounds that potentiate fluconazole action in pathogenic Candida and Cryptococcus strains and the model yeast Saccharomyces. Many compounds exhibited species- or genus-specific synergism, and often improved fluconazole from fungistatic to fungicidal activity. Mode of action studies revealed two classes of synergistic compound, which either perturbed membrane permeability or inhibited sphingolipid biosynthesis. Synergistic drug interactions were rationalized by global genetic interaction networks and, notably, higher order drug combinations further potentiated the activity of fluconazole. Synergistic combinations were active against fluconazole-resistant clinical isolates and an in vivo model of Cryptococcus infection. The systematic repurposing of approved drugs against a spectrum of pathogens thus identifies network vulnerabilities that may be exploited to increase the activity and repertoire of antifungal agents.
doi:10.1038/msb.2011.31
PMCID: PMC3159983  PMID: 21694716
antifungal; combination; pathogen; resistance; synergism
3.  Effects of Fluconazole on the Secretome, the Wall Proteome, and Wall Integrity of the Clinical Fungus Candida albicans ▿ † 
Eukaryotic Cell  2011;10(8):1071-1081.
Fluconazole is a commonly used antifungal drug that inhibits Erg11, a protein responsible for 14α-demethylation during ergosterol synthesis. Consequently, ergosterol is depleted from cellular membranes and replaced by toxic 14α-methylated sterols, which causes increased membrane fluidity and drug permeability. Surface-grown and planktonic cultures of Candida albicans responded similarly to fluconazole at 0.5 mg/liter, showing reduced biomass formation, severely reduced ergosterol levels, and almost complete inhibition of hyphal growth. There was no evidence of cell leakage. Mass spectrometric analysis of the secretome showed that its composition was strongly affected and included 17 fluconazole-specific secretory proteins. Relative quantification of 14N-labeled query walls relative to a reference standard mixture of 15N-labeled yeast and hyphal walls in combination with immunological analysis revealed considerable fluconazole-induced changes in the wall proteome as well. They were, however, similar for both surface-grown and planktonic cultures. Two major trends emerged: (i) decreased incorporation of hypha-associated wall proteins (Als3, Hwp1, and Plb5), consistent with inhibition of hyphal growth, and (ii) increased incorporation of putative wall repair-related proteins (Crh11, Pga4, Phr1, Phr2, Pir1, and Sap9). As exposure to the wall-perturbing drug Congo red led to a similar response, these observations suggested that fluconazole affects the wall. In keeping with this, the resistance of fluconazole-treated cells to wall-perturbing compounds decreased. We propose that fluconazole affects the integrity of both the cellular membranes and the fungal wall and discuss its potential consequences for antifungal therapy. We also present candidate proteins from the secretome for clinical marker development.
doi:10.1128/EC.05011-11
PMCID: PMC3165447  PMID: 21622905
4.  A Role for the Unfolded Protein Response (UPR) in Virulence and Antifungal Susceptibility in Aspergillus fumigatus 
PLoS Pathogens  2009;5(1):e1000258.
Filamentous fungi rely heavily on the secretory pathway, both for the delivery of cell wall components to the hyphal tip and the production and secretion of extracellular hydrolytic enzymes needed to support growth on polymeric substrates. Increased demand on the secretory system exerts stress on the endoplasmic reticulum (ER), which is countered by the activation of a coordinated stress response pathway termed the unfolded protein response (UPR). To determine the contribution of the UPR to the growth and virulence of the filamentous fungal pathogen Aspergillus fumigatus, we disrupted the hacA gene, encoding the major transcriptional regulator of the UPR. The ΔhacA mutant was unable to activate the UPR in response to ER stress and was hypersensitive to agents that disrupt ER homeostasis or the cell wall. Failure to induce the UPR did not affect radial growth on rich medium at 37°C, but cell wall integrity was disrupted at 45°C, resulting in a dramatic loss in viability. The ΔhacA mutant displayed a reduced capacity for protease secretion and was growth-impaired when challenged to assimilate nutrients from complex substrates. In addition, the ΔhacA mutant exhibited increased susceptibility to current antifungal agents that disrupt the membrane or cell wall and had attenuated virulence in multiple mouse models of invasive aspergillosis. These results demonstrate the importance of ER homeostasis to the growth and virulence of A. fumigatus and suggest that targeting the UPR, either alone or in combination with other antifungal drugs, would be an effective antifungal strategy.
Author Summary
The pathogenic mold Aspergillus fumigatus is the leading cause of airborne fungal infections in immunocompromised patients. The fungus normally resides in compost, an environment that challenges the organism to obtain nutrients by degrading complex organic polymers. This is accomplished by secreted enzymes, some of which may also contribute to nutrient acquisition during infection. Extracellular enzymes are folded in the endoplasmic reticulum (ER) prior to secretion. If the folding capacity of the ER is overwhelmed by increased secretory demand, the resulting ER stress triggers an adaptive response termed the unfolded protein response (UPR). In this study, we uncover a previously unknown function for the master transcriptional regulator of the UPR, HacA, in fungal virulence. In the absence of HacA, A. fumigatus was unable to secrete high levels of proteins and had reduced virulence in mice. In addition, loss of HacA caused a cell wall defect and increased susceptibility to two major classes of antifungal drugs used for the treatment of aspergillosis. These findings demonstrate that A. fumigatus relies on HacA for growth in the host environment and suggest that therapeutic targeting of the UPR could have merit against A. fumigatus, as well as other eukaryotic pathogens with highly developed secretory systems.
doi:10.1371/journal.ppat.1000258
PMCID: PMC2606855  PMID: 19132084
5.  Monoclonal Antibodies to Hyphal Exoantigens Derived from the Opportunistic Pathogen Aspergillus terreus ▿ 
Aspergillus terreus has been difficult to identify in cases of aspergillosis, and clinical identification has been restricted to the broad identification of aspergillosis lesions in affected organs or the detection of fungal carbohydrates. As a result, there is a clinical need to identify species-specific biomarkers that can be used to detect invasive A. terreus disease. Monoclonal antibodies (MAbs) were developed to a partially purified preparation of cytolytic hyphal exoantigens (HEA) derived from A. terreus culture supernatant (CSN). Twenty-three IgG1 isotype murine MAbs were developed and tested for cross-reactivity against hyphal extracts of 54 fungal species. Sixteen MAbs were shown to be specific for A. terreus. HEA were detected in conidia, hyphae, and in CSN of A. terreus. HEA were expressed in high levels in the hyphae during early stages of A. terreus growth at 37°C, whereas at room temperature the expression of HEA peaked by days 4 to 5. Expression kinetics of HEA in CSN showed a lag, with peak levels at later time points at room temperature and 37°C than in hyphal extracts. Serum spiking experiments demonstrated that human serum components do not inhibit detection of the HEA epitopes by MAb enzyme-linked immunosorbent assay (ELISA). Immunoprecipitation and proteomic analysis demonstrated that MAbs 13E11 and 12C4 immunoprecipitated a putative uncharacterized leucine aminopeptidase (Q0CAZ7), while MAb 19B2 recognized a putative dipeptidyl-peptidase V (DPP5). Studies using confocal laser scanning microscopy showed that the uncharacterized leucine aminopeptidase mostly localized to extracellular matrix structures while dipeptidyl-peptidase V was mostly confined to the cytoplasm.
doi:10.1128/CVI.05163-11
PMCID: PMC3165237  PMID: 21734068
6.  Alginate Oligosaccharides Inhibit Fungal Cell Growth and Potentiate the Activity of Antifungals against Candida and Aspergillus spp 
PLoS ONE  2014;9(11):e112518.
The oligosaccharide OligoG, an alginate derived from seaweed, has been shown to have anti-bacterial and anti-biofilm properties and potentiates the activity of selected antibiotics against multi-drug resistant bacteria. The ability of OligoG to perturb fungal growth and potentiate conventional antifungal agents was evaluated using a range of pathogenic fungal strains. Candida (n = 11) and Aspergillus (n = 3) spp. were tested using germ tube assays, LIVE/DEAD staining, scanning electron microscopy (SEM), atomic force microscopy (AFM) and high-throughput minimum inhibition concentration assays (MICs). In general, the strains tested showed a significant dose-dependent reduction in cell growth at ≥6% OligoG as measured by optical density (OD600; P<0.05). OligoG (>0.5%) also showed a significant inhibitory effect on hyphal growth in germ tube assays, although strain-dependent variations in efficacy were observed (P<0.05). SEM and AFM both showed that OligoG (≥2%) markedly disrupted fungal biofilm formation, both alone, and in combination with fluconazole. Cell surface roughness was also significantly increased by the combination treatment (P<0.001). High-throughput robotic MIC screening demonstrated the potentiating effects of OligoG (2, 6, 10%) with nystatin, amphotericin B, fluconazole, miconazole, voriconazole or terbinafine with the test strains. Potentiating effects were observed for the Aspergillus strains with all six antifungal agents, with an up to 16-fold (nystatin) reduction in MIC. Similarly, all the Candida spp. showed potentiation with nystatin (up to 16-fold) and fluconazole (up to 8-fold). These findings demonstrate the antifungal properties of OligoG and suggest a potential role in the management of fungal infections and possible reduction of antifungal toxicity.
doi:10.1371/journal.pone.0112518
PMCID: PMC4237368  PMID: 25409186
7.  Requirement for Ergosterol in V-ATPase Function Underlies Antifungal Activity of Azole Drugs 
PLoS Pathogens  2010;6(6):e1000939.
Ergosterol is an important constituent of fungal membranes. Azoles inhibit ergosterol biosynthesis, although the cellular basis for their antifungal activity is not understood. We used multiple approaches to demonstrate a critical requirement for ergosterol in vacuolar H+-ATPase function, which is known to be essential for fungal virulence. Ergosterol biosynthesis mutants of S. cerevisiae failed to acidify the vacuole and exhibited multiple vma− phenotypes. Extraction of ergosterol from vacuolar membranes also inactivated V-ATPase without disrupting membrane association of its subdomains. In both S. cerevisiae and the fungal pathogen C. albicans, fluconazole impaired vacuolar acidification, whereas concomitant ergosterol feeding restored V-ATPase function and cell growth. Furthermore, fluconazole exacerbated cytosolic Ca2+ and H+ surges triggered by the antimicrobial agent amiodarone, and impaired Ca2+ sequestration in purified vacuolar vesicles. These findings provide a mechanistic basis for the synergy between azoles and amiodarone observed in vitro. Moreover, we show the clinical potential of this synergy in treatment of systemic fungal infections using a murine model of Candidiasis. In summary, we demonstrate a new regulatory component in fungal V-ATPase function, a novel role for ergosterol in vacuolar ion homeostasis, a plausible cellular mechanism for azole toxicity in fungi, and preliminary in vivo evidence for synergism between two antifungal agents. New insights into the cellular basis of azole toxicity in fungi may broaden therapeutic regimens for patient populations afflicted with systemic fungal infections.
Author Summary
Systemic fungal infections impose a significant threat to public health and therapeutic options to treat these diseases remain limited. Azoles represent the largest category of anti-fungal drugs and repress fungal growth by inhibiting biosynthesis of ergosterol, an important constituent of fungal membranes. Despite the wide use of azoles in the clinic for decades, the cellular basis for their antifungal mechanism remains elusive. In this study, we use a range of genetic, cellular and biochemical approaches to reveal a requirement for ergosterol in vacuolar H+-ATPase function. V-ATPase plays essential roles in diverse cellular processes, and is required for fungal virulence. Concomitant ergosterol feeding restores vacuolar acidification and growth in cells treated with fluconazole. These results suggest that the critical requirement for ergosterol in V-ATPase function may underlie the antifungal activity of azoles. Moreover, we show in a mouse Candidiasis model that combining an ion homeostasis-disruptive drug with azole is an effective approach to treat fungal infections.
doi:10.1371/journal.ppat.1000939
PMCID: PMC2880581  PMID: 20532216
8.  In Vitro Analyses of Mild Heat Stress in Combination with Antifungal Agents against Aspergillus fumigatus Biofilm 
Aspergillus fumigatus biofilms still present a challenge for effective treatment in clinical settings. While mild heat stress has been introduced as a treatment for infectious diseases, the effectiveness of mild heat stress on A. fumigatus biofilm formation and antifungal susceptibility is still unknown. In the present study, confocal laser scanning microscopy (CLSM) was used to image and quantify Aspergillus fumigatus biofilm formation under three different regimens of continuous mild heat stress: at 37, 39, and 41°C. Furthermore, fungal growth has been investigated under the above conditions in combination with antifungal drugs (amphotericin B [AMB], micafungin [MCF], and voriconazole [VOC]) at early and late stages. CLSM analysis showed that higher temperatures induce earlier germination and greater hyphal elongation but poorer polar growth and reduced biofilm thickness. In the early stage of biofilm formation, the combination of treatment at 39 or 41°C with MCF or VOC produced no visible difference in biomass formation from similar treatments at 37°C with the same drug. Interestingly, AMB treatment at 37°C inhibited early stage biofilm formation to a much greater extent than at 39 and 41°C. At the late stage of biofilm formation, the mild heat treatments at 39 and 41°C with AMB, MCF, and VOC inhibited biomass formation compared to that at 37°C. The present data show that mild heat stress has a negative regulatory effect on biofilm formation in vitro, and antifungal drug improvement with mild heat treatment at late-stage biofilm formation provides useful indications of possible effective strategies for clinical management of aspergillosis.
doi:10.1128/AAC.01007-13
PMCID: PMC3957873  PMID: 24342649
9.  ChLae1 and ChVel1 Regulate T-toxin Production, Virulence, Oxidative Stress Response, and Development of the Maize Pathogen Cochliobolus heterostrophus 
PLoS Pathogens  2012;8(2):e1002542.
LaeA and VeA coordinate secondary metabolism and differentiation in response to light signals in Aspergillus spp. Their orthologs, ChLae1 and ChVel1, were identified in the maize pathogen Cochliobolus heterostrophus, known to produce a wealth of secondary metabolites, including the host selective toxin, T-toxin. Produced by race T, T-toxin promotes high virulence to maize carrying Texas male sterile cytoplasm (T-cms). T-toxin production is significantly increased in the dark in wild type (WT), whereas Chvel1 and Chlae1 mutant toxin levels are much reduced in the dark compared to WT. Correspondingly, expression of T-toxin biosynthetic genes (Tox1) is up-regulated in the dark in WT, while dark-induced expression is much reduced/minimal in Chvel1 and Chlae1 mutants. Toxin production and Tox1 gene expression are increased in ChVEL1 overexpression (OE) strains grown in the dark and in ChLAE1 strains grown in either light or dark, compared to WT. These observations establish ChLae1 and ChVel1 as the first factors known to regulate host selective toxin production. Virulence of Chlae1 and Chvel1 mutants and OE strains is altered on both T-cms and normal cytoplasm maize, indicating that both T-toxin mediated super virulence and basic pathogenic ability are affected. Deletion of ChLAE1 or ChVEL1 reduces tolerance to H2O2. Expression of CAT3, one of the three catalase genes, is reduced in the Chvel1 mutant. Chlae1 and Chvel1 mutants also show decreased aerial hyphal growth, increased asexual sporulation and female sterility. ChLAE1 OE strains are female sterile, while ChVEL1 OE strains are more fertile than WT. ChLae1 and ChVel1 repress expression of 1,8-dihydroxynaphthalene (DHN) melanin biosynthesis genes, and, accordingly, melanization is enhanced in Chlae1 and Chvel1 mutants, and reduced in OE strains. Thus, ChLae1 and ChVel1 positively regulate T-toxin biosynthesis, pathogenicity and super virulence, oxidative stress responses, sexual development, and aerial hyphal growth, and negatively control melanin biosynthesis and asexual differentiation.
Author Summary
Filamentous fungi produce chemically diverse metabolites that broker positive and negative interactions with other organisms, manage host pathogenicity/virulence, nutritional and environmental stresses, and differentiation of the fungus. The maize pathogen Cochliobolus heterostrophus is notorious as the causal agent of the most economically devastating epidemic to date, in 1970. Disease severity was associated with appearance of a new race, producing T-toxin, a host selective toxin promoting high virulence to Texas male sterile cytoplasm maize, widely planted at the time. LaeA and VeA are central regulators of secondary metabolism in Aspergillus, coordinating metabolite production and differentiation in response to light. Given the significance of effector-type host selective toxins in pathogenic interactions, we characterized ChLae1 and ChVel1 and found that deletion and overexpression affect T-toxin production in planta and in vitro. Both chlorosis due to T-toxin and necrotic lesion formation are altered, establishing these as the first factors known to regulate both super virulence conferred by T-toxin, and basic pathogenicity, due to unknown factors. The mutants are also altered in oxidative stress responses, key to success in the infection court, asexual and sexual development, essential for fungal dissemination in the field, aerial hyphal growth, and pigment biosynthesis, essential for survival in the field.
doi:10.1371/journal.ppat.1002542
PMCID: PMC3285592  PMID: 22383877
10.  Functional Characterization of Aspergillus nidulans ypkA, a Homologue of the Mammalian Kinase SGK 
PLoS ONE  2013;8(3):e57630.
The serum- and glucocorticoid-regulated protein kinase (SGK) is an AGC kinase involved in signal cascades regulated by glucocorticoid hormones and serum in mammals. The Saccharomyces cerevisiae ypk1 and ypk2 genes were identified as SGK homologues and Ypk1 was shown to regulate the balance of sphingolipids between the inner and outer plasma membrane. This investigation characterized the Aspergillus nidulans YPK1 homologue, YpkA, representing the first filamentous fungal YPK1 homologue. Two conditional mutant strains were constructed by replacing the endogenous ypk1 promoter with two different regulatable promoters, alcA (from the alcohol dehydrogenase gene) and niiA (from the nitrate reductase gene). Both constructs confirmed that ypkA was an essential gene in A. nidulans. Repression of ypkA caused decreased radial growth, a delay in conidial germination, deficient polar axis establishment, intense branching during late stages of growth, a lack of asexual spores, and a terminal phenotype. Membrane lipid polarization, endocytosis, eisosomes and vacuolar distribution were also affected by ypkA repression, suggesting that YpkA plays a role in hyphal morphogenesis via coordinating the delivery of cell membrane and wall constituents to the hyphal apex. The A. nidulans Pkh1 homologue pkhA was also shown to be an essential gene, and preliminary genetic analysis suggested that the ypkA gene is not directly downstream of pkhA or epistatic to pkhA, rather, ypkA and pkhA are genetically independent or in parallel. BarA is a homologue of the yeast Lag1 acyl-CoA-dependent ceramide synthase, which catalyzes the condensation of phytosphingosine with a fatty acyl-CoA to form phytoceramide. When barA was absent, ypkA repression was lethal to the cell. Therefore, there appears to be a genetic interaction between ypkA, barA, and the sphingolipid synthesis. Transcriptional profiling of ypkA overexpression and down-regulation revealed several putative YpkA targets associated with the observed phenotypes.
doi:10.1371/journal.pone.0057630
PMCID: PMC3589345  PMID: 23472095
11.  A GATA Transcription Factor Recruits Hda1 in Response to Reduced Tor1 Signaling to Establish a Hyphal Chromatin State in Candida albicans 
PLoS Pathogens  2012;8(4):e1002663.
Candida albicans is an important opportunistic fungal pathogen of immunocompromised individuals. One critical virulence attribute is its morphogenetic plasticity. Hyphal development requires two temporally linked changes in promoter chromatin, which is sequentially regulated by temporarily clearing the transcription inhibitor Nrg1 upon activation of the cAMP/PKA pathway and promoter recruitment of the histone deacetylase Hda1 under reduced Tor1 signaling. Molecular mechanisms for the temporal connection and the link to Tor1 signaling are not clear. Here, through a forward genetic screen, we report the identification of the GATA family transcription factor Brg1 as the factor that recruits Hda1 to promoters of hypha-specific genes during hyphal elongation. BRG1 expression requires both the removal of Nrg1 and a sub-growth inhibitory level of rapamycin; therefore, it is a sensitive readout of Tor1 signaling. Interestingly, promoters of hypha-specific genes are not accessible to Brg1 in yeast cells. Furthermore, ectopic expression of Brg1 cannot induce hyphae, but can sustain hyphal development. Nucleosome mapping of a hypha-specific promoter shows that Nrg1 binding sites are in nucleosome free regions in yeast cells, whereas Brg1 binding sites are occupied by nucleosomes. Nucleosome disassembly during hyphal initiation exposes the binding sites for both regulators. During hyphal elongation, Brg1-mediated Hda1 recruitment causes nucleosome repositioning and occlusion of Nrg1 binding sites. We suggest that nucleosome repositioning is the underlying mechanism for the yeast-hyphal transition. The hypha-specific regulator Ume6 is a key downstream target of Brg1 and functions after Brg1 as a built-in positive feedback regulator of the hyphal transcriptional program to sustain hyphal development. With the levels of Nrg1 and Brg1 dynamically and sensitively controlled by the two major cellular growth pathways, temporal changes in nucleosome positioning during the yeast-to-hypha transition provide a mechanism for signal integration and cell fate specification. This mechanism is likely used broadly in development.
Author Summary
Candida is part of the gut microflora in healthy individuals, but can disseminate and cause systemic disease when the host's immune system is suppressed. Its ability to grow as yeast and hyphae in response to environmental cues is a major virulence attribute. Hyphal development requires temporary clearing of the transcription inhibitor Nrg1 upon activation of cAMP/PKA for initiation and promoter recruitment of the histone deacetylase Hda1 under reduced Tor1 signaling for maintenance. Here, we show that, during hyphal initiation when Nrg1 is gone, expression of the GATA family transcription factor Brg1 is activated under reduced Tor1 signaling. Accumulated Brg1 recruits Hda1 to hyphal promoters to reposition nucleosomes, leading to obstruction of Nrg1 binding sites and sustained hyphal development. The nucleosome repositioning during the yeast-hyphal transition provides a mechanism for temporal integration of extracellular signals and cell-fate specification. The hypha-specific transcription factor Ume6 functions after Brg1 in this succession of feed-forward regulation of hyphal development. Since misregulation of either Nrg1 or Ume6 causes altered virulence, and Brg1 regulates both Nrg1 accessibility and Ume6 transcription, our findings should provide a better understanding of how Candida controls its morphological program in different host niches to exist as a commensal and a pathogen.
doi:10.1371/journal.ppat.1002663
PMCID: PMC3334898  PMID: 22536157
12.  The Putative α-1,2-Mannosyltransferase AfMnt1 of the Opportunistic Fungal Pathogen Aspergillus fumigatus Is Required for Cell Wall Stability and Full Virulence▿  
Eukaryotic Cell  2008;7(10):1661-1673.
Proteins entering the eukaryotic secretory pathway commonly are glycosylated. Important steps in this posttranslational modification are carried out by mannosyltransferases. In this study, we investigated the putative α-1,2-mannosyltransferase AfMnt1 of the human pathogenic mold Aspergillus fumigatus. AfMnt1 belongs to a family of enzymes that comprises nine members in Saccharomyces cerevisiae but only three in A. fumigatus. A Δafmnt1 mutant is viable and grows normally at 37°C, but its hyphal cell wall appears to be thinner than that of the parental strain. The lack of AfMnt1 leads to a higher sensitivity to calcofluor white and Congo red but not to sodium dodecyl sulfate. The growth of the mutant is abrogated at 48°C but can be restored by osmotic stabilization. The resulting colonies remain white due to a defect in the formation of conidia. Electron and immunofluorescence microscopy further revealed that the observed growth defect of the mutant at 48°C can be attributed to cell wall instability resulting in leakage at the hyphal tips. Using a red fluorescence fusion protein, we localized AfMnt1 in compact, brefeldin A-sensitive organelles that most likely represent fungal Golgi equivalents. The tumor necrosis factor alpha response of murine macrophages to hyphae was not affected by the lack of the afmnt1 gene, but the corresponding mutant was attenuated in a mouse model of infection. This and the increased sensitivity of the Δafmnt1 mutant to azoles, antifungal agents that currently are used to treat Aspergillus infections, suggest that α-1,2-mannosyltransferases are interesting targets for novel antifungal drugs.
doi:10.1128/EC.00221-08
PMCID: PMC2568062  PMID: 18708564
13.  Heat Shock Protein 90 Is Required for Conidiation and Cell Wall Integrity in Aspergillus fumigatus 
Eukaryotic Cell  2012;11(11):1324-1332.
Heat shock protein 90 (Hsp90) is a eukaryotic molecular chaperone. Its involvement in the resistance of Candida albicans to azole and echinocandin antifungals is well established. However, little is known about Hsp90's function in the filamentous fungal pathogen Aspergillus fumigatus. We investigated the role of Hsp90 in A. fumigatus by genetic repression and examined its cellular localization under various stress conditions. Failure to generate a deletion strain of hsp90 suggested that it is essential. Genetic repression of Hsp90 was achieved by an inducible nitrogen-dependent promoter (pniiA-Hsp90) and led to decreased spore viability, decreased hyphal growth, and severe defects in germination and conidiation concomitant with the downregulation of the conidiation-specific transcription factors brlA, wetA, and abaA. Hsp90 repression potentiated the effect of cell wall inhibitors affecting the β-glucan structure of the cell wall (caspofungin, Congo red) and of the calcineurin inhibitor FK506, supporting a role in regulating cell wall integrity pathways. Moreover, compromising Hsp90 abolished the paradoxical effect of caspofungin. Pharmacological inhibition of Hsp90 by geldanamycin and its derivatives (17-AAG and 17-DMAG) resulted in similar effects. C-terminal green fluorescent protein (GFP) tagging of Hsp90 revealed mainly cytosolic distribution under standard growth conditions. However, treatment with caspofungin resulted in Hsp90 accumulation at the cell wall and at sites of septum formation, further highlighting its role in cell wall stress compensatory mechanisms. Targeting Hsp90 with fungal-specific inhibitors to cripple stress response compensatory pathways represents an attractive new antifungal strategy.
doi:10.1128/EC.00032-12
PMCID: PMC3486032  PMID: 22822234
14.  Extrinsic extracellular DNA leads to biofilm formation and colocalizes with matrix polysaccharides in the human pathogenic fungus Aspergillus fumigatus 
The environmentally acquired fungal pathogen Aspergillus fumigatus causes a variety of severe diseases. Furthermore, it is often found colonizing the respiratory tract of patients suffering from cystic fibrosis. Conidia of this filamentous fungus adhere to substrate surfaces and germinate to form biofilms comprised of dense hyphal networks embedded in an adhesive extracellular matrix (ECM), built predominantly of polysaccharides. These fungal microconsortia are likely to be of clinical relevance, as they have also been observed during growth in the host and they confer drastically reduced susceptibility to antifungals. Little is known about environmental factors or signals contributing to the formation and structural organization of this polysaccharide matrix. Extracellular DNA (eDNA) is an abundant molecule in the mucus-rich surfaces in the lungs of cystic fibrosis patients. Here, we studied its influence on the biofilm establishment and progression of A. fumigatus. Using an in vitro biofilm model eDNA was identified as an efficient biofilm inducer promoting conidial surface adhesion and polysaccharide ECM production. Confocal laser scanning microscopy revealed entirely different ECM architectures depending on the substrates used for biofilm induction. In the presence of serum, adhesive polysaccharides were mainly localized to the hyphal tips appearing as cohesive threads or “halo” areas agglutinating the hyphae. Exogenous DNA altered the structural organization of the biofilm specifically by colocalizing to a grid-like bottom layer of ECM. These results indicate that biofilm formation in A. fumigatus is shaped by certain substrates and in response to host environmental signals.
doi:10.3389/fmicb.2013.00141
PMCID: PMC3674311  PMID: 23760756
aspergillus; cystic fibrosis; extracellular DNA; extracellular matrix; fungal biofilm
15.  In Vitro Activity of Caspofungin Combined with Sulfamethoxazole against Clinical Isolates of Aspergillus spp. 
Caspofungin (CAS) inhibits fungal cell wall synthesis. Sulfamethoxazole (SMX) inhibits folate biosynthesis and is active in vitro against Aspergillus spp. We studied the activities of the combination of CAS and SMX against 31 Aspergillus isolates and compared them with that of SMX combined with amphotericin B (AMB) or itraconazole (ITC). MICs and minimal effective concentrations (MECs) were determined by the NCCLS broth microdilution method. With MIC endpoints, the combination of SMX and CAS showed synergy or synergy to additivity against 29 of 31 isolates. With MEC endpoints, synergy to additivity was found against 12 of 31 isolates and indifference was displayed against the rest of them. SMX in combination with AMB or ITC was not truly synergistic, while synergy to additivity was found for SMX-AMB and SMX-ITC against 17 of 31 and 3 of 12 isolates, respectively. No antagonism was found with any of the drug combinations. Further analysis of the synergy of CAS and SMX was performed by detailed measurement of hyphal length by microscopy and time-dependent 2,3-bis(2-methoxy-4-nitro-5-[(sulfenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT)-based hyphal damage experiments. With MEC endpoints, the combination of CAS and SMX was characterized by a greater than 50% decrease in hyphal length compared to the hyphal lengths achieved with double the concentration of each drug alone. The XTT-based hyphal damage studies showed a statistically significant (P < 0.05) reduction in viability with CAS and SMX in combination compared to the viabilities achieved with double the concentration of each drug alone. These findings support the synergy results found by using MIC endpoints and suggest that visual MEC measurements may not be sufficient to identify the synergistic interactions seen by more sensitive, quantitative methods. Animal models are required to validate the significance of the synergy of CAS and SMX against Aspergillus spp. observed in vitro.
doi:10.1128/AAC.48.9.3279-3283.2004
PMCID: PMC514744  PMID: 15328085
16.  Colletotrichum trifolii TB3 Kinase, a COT1 Homolog, Is Light Inducible and Becomes Localized in the Nucleus during Hyphal Elongation 
Eukaryotic Cell  2002;1(4):626-633.
Colletotrichum trifolii is a fungal pathogen responsible for anthracnose disease of alfalfa. Previously, a serine/threonine protein kinase gene from this fungus (TB3), which is a functional homolog of the Neurospora crassa COT1 kinase, has been isolated in our laboratory and appears to be associated with hyphal elongation and branching. In this report we show that light treatment rapidly induces TB3 expression and hyphal branching frequency. Western analysis showed TB3 localization in both the cytoplasm and nucleus, but not in membranes. Moreover, indirect immunofluorescence indicated that TB3 levels were most abundant in the nucleus. To further evaluate the subcellular distribution of TB3, a TB3::GFP fusion construct was inserted into C. trifolii. Results indicated that the cellular location of TB3 changed during fungal growth and development. Consistent with previous observations, TB3 was localized in both the cytoplasm and the nucleus but was preferentially localized in the nucleus during extended hyphal growth. The amino terminus of TB3 contains two relatively long polyglutamine repeats. Yeast-based assays showed that these polyglutamine tracts can activate transcription. These results suggest that TB3 may be positioned in a signaling cascade regulating proper hyphal growth and development by functioning as a transcription factor.
doi:10.1128/EC.1.4.626-633.2002
PMCID: PMC117997  PMID: 12456010
17.  Antibiosis interaction of Staphylococccus aureus on Aspergillus fumigatus assessed in vitro by mixed biofilm formation 
BMC Microbiology  2015;15:33.
Background
Microorganisms of different species interact in several ecological niches, even causing infection. During the infectious process, a biofilm of single or multispecies can develop. Aspergillus fumigatus and Staphyloccocus aureus are etiologic agents that can cause infectious keratitis. We analyzed in vitro single A. fumigatus and S. aureus, and mixed A. fumigatus-S. aureus biofilms. Both isolates were from patients with infectious keratitis. Structure of the biofilms was analyzed through microscopic techniques including scanning electron microscopy (SEM), transmission electron microscopy (TEM), confocal, and fluorescence microscopy (CLSM) in mixed biofilm as compared with the single A. fumigatus biofilm.
Results
To our knowledge, this is the first time that the structural characteristics of the mixed biofilm A. fumigatus-A. fumigatus were described and shown. S. aureus sharply inhibited the development of biofilm formed by A. fumigatus, regardless of the stage of biofilm formation and bacterial inoculum. Antibiosis effect of bacterium on fungus was as follows: scarce production of A. fumigatus biofilm; disorganized fungal structures; abortive hyphae; and limited hyphal growth; while conidia also were scarce, have modifications in their surface and presented lyses. Antagonist effect did not depend on bacterial concentration, which could probably be due to cell-cell contact interactions and release of bacterial products. In addition, we present images about the co-localization of polysaccharides (glucans, mannans, and chitin), and DNA that form the extracellular matrix (ECM). In contrast, single biofilms showed extremely organized structures: A. fumigatus showed abundant hyphal growth, hyphal anastomosis, and channels, as well as some conidia, and ECM. S. aureus showed microcolonies and cell-to-cell bridges and ECM.
Conclusions
Herein we described the antibiosis relationship of S. aureus against A. fumigatus during in vitro biofilm formation, and report the composition of the ECM formed.
doi:10.1186/s12866-015-0363-2
PMCID: PMC4335557
Biofilm; Extracellular matrix; Antibiosis of Staphylococcus aureus against Aspergillus fumigatus; Fungus-bacteria interaction
18.  Screening for Antifungal Peptides and Their Modes of Action in Aspergillus nidulans▿ †  
Applied and Environmental Microbiology  2010;76(21):7102-7108.
Many short cationic peptides have been identified as potent antimicrobial agents, but their modes of action are not well understood. Peptide synthesis on cellulose membranes has resulted in the generation of peptide libraries, while high-throughput assays have been developed to test their antibacterial activities. In this paper a microtiter plate-based screening method for fungi has been developed and used to test nine antibacterial peptides against the model fungus Aspergillus nidulans. Microscopical studies using sublethal peptide concentrations caused defects in polarized growth, including increased branch formation and depolarized hyphae. We characterized the mode of action for one of our target peptides, Sub5 (12 amino acids), which has already been shown to possess pharmacological potential as an antibacterial agent and is able to interact with ATP and ATP-dependent enzymes. The MIC for A. nidulans is 2 μg/ml, which is in the same range as the MICs reported for bacteria. Fluorescein isothiocyanate (FITC)-labeled Sub5 targeted the cytoplasmic membrane, particularly hyphal tips, and entered the cytoplasm after prolonged exposure, independent of endocytosis. Interestingly, Sub5 peptide treatment disturbed sterol-rich membrane domains, important for tip growth, at hyphal tips. A very similar peptide, FITC-P7, also accumulated on the cell membrane but did not have antibacterial or antifungal activity, suggesting that the cytoplasmic membrane is a first target for the Sub5 peptide; however, the antifungal activity seems to be correlated with the ability to enter the cytoplasm, where the peptides might act on other targets.
doi:10.1128/AEM.01560-10
PMCID: PMC2976221  PMID: 20833782
19.  Comparison of Six DNA Extraction Methods for Recovery of Fungal DNA as Assessed by Quantitative PCR 
Journal of Clinical Microbiology  2005;43(10):5122-5128.
The detection of fungal pathogens in clinical samples by PCR requires the use of extraction methods that efficiently lyse fungal cells and recover DNA suitable for amplification. We used quantitative PCR assays to measure the recovery of DNA from two important fungal pathogens subjected to six DNA extraction methods. Aspergillus fumigatus conidia or Candida albicans yeast cells were added to bronchoalveolar lavage fluid and subjected to DNA extraction in order to assess the recovery of DNA from a defined number of fungal propagules. In order to simulate hyphal growth in tissue, Aspergillus fumigatus conidia were allowed to form mycelia in tissue culture media and then harvested for DNA extraction. Differences among the DNA yields from the six extraction methods were highly significant (P < 0.0001) in each of the three experimental systems. An extraction method based on enzymatic lysis of fungal cell walls (yeast cell lysis plus the use of GNOME kits) produced high levels of fungal DNA with Candida albicans but low levels of fungal DNA with Aspergillus fumigatus conidia or hyphae. Extraction methods employing mechanical agitation with beads produced the highest yields with Aspergillus hyphae. The MasterPure yeast method produced high levels of DNA from C. albicans but only moderate yields from A. fumigatus. A reagent from one extraction method was contaminated with fungal DNA, including DNA from Aspergillus and Candida species. In conclusion, the six extraction methods produce markedly differing yields of fungal DNA and thus can significantly affect the results of fungal PCR assays. No single extraction method was optimal for all organisms.
doi:10.1128/JCM.43.10.5122-5128.2005
PMCID: PMC1248488  PMID: 16207973
20.  Calcineurin Plays Key Roles in the Dimorphic Transition and Virulence of the Human Pathogenic Zygomycete Mucor circinelloides 
PLoS Pathogens  2013;9(9):e1003625.
Many pathogenic fungi are dimorphic and switch between yeast and filamentous states. This switch alters host-microbe interactions and is critical for pathogenicity. However, in zygomycetes, whether dimorphism contributes to virulence is a central unanswered question. The pathogenic zygomycete Mucor circinelloides exhibits hyphal growth in aerobic conditions but switches to multi-budded yeast growth under anaerobic/high CO2 conditions. We found that in the presence of the calcineurin inhibitor FK506, Mucor exhibits exclusively multi-budded yeast growth. We also found that M. circinelloides encodes three calcineurin catalytic A subunits (CnaA, CnaB, and CnaC) and one calcineurin regulatory B subunit (CnbR). Mutations in the latch region of CnbR and in the FKBP12-FK506 binding domain of CnaA result in hyphal growth of Mucor in the presence of FK506. Disruption of the cnbR gene encoding the sole calcineurin B subunit necessary for calcineurin activity yielded mutants locked in permanent yeast phase growth. These findings reveal that the calcineurin pathway plays key roles in the dimorphic transition from yeast to hyphae. The cnbR yeast-locked mutants are less virulent than the wild-type strain in a heterologous host system, providing evidence that hyphae or the yeast-hyphal transition are linked to virulence. Protein kinase A activity (PKA) is elevated during yeast growth under anaerobic conditions, in the presence of FK506, or in the yeast-locked cnbR mutants, suggesting a novel connection between PKA and calcineurin. cnaA mutants lacking the CnaA catalytic subunit are hypersensitive to calcineurin inhibitors, display a hyphal polarity defect, and produce a mixture of yeast and hyphae in aerobic culture. The cnaA mutants also produce spores that are larger than wild-type, and spore size is correlated with virulence potential. Our results demonstrate that the calcineurin pathway orchestrates the yeast-hyphal and spore size dimorphic transitions that contribute to virulence of this common zygomycete fungal pathogen.
Author Summary
Calcineurin is a Ca2+/calmodulin-dependent, serine/threonine-specific protein phosphatase. In pathogenic fungi, calcineurin is involved in morphogenesis and virulence. Therefore, calcineurin is an attractive antifungal drug target. The roles of calcineurin in virulence have been established in both major human pathogenic fungi (Candida species, Cryptococcus neoformans/gattii, Aspergillus fumigatus) and in plant pathogenic fungi (Magnaporthe oryzae, Ustilago maydis/hordei). However, the role of calcineurin is currently unknown in pathogenic zygomycetes. We found that the calcineurin inhibitors FK506 and cyclosporine A inhibit the growth of a prevalent zygomycete pathogen, Mucor. This fungus grows as multi-budded yeast under anaerobic conditions, and we have found that even in aerated culture (which without FK506 would result in abundant hyphal growth), Mucor exhibits yeast growth when exposed to FK506. Mucor cnbR mutants that lack the calcineurin regulatory subunit essential for calcineurin activity, are locked in perpetual yeast phase growth, indicating that calcineurin is required for hyphal growth. We further demonstrated that these yeast-locked mutants are attenuated for virulence, illustrating that hyphae or the yeast-hyphal transition are linked to virulence. These findings indicate that: 1) calcineurin governs the yeast/hyphae morphogenic transition; 2) a link exists between respiration and the calcineurin pathway; and 3) calcineurin inhibitors are attractive anti-mucormycosis drug candidates.
doi:10.1371/journal.ppat.1003625
PMCID: PMC3764228  PMID: 24039585
21.  Use of Fluorescent Probes To Determine MICs of Amphotericin B and Caspofungin against Candida spp. and Aspergillus spp. 
Journal of Clinical Microbiology  2005;43(8):3788-3792.
We investigated the utility of mechanism-based fluorescent probes for determination of MICs (FMICs) of amphotericin B and caspofungin against Candida spp. and Aspergillus spp. Amphotericin B was selected as a membrane-active antifungal agent, and caspofungin was selected as a cell wall-active agent. FMICs were also compared to the MIC determined by CLSI (formerly NCCLS) methods. Five isolates per species of Candida albicans, Candida glabrata, Candida parapsilosis, Aspergillus fumigatus, and Aspergillus terreus were studied with either amphotericin B or caspofungin. The fluorescent probes, carboxyfluorescein diacetate (CFDA) for cytoplasmic esterase activity and dihexyloxacarbocyanine iodide (DiOC6) for cell membrane potential, were each added to their respective plates. MICs and FMICs were determined in at least three separate experiments (in duplicate). Fluorescence was measured using a 96-well plate fluorometer. For amphotericin B and caspofungin, the FMIC end point was the lowest concentration of drug at which the percent growth inhibition from treated organisms versus control organisms displayed 80% inhibition for amphotericin B and 50% inhibition for caspofungin as measured by a fluorescent signal. The MIC for amphotericin B was defined as the lowest concentration of antifungal displaying no visible growth for both Aspergillus and Candida spp. The MIC for caspofungin was the lowest concentration of drug that displayed a minimum effective concentration for Aspergillus spp. For Candida spp., the MIC for caspofungin was defined as the concentration at which the antifungal agent significantly inhibits the organism. The FMICs of both antifungals, as measured by the DiOC6 membrane probe, showed good agreement (83% to 100%), within one well dilution, with the MICs against amphotericin B and caspofungin for all species. Also, the FMICs measured by the CFDA cytoplasmic esterase probe reflecting damage due to cell wall or cell membrane showed strong agreement (79 to 100%) with the MICs of both amphotericin B and caspofungin for all species. There was no significant difference in comparisons of MIC and FMIC values (P ≥ 0.05). The use of fluorescent probes provides a mechanism-based method of determination of MICs of amphotericin B and caspofungin against Candida spp. and Aspergillus spp. that correlates well with standard methods.
doi:10.1128/JCM.43.8.3788-3792.2005
PMCID: PMC1233965  PMID: 16081911
22.  IL-1α Signaling Is Critical for Leukocyte Recruitment after Pulmonary Aspergillus fumigatus Challenge 
PLoS Pathogens  2015;11(1):e1004625.
Aspergillus fumigatus is a mold that causes severe pulmonary infections. Our knowledge of how A. fumigatus growth is controlled in the respiratory tract is developing, but still limited. Alveolar macrophages, lung resident macrophages, and airway epithelial cells constitute the first lines of defense against inhaled A. fumigatus conidia. Subsequently, neutrophils and inflammatory CCR2+ monocytes are recruited to the respiratory tract to prevent fungal growth. However, the mechanism of neutrophil and macrophage recruitment to the respiratory tract after A. fumigatus exposure remains an area of ongoing investigation. Here we show that A. fumigatus pulmonary challenge induces expression of the inflammasome-dependent cytokines IL-1β and IL-18 within the first 12 hours, while IL-1α expression continually increases over at least the first 48 hours. Strikingly, Il1r1-deficient mice are highly susceptible to pulmonary A. fumigatus challenge exemplified by robust fungal proliferation in the lung parenchyma. Enhanced susceptibility of Il1r1-deficient mice correlated with defects in leukocyte recruitment and anti-fungal activity. Importantly, IL-1α rather than IL-1β was crucial for optimal leukocyte recruitment. IL-1α signaling enhanced the production of CXCL1. Moreover, CCR2+ monocytes are required for optimal early IL-1α and CXCL1 expression in the lungs, as selective depletion of these cells resulted in their diminished expression, which in turn regulated the early accumulation of neutrophils in the lung after A. fumigatus challenge. Enhancement of pulmonary neutrophil recruitment and anti-fungal activity by CXCL1 treatment could limit fungal growth in the absence of IL-1α signaling. In contrast to the role of IL-1α in neutrophil recruitment, the inflammasome and IL-1β were only essential for optimal activation of anti-fungal activity of macrophages. As such, Pycard-deficient mice are mildly susceptible to A. fumigatus infection. Taken together, our data reveal central, non-redundant roles for IL-1α and IL-1β in controlling A. fumigatus infection in the murine lung.
Author Summary
Aspergillus spp. are ubiquitous in the environment, and even though individuals are regularly exposed to fungal spores clinical invasive disease is a rare manifestation. In contrast, individuals with weakened immune systems develop severe disease, such as invasive pulmonary aspergillosis (IPA). IPA is associated with extremely poor prognoses and unacceptably high mortality rates. Knowledge gained from understanding how immunocompetent mammals control Aspergillus challenge will help develop new immunomodulatory strategies aimed at improving patient outcomes. It is well known that neutrophils and monocytes are crucial immune cells that act to limit fungal growth. Our work demonstrates a central role for the cytokine IL-1α in orchestrating the optimal recruitment of neutrophils and monocytes, whereas IL-1β and the inflammasome are more important in activation of anti-fungal activity of the monocytes. Moreover, our studies indicate that CCR2+ monocytes are required for optimal production of IL-1α in the lungs of A. fumigatus challenged mice. Thus, our data highlight a crucial role of the IL-1 cytokine in mediating anti-fungal immunity which might be harnessed to treat clinical cases of IPA.
doi:10.1371/journal.ppat.1004625
PMCID: PMC4309569  PMID: 25629406
23.  Inducible expression of beta defensins by human respiratory epithelial cells exposed to Aspergillus fumigatus organisms 
BMC Microbiology  2009;9:33.
Background
Aspergillus fumigatus, a saprophytic mould, is responsible for life-threatening, invasive pulmonary diseases in immunocompromised hosts. The role of the airway epithelium involves a complex interaction with the inhaled pathogen. Antimicrobial peptides with direct antifungal and chemotactic activities may boost antifungal immune response.
Results
The inducible expression of defensins by human bronchial epithelial 16HBE cells and A549 pneumocyte cells exposed to A. fumigatus was investigated. Using RT-PCR and real time PCR, we showed an activation of hBD2 and hBD9 defensin genes: the expression was higher in cells exposed to swollen conidia (SC), compared to resting conidia (RC) or hyphal fragments (HF). The kinetics of defensin expression was different for each one, evoking a putative distinct function for each investigated defensin. The decrease of defensin expression in the presence of heat-inactivated serum indicated a possible link between defensins and the proteins of the host complement system. The presence of defensin peptide hBD2 was revealed using immunofluorescence that showed a punctual cytoplasmic and perinuclear staining. Quantification of the cells stained with anti hBD2 antibody demonstrated that SC induced a greater number of cells that synthesized hBD2, compared to RC or HF. Labelling of the cells with anti-hBD-2 antibody showed a positive immunofluorescence signal around RC or SC in contrast to HF. This suggests co-localisation of hBD2 and digested conidia. The HBD2 level was highest in the supernatants of cells exposed to SC, as was determined by sandwich ELISA. Experiments using neutralising anti-interleukine-1β antibody reflect the autocrine mechanism of defensin expression induced by SC. Investigation of defensin expression at transcriptional and post-transcriptional levels demonstrated the requirement of transcription as well as new protein synthesis during A. fumigatus defensin induction. Finally, induced defensin expression in primary culture of human respiratory cells exposed to A. fumigatus points to the biological significance of described phenomena.
Conclusion
Our findings provide evidence that respiratory epithelium might play an important role in the immune response during Aspergillus infection. Understanding the mechanisms of regulation of defensin expression may thus lead to new approaches that could enhance expression of antimicrobial peptides for potential therapeutic use during aspergillosis treatment.
doi:10.1186/1471-2180-9-33
PMCID: PMC2653505  PMID: 19208266
24.  The Plasma Membrane Proton Pump PMA-1 Is Incorporated into Distal Parts of the Hyphae Independently of the Spitzenkörper in Neurospora crassa 
Eukaryotic Cell  2013;12(8):1097-1105.
Most models for fungal growth have proposed a directional traffic of secretory vesicles to the hyphal apex, where they temporarily aggregate at the Spitzenkörper before they fuse with the plasma membrane (PM). The PM H+-translocating ATPase (PMA-1) is delivered via the classical secretory pathway (endoplasmic reticulum [ER] to Golgi) to the cell surface, where it pumps H+ out of the cell, generating a large electrochemical gradient that supplies energy to H+-coupled nutrient uptake systems. To characterize the traffic and delivery of PMA-1 during hyphal elongation, we have analyzed by laser scanning confocal microscopy (LSCM) strains of Neurospora crassa expressing green fluorescent protein (GFP)-tagged versions of the protein. In conidia, PMA-1-GFP was evenly distributed at the PM. During germination and germ tube elongation, PMA-1-GFP was found all around the conidial PM and extended to the germ tube PM, but fluorescence was less intense or almost absent at the tip. Together, the data indicate that the electrochemical gradient driving apical nutrient uptake is generated from early developmental stages. In mature hyphae, PMA-1-GFP localized at the PM at distal regions (>120 μm) and in completely developed septa, but not at the tip, indicative of a distinct secretory route independent of the Spitzenkörper occurring behind the apex.
doi:10.1128/EC.00328-12
PMCID: PMC3754540  PMID: 23729384
25.  The pH-Responsive PacC Transcription Factor of Aspergillus fumigatus Governs Epithelial Entry and Tissue Invasion during Pulmonary Aspergillosis 
PLoS Pathogens  2014;10(10):e1004413.
Destruction of the pulmonary epithelium is a major feature of lung diseases caused by the mould pathogen Aspergillus fumigatus. Although it is widely postulated that tissue invasion is governed by fungal proteases, A. fumigatus mutants lacking individual or multiple enzymes remain fully invasive, suggesting a concomitant requirement for other pathogenic activities during host invasion. In this study we discovered, and exploited, a novel, tissue non-invasive, phenotype in A. fumigatus mutants lacking the pH-responsive transcription factor PacC. Our study revealed a novel mode of epithelial entry, occurring in a cell wall-dependent manner prior to protease production, and via the Dectin-1 β-glucan receptor. ΔpacC mutants are defective in both contact-mediated epithelial entry and protease expression, and significantly attenuated for pathogenicity in leukopenic mice. We combined murine infection modelling, in vivo transcriptomics, and in vitro infections of human alveolar epithelia, to delineate two major, and sequentially acting, PacC-dependent processes impacting epithelial integrity in vitro and tissue invasion in the whole animal. We demonstrate that A. fumigatus spores and germlings are internalised by epithelial cells in a contact-, actin-, cell wall- and Dectin-1 dependent manner and ΔpacC mutants, which aberrantly remodel the cell wall during germinative growth, are unable to gain entry into epithelial cells, both in vitro and in vivo. We further show that PacC acts as a global transcriptional regulator of secreted molecules during growth in the leukopenic mammalian lung, and profile the full cohort of secreted gene products expressed during invasive infection. Our study reveals a combinatorial mode of tissue entry dependent upon sequential, and mechanistically distinct, perturbations of the pulmonary epithelium and demonstrates, for the first time a protective role for Dectin-1 blockade in epithelial defences. Infecting ΔpacC mutants are hypersensitive to cell wall-active antifungal agents highlighting the value of PacC signalling as a target for antifungal therapy.
Author Summary
Inhaled spores of the pathogenic mould Aspergillus fumigatus cause fungal lung infections in humans having immune defects. A. fumigatus spores germinate within the immunocompromised lung, producing invasively growing, elongated cells called hyphae. Hyphae degrade the surrounding pulmonary tissue, a process thought to be caused by secreted fungal enzymes; however, A. fumigatus mutants lacking one or more protease activities retain fully invasive phenotypes in mouse models of disease. Here we report the first discovery of a non-invasive A. fumigatus mutant, which lacks a pH-responsive transcription factor PacC. Using global transcriptional profiling of wild type and mutant isolates, and in vitro pulmonary invasion assays, we established that loss of PacC leads to a compound non-invasive phenotype characterised by deficits in both contact-mediated epithelial entry and protease expression. Consistent with an important role for epithelial entry in promoting invasive disease in mammalian tissues, PacC mutants remain surface-localised on mammalian epithelia, both in vitro and in vivo. Our study sets a new precedent for involvement of both host and pathogen activities in promoting epithelial invasion by A. fumigatus and supports a model wherein fungal protease activity acting subsequently to, or in parallel with, host-mediated epithelial entry provides the mechanistic basis for tissue invasion.
doi:10.1371/journal.ppat.1004413
PMCID: PMC4199764  PMID: 25329394

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