Opportunistic Pathogen Aspergillus Fumigatus Research Articles

The Transcriptome Response to Azole Compounds in Aspergillus fumigatus Shows Differential Gene Expression across Pathways Essential for Azole Resistance and Cell Survival.

The opportunistic pathogen Aspergillus fumigatus is found on all continents and thrives in soil and agricultural environments. Its ability to readily adapt to novel environments and to produce billions of spores led to the spread of azole-resistant A. fumigatus across the globe, posing a threat to many immunocompromised patients, including critically ill patients with severe influenza or COVID-19. In our study, we sought to compare the adaptational response to azoles from A. fumigatus isolates that differ in azole susceptibility and genetic background. To gain more insight into how short-term adaptation to stressful azole compounds is managed through gene expression, we conducted an RNA-sequencing study on the response of A. fumigatus to itraconazole and the newest clinically approved azole, isavuconazole. We observed many similarities in ergosterol biosynthesis up-regulation across isolates, with the exception of the pan-azole-resistant isolate, which showed very little differential regulation in comparison to other isolates. Additionally, we found differential regulation of membrane efflux transporters, secondary metabolites, iron metabolism, and various stress response and cell signaling mechanisms.

Evaluation of liposomal hydrocolloidal NPs loaded by tea tree oil as antifungal agent in vitro and in vivo investigations: Preclinical studies

Fungi have a great ability and a wide variety of mechanisms to endure the toxicity of current antifungal agents. Researchers are working to find new therapeutic agents to combat the resistance ability of fungi. Almost all commercial antifungal agents have a wide variety of side effects on human health. This study aims to introduce tea tree oil nanoparticles as antifungal delivery termed TNSAD, which contains hybrid tea tree oil attached chitosan in a liposomal formulation, and assess its antifungal activity using in vitro and in vivo infection models. Tea tree oil was first coated by chitosan, then inserted inside liposomal bilayers, and finally functionalized by a layer of chitosan, forming the TNSAD. The antifungal activity was evaluated against four different invasive, opportunistic, and zoonotic fungal pathogens (Aspergillus flavus, Aspergillus fumigatus, Microsporum gypsum, and Fusarium oxysporum). The cytotoxicity of TNSAD was then tested against the HEp-2 cell line. Finally, the antifungal activity against Aspergillus fumigatus and Microsporum gypsum was assessed in vivo in a rat model. The in vitro results confirm the potency of TNSAD against fungi and its safety at a concentration of ≤ 5 mg/ml. In vivo results revealed that fungal cells were destroyed within the tissue when used systematically. we have described here a natural remarkable design that represents a potential antifungal agent and provided evidence for its efficiency and safety, which makes it a promising antifungal agent for the treatment of systemic and topical fungal infections.

Requirement of a putative mitochondrial GTPase, GemA, for azole susceptibility, virulence, and cell wall integrity in Aspergillus fumigatus.

Drug resistance in fungal pathogens is a new challenge in clinical aspergillosis treatment. Mitochondria as dynamic organelles are involved in numerous biological processes in fungi, including drug resistance. However, little is known about the mechanism underlying mitochondrial regulation of the response of fungal pathogens to antifungal drugs. Here, we showed that a putative mitochondrial GTPase, GemA, a yeast Gem1 homolog, is crucial for the azole response and cell wall integrity in the opportunistic pathogen Aspergillus fumigatus. The fluorescence observation showed that GFP-labeled GemA is located in mitochondria, and loss of gemA results in aberrant giant mitochondrial morphology and abnormal mitochondrial membrane potential. Moreover, a ΔgemA mutant attenuates fungal virulence in the Galleria mellonella model of aspergillosis. Furthermore, gemA loss increases resistance to azoles and terbinafine but not to amphotericin B. Of note, RNA-seq combined with RT-qPCR showed that a series of drug efflux pumps were upregulated in the gemA deletion mutant. Deleting mdr1 or inhibiting the expression of drug efflux pumps can partially decrease the resistance to azoles resulting from the gemA mutant, implying that GemA influences azole response by affecting the expression of drug efflux pumps. Importantly, the ΔgemA mutant is susceptible to the cell wall-perturbing reagent CR, but not to CFW, and this defect can be partly rescued by hyperosmotic stress. TEM revealed that the cell wall of ΔgemA was thicker than that of the WT strain, demonstrating that GemA plays a role in cell wall composition and integrity. Collectively, we identified a putative mitochondrial GTPase, GemA, which is critical for hyphal growth, virulence, azole susceptibility, and cell wall integrity and acts by affecting mitochondrial function.

Fusarium fruiting body microbiome member Pantoea agglomerans inhibits fungal pathogenesis by targeting lipid rafts

Plant-pathogenic fungi form intimate interactions with their associated bacterial microbiota during their entire life cycle. However, little is known about the structure, functions and interaction mechanisms of bacterial communities associated with fungal fruiting bodies (perithecia). Here we examined the bacterial microbiome of perithecia formed by Fusarium graminearum, the major pathogenic fungus causing Fusarium head blight in cereals. A total of 111 shared bacterial taxa were identified in the microbiome of 65 perithecium samples collected from 13 geographic locations. Within a representative culture collection, 113 isolates exhibited antagonistic activity against F. graminearum, with Pantoea agglomerans ZJU23 being the most efficient in reducing fungal growth and infectivity. Herbicolin A was identified as the key antifungal compound secreted by ZJU23. Genetic and chemical approaches led to the discovery of its biosynthetic gene cluster. Herbicolin A showed potent in vitro and in planta efficacy towards various fungal pathogens and fungicide-resistant isolates, and exerted a fungus-specific mode of action by directly binding and disrupting ergosterol-containing lipid rafts. Furthermore, herbicolin A exhibited substantially higher activity (between 5- and 141-fold higher) against the human opportunistic fungal pathogens Aspergillus fumigatus and Candida albicans in comparison with the clinically used fungicides amphotericin B and fluconazole. Its mode of action, which is distinct from that of other antifungal drugs, and its efficacy make herbicolin A a promising antifungal drug to combat devastating fungal pathogens, both in agricultural and clinical settings.

Growth of Aspergillus fumigatus in Biofilms in Comparison to Candida albicans.

Biofilm formation during infections with the opportunistic pathogen Aspergillus fumigatus can be very problematic in clinical settings, since it provides the fungal cells with a protective environment. Resistance against drug treatments, immune recognition as well as adaptation to the host environment allows fungal survival in the host. The exact molecular mechanisms behind most processes in the formation of biofilms are unclear. In general, the formation of biofilms can be categorized roughly in a few stages; adhesion, conidial germination and development of hyphae, biofilm maturation and cell dispersion. Fungi in biofilms can adapt to the in-host environment. These adaptations can occur on a level of phenotypic plasticity via gene regulation. However, also more substantial genetic changes of the genome can result in increased resistance and adaptation in the host, enhancing the survival chances of fungi in biofilms. Most research has focused on the development of biofilms. However, to tackle developing microbial resistance and adaptation in biofilms, more insight in mechanisms behind genetic adaptations is required to predict which defense mechanisms can be expected. This can be helpful in the development of novel and more targeted antifungal treatments to combat fungal infections.

Emerging Fungal Infections: New Species, New Names, and Antifungal Resistance.

BackgroundInfections caused by fungi can be important causes of morbidity and mortality in certain patient populations, including those who are highly immunocompromised or critically ill. Invasive mycoses can be caused by well-known species, as well as emerging pathogens, including those that are resistant to clinically available antifungals.ContentThis review highlights emerging fungal infections, including newly described species, such as Candida auris, and those that having undergone taxonomic classification and were previously known by other names, including Blastomyces and Emergomyces species, members of the Rasamsonia argillacea species complex, Sporothrix brasiliensis, and Trichophyton indotinae. Antifungal resistance also is highlighted in several of these emerging species, as well as in the well-known opportunistic pathogen Aspergillus fumigatus. Finally, the increased recognition and importance of fungal co-infections with respiratory pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is discussed.SummaryBoth clinicians and clinical microbiology laboratories should remain vigilant regarding emerging fungal infections. These may be difficult both to diagnose and treat due to the lack of experience of clinicians and laboratory personnel with these organisms and the infections they may cause. Many of these fungal infections have been associated with poor clinical outcomes, either due to inappropriate therapy or the development of antifungal resistance.

Antifungal Siderophore Conjugates for Theranostic Applications in Invasive Pulmonary Aspergillosis Using Low-Molecular TAFC Scaffolds.

Invasive pulmonary aspergillosis (IPA) is a life-threatening form of fungal infection, primarily in immunocompromised patients and associated with significant mortality. Diagnostic procedures are often invasive and/or time consuming and existing antifungals can be constrained by dose-limiting toxicity and drug interaction. In this study, we modified triacetylfusarinine C (TAFC), the main siderophore produced by the opportunistic pathogen Aspergillus fumigatus (A. fumigatus), with antifungal molecules to perform antifungal susceptibility tests and molecular imaging. A variation of small organic molecules (eflornithine, fludioxonil, thiomersal, fluoroorotic acid (FOA), cyanine 5 (Cy5) with antifungal activity were coupled to diacetylfusarinine C (DAFC), resulting in a “Trojan horse” to deliver antifungal compounds specifically into A. fumigatus hyphae by the major facilitator transporter MirB. Radioactive labeling with gallium-68 allowed us to perform in vitro characterization (distribution coefficient, stability, uptake assay) as well as biodistribution experiments and PET/CT imaging in an IPA rat infection model. Compounds chelated with stable gallium were used for antifungal susceptibility tests. [Ga]DAFC-fludioxonil, -FOA, and -Cy5 revealed a MirB-dependent active uptake with fungal growth inhibition at 16 µg/mL after 24 h. Visualization of an A. fumigatus infection in lungs of a rat was possible with gallium-68-labeled compounds using PET/CT. Heterogeneous biodistribution patterns revealed the immense influence of the antifungal moiety conjugated to DAFC. Overall, novel antifungal siderophore conjugates with promising fungal growth inhibition and the possibility to perform PET imaging combine both therapeutic and diagnostic potential in a theranostic compound for IPA caused by A. fumigatus.

Antifungal Liposomes Directed by Dectin-2 Offer a Promising Therapeutic Option for Pulmonary Aspergillosis.

ABSTRACTInvasive fungal diseases cause millions of deaths each year. There are currently approximately 300,000 acute cases of aspergillosis, most of which result from a pulmonary infection of immunocompromised patients by the common soil organism and opportunistic pathogen Aspergillus fumigatus. Patients are treated with antifungal drugs, such as amphotericin B (AmB). However, AmB has serious limitations due to human organ toxicity. AmB is slightly less toxic if loaded in liposomes, such as AmBisome or AmB-loaded liposomes (AmB-LLs). Even with antifungal therapy, recurrent infections are common, and 1-year fatality rates may exceed 50%. We have previously shown that coating AmB-LLs with the extracellular oligomannan-binding domain of the C-type lectin receptor Dectin-2 (DEC2-AmB-LLs) effectively targets DEC2-AmB-LLs to cell walls, exopolysaccharide matrices, and biofilms of fungal pathogens in vitro. In vitro, DEC2-AmB-LLs reduce the effective dose of AmB for 95% inhibition and killing of A. fumigatus 10-fold compared to that of untargeted AmB-LLs. Herein we tested the antifungal activity of DEC2-AmB-LLs relative to that of untargeted AmB-LLs in immunosuppressed mice with pulmonary aspergillosis. Remarkably, DEC2-AmB-LLs bound 30-fold more efficiently to A. fumigatus at sites of infection in the lungs. Furthermore, Dectin-2-targeted liposomes delivering AmB at a dose of 0.2 mg/kg of body weight significantly reduced the fungal burden in lungs compared to results with untargeted AmB-LLs at 0.2 mg/kg and micellar voriconazole at 20 mg/kg and prolonged mouse survival. By dramatically increasing the efficacy of antifungal drugs at low doses, targeted liposomes have the potential to create a new clinical paradigm to treat diverse fungal diseases.

The fungal-specific histone acetyltransferase Rtt109 regulates development, DNA damage response, and virulence in Aspergillus fumigatus.

In eukaryotes, histone acetylation catalyzed by histone acetyltransferase (HAT) has been demonstrated to be critical for various physiological processes. However, the biological functions of HAT and the underlying mechanism by which HAT-regulated processes are involved in fungal development and virulence in the human opportunistic pathogen Aspergillus fumigatus remain largely unexplored. Here, we functionally characterized the roles of Rtt109 in A. fumigatus, an ortholog of Saccharomyces cerevisiae histone acetyltransferase Rtt109. In vivo and in vitro HAT assays revealed that AfRtt109 functions as a canonical histone acetyltransferase, acetylating lysines 9 and 56 of histone H3. Deletion of Afrtt109 leads to severe defects in vegetative growth, conidiation, and causes reduced virulence in the Galleria mellonella model, as well as hypersensitivity to genotoxic agents. Moreover, site-directed mutagenesis revealed that the conserved arginine residues R265 and R306 of Rtt109 are required for the H3K9 and H3K56 acetylation and virulence of A. fumigatus. Unexpectedly, R265E and R306E mutants did not exhibit any detectable phenotypic defects, implying that A. fumigatus Rtt109 regulates fungal development via histone acetylation-independent mechanisms. Together, our results revealed the critical role of fungal-specific HAT Rtt109 in regulating fungal development and virulence, and suggested that it may serve as a unique target for antifungal therapies.

Proinflammatory effects of environmental cadmium boost resistance to opportunistic pathogen Aspergillus fumigatus: Implications for sustained low-level pulmonary inflammation?

Cadmium (Cd) is one of the most toxic environmental heavy metals to which the general population is exposed mainly via the oral route. Owing to its immunomodulatory potential, orally acquired Cd affects antimicrobial immune defense in several organs, including the lungs. While there are data concerning Cd and viral and bacterial pulmonary infections, effects on fungal infections are not studied yet. In the present study, the effect of the Cd (5 mg/L for 30 days, in drinking water, the average daily Cd intake 0.641 ± 0.089 mg/kg) on the immune response of rats to pulmonary A. fumigatus infection was examined. Data obtained showed that orally acquired cadmium does not affect the elimination of the fungus in immunocompetent rats owing to the preservation of some aspects of innate immune responses (lung leukocyte infiltration and NBT reduction) and an increase in other (increased numbers of mucus-producing goblet cells, MPO release). Cd does not affect an IFN-γ response in lung leukocytes during the infection (despite suppression of cytokine production in cells of lung-draining lymph nodes), while it stimulates IL-17 and suppresses IL-10 response to the fungus. As a result, the elimination of the fungus occurs in a milieu with the prevailing proinflammatory response in Cd-exposed animals that preserved fungal elimination from the lungs, though with more intense injury to the lung tissue. Therefore, the proinflammatory microenvironment in the lungs created by Cd that sustains inflammatory/immune response to the fungus to which humans are exposed for a lifetime, raises a concern of orally acquired Cd as a risk factor for the development of chronic low-grade pulmonary inflammation.

Global Sexual Fertility in the Opportunistic Pathogen Aspergillus fumigatus and Identification of New Supermater Strains.

A sexual cycle in Aspergillus fumigatus was first described in 2009 with isolates from Dublin, Ireland. However, the extent to which worldwide isolates can undergo sexual reproduction has remained unclear. In this study a global collection of 131 isolates was established with a near 1:1 ratio of mating types. All isolates were crossed to MAT1-1 or MAT1-2 Irish strains, and a subset of isolates from different continents were crossed together. Ninety seven percent of isolates were found to produce cleistothecia with at least one mating partner, showing that sexual fertility is not limited to the Irish population but is a characteristic of global A. fumigatus. However, large variation was seen in numbers of cleistothecia produced per cross, suggesting differences in the possibility for genetic exchange between strains in nature. The majority of crosses produced ascospores with >50% germination rates, but with wide variation evident. A high temperature heat shock was required to induce ascospore germination. Finally, a new set of highly fertile MAT1-1 and MAT1-2 supermater strains were identified and pyrimidine auxotrophs generated for community use. Results provide insights into the potential for the A. fumigatus sexual cycle to generate genetic variation and allow gene flow of medically important traits.

Uncovering New Mutations Conferring Azole Resistance in the Aspergillus fumigatus cyp51A Gene.

The opportunistic pathogen Aspergillus fumigatus has developed worldwide resistance to azoles largely through mutations in cytochromeP450 enzyme Cyp51. In this study, we indicated that in vitro azole situation results in emergence of azole-resistant mutations. There are previously identified azole-resistant cyp51A mutations (M220K, M220I, M220R, G54E and G54W mutations) and we successfully identified in this study two new mutations (N248K/V436A, Y433N substitution) conferring azole resistance among 18 independent stable azole-resistant isolates. The Galleria mellonella model of A. fumigatus infection experiment verified that Cyp51A mutations N248K/V436A and Y433N reduce efficacy of azole therapy. In addition, a predicted Cyp51A 3D structural model suggested that Y433N mutation causes the reduced affinities between drug target Cyp51A and azole antifungals. This study suggests that drug selection pressure make it possible to isolate unidentified cyp51A mutations conferring azole resistance in A. fumigatus.

RgsA Attenuates the PKA Signaling, Stress Response, and Virulence in the Human Opportunistic Pathogen Aspergillus fumigatus.

The regulator of G-protein signaling (RGS) proteins play an important role in upstream control of heterotrimeric G-protein signaling pathways. In the genome of the human opportunistic pathogenic fungus Aspergillus fumigatus, six RGS protein-encoding genes are present. To characterize the rgsA gene predicted to encode a protein with an RGS domain, we generated an rgsA null mutant and observed the phenotypes of the mutant. The deletion (Δ) of rgsA resulted in increased radial growth and enhanced asexual sporulation in both solid and liquid culture conditions. Accordingly, transcripts levels of the key asexual developmental regulators abaA, brlA, and wetA are elevated in the ΔrgsA mutant. Moreover, ΔrgsA resulted in elevated spore germination rates in the absence of a carbon source. The activity of cAMP-dependent protein kinase A (PKA) and mRNA levels of genes encoding PKA signaling elements are elevated by ΔrgsA. In addition, mRNA levels of genes associated with stress-response signaling increased with the lack of rgsA, and the ΔrgsA spores showed enhanced tolerance against oxidative stressors. Comparative transcriptomic analyses revealed that the ΔrgsA mutant showed higher mRNA levels of gliotoxin (GT) biosynthetic genes. Accordingly, the rgsA null mutant exhibited increased production of GT and elevated virulence in the mouse. Conversely, the majority of genes encoding glucan degrading enzymes were down-regulated by ΔrgsA, and endoglucanase activities were reduced. In summary, RgsA plays multiple roles, governing growth, development, stress responses, virulence, and external polymer degradation—likely by attenuating PKA signaling.

Elevated Prevalence of Azole-Resistant Aspergillus fumigatus in Urban versus Rural Environments in the United Kingdom.

Azole resistance in the opportunistic pathogen Aspergillus fumigatus is increasing, dominated primarily by the following two environmentally associated resistance alleles: TR34/L98H and TR46/Y121F/T289A. By sampling soils across the South of England, we assess the prevalence of azole-resistant A. fumigatus (ARAf) in samples collected in both urban and rural locations. We characterize the susceptibility profiles of the resistant isolates to three medical azoles, identify the underlying genetic basis of resistance, and investigate their genetic relationships. ARAf was detected in 6.7% of the soil samples, with a higher prevalence in urban (13.8%) than rural (1.1%) locations. Twenty isolates were confirmed to exhibit clinical breakpoints for resistance to at least one of three medical azoles, with 18 isolates exhibiting resistance to itraconazole, 6 to voriconazole, and 2 showing elevated minimum inhibitory concentrations to posaconazole. Thirteen of the resistant isolates harbored the TR34/L98H resistance allele, and six isolates carried the TR46/Y121F/T289A allele. The 20 azole-resistant isolates were spread across five csp1 genetic subtypes, t01, t02, t04B, t09, and t18 with t02 being the predominant subtype. Our study demonstrates that ARAf can be easily isolated in the South of England, especially in urban city centers, which appear to play an important role in the epidemiology of environmentally linked drug-resistant A. fumigatus.

Pangloss: A Tool for Pan-Genome Analysis of Microbial Eukaryotes.

Although the pan-genome concept originated in prokaryote genomics, an increasing number of eukaryote species pan-genomes have also been analysed. However, there is a relative lack of software intended for eukaryote pan-genome analysis compared to that available for prokaryotes. In a previous study, we analysed the pan-genomes of four model fungi with a computational pipeline that constructed pan-genomes using the synteny-dependent Pan-genome Ortholog Clustering Tool (PanOCT) approach. Here, we present a modified and improved version of that pipeline which we have called Pangloss. Pangloss can perform gene prediction for a set of genomes from a given species that the user provides, constructs and optionally refines a species pan-genome from that set using PanOCT, and can perform various functional characterisation and visualisation analyses of species pan-genome data. To demonstrate Pangloss’s capabilities, we constructed and analysed a species pan-genome for the oleaginous yeast Yarrowia lipolytica and also reconstructed a previously-published species pan-genome for the opportunistic respiratory pathogen Aspergillus fumigatus. Pangloss is implemented in Python, Perl and R and is freely available under an open source GPLv3 licence via GitHub.

Chemical shifts assignments of the RodA hydrophobin from the opportunistic pathogen Aspergillus fumigatus

Chemical shifts assignments of the RodA hydrophobin from the opportunistic pathogen Aspergillus fumigatus

Mitochondrial Fragmentation in Aspergillus fumigatus as Early Marker of Granulocyte Killing Activity.

The host's defense against invasive mold infections relies on diverse antimicrobial activities of innate immune cells. However, studying these mechanisms in vitro is complicated by the filamentous nature of such pathogens that typically form long, branched, multinucleated and compartmentalized hyphae. Here we describe a novel method that allows for the visualization and quantification of the antifungal killing activity exerted by human granulocytes against hyphae of the opportunistic pathogen Aspergillus fumigatus. The approach relies on the distinct impact of fungal cell death on the morphology of mitochondria that were visualized with green fluorescent protein (GFP). We show that oxidative stress induces complete fragmentation of the tubular mitochondrial network which correlates with cell death of affected hyphae. Live cell microscopy revealed a similar and non-reversible disruption of the mitochondrial morphology followed by fading of fluorescence in Aspergillus hyphae that were killed by human granulocytes. Quantitative microscopic analysis of fixed samples was subsequently used to estimate the antifungal activity. By utilizing this assay, we demonstrate that lipopolysaccharides as well as human serum significantly increase the killing efficacy of the granulocytes. Our results demonstrate that evaluation of the mitochondrial morphology can be utilized to assess the fungicidal activity of granulocytes against A. fumigatus hyphae.

Comparative systems analysis of the secretome of the opportunistic pathogen Aspergillus fumigatus and other Aspergillus species

Aspergillus fumigatus and multiple other Aspergillus species cause a wide range of lung infections, collectively termed aspergillosis. Aspergilli are ubiquitous in environment with healthy immune systems routinely eliminating inhaled conidia, however, Aspergilli can become an opportunistic pathogen in immune-compromised patients. The aspergillosis mortality rate and emergence of drug-resistance reveals an urgent need to identify novel targets. Secreted and cell membrane proteins play a critical role in fungal-host interactions and pathogenesis. Using a computational pipeline integrating data from high-throughput experiments and bioinformatic predictions, we have identified secreted and cell membrane proteins in ten Aspergillus species known to cause aspergillosis. Small secreted and effector-like proteins similar to agents of fungal-plant pathogenesis were also identified within each secretome. A comparison with humans revealed that at least 70% of Aspergillus secretomes have no sequence similarity with the human proteome. An analysis of antigenic qualities of Aspergillus proteins revealed that the secretome is significantly more antigenic than cell membrane proteins or the complete proteome. Finally, overlaying an expression dataset, four A. fumigatus proteins upregulated during infection and with available structures, were found to be structurally similar to known drug target proteins in other organisms, and were able to dock in silico with the respective drug.

Microbial glycoside hydrolases as antibiofilm agents with cross-kingdom activity

Galactosaminogalactan and Pel are cationic heteropolysaccharides produced by the opportunistic pathogens Aspergillus fumigatus and Pseudomonas aeruginosa, respectively. These exopolysaccharides both contain 1,4-linked N-acetyl-d-galactosamine and play an important role in biofilm formation by these organisms. Proteins containing glycoside hydrolase domains have recently been identified within the biosynthetic pathway of each exopolysaccharide. Recombinant hydrolase domains from these proteins (Sph3h from A. fumigatus and PelAh from P. aeruginosa) were found to degrade their respective polysaccharides in vitro. We therefore hypothesized that these glycoside hydrolases could exhibit antibiofilm activity and, further, given the chemical similarity between galactosaminogalactan and Pel, that they might display cross-species activity. Treatment of A. fumigatus with Sph3h disrupted A. fumigatus biofilms with an EC50 of 0.4 nM. PelAh treatment also disrupted preformed A. fumigatus biofilms with EC50 values similar to those obtained for Sph3h In contrast, Sph3h was unable to disrupt P. aeruginosa Pel-based biofilms, despite being able to bind to the exopolysaccharide. Treatment of A. fumigatus hyphae with either Sph3h or PelAh significantly enhanced the activity of the antifungals posaconazole, amphotericin B, and caspofungin, likely through increasing antifungal penetration of hyphae. Both enzymes were noncytotoxic and protected A549 pulmonary epithelial cells from A. fumigatus-induced cell damage for up to 24 h. Intratracheal administration of Sph3h was well tolerated and reduced pulmonary fungal burden in a neutropenic mouse model of invasive aspergillosis. These findings suggest that glycoside hydrolases can exhibit activity against diverse microorganisms and may be useful as therapeutic agents by degrading biofilms and attenuating virulence.

202 - Biochemical and Structural Characterization of 1-Cys Peroxiredoxin from the Human Opportunistic Pathogen Aspergillus Fumigatus

Peroxiredoxin (Prx) are Cys-based, thiol-dependent peroxidases that contain one or two conserved Cys residues. To date, all characterized Prx enzymes display the universal motif PxxxT/SxxC. Here, we aim to structurally and biochemically characterize AfPrxA, a 1-Cys Prx (Prx6 subfamily) from the human opportunistic pathogen Aspergillus fumigatus . Remarkably, AfPrxA presents a unique motif among Prx enzymes (SxxxHxxC) and still displays an extraordinary reactivity towards H 2 O 2 . Indeed, the reactivity of AfPrxA towards H 2 O 2 was 3.7×10 7 M -1 s -1 as determined through a HRP competitive assay. Therefore, we are now attempting to obtain the crystal structure of AfPrxA. To date, the best diffraction pattern was obtained with AfPrxA pre-reduced, alkylated with iodoacetamide and treated with trypsin (resolution 3A); the refinement is in progress. Additionally, we are investigating the reducing system of AfPrxA, which varies for Prx6 type of enzymes in distinct organisms. Therefore, we produced recombinant enzymes of the thioredoxin (Trx) system from Aspergillus fumigatus and none of them could reduce AfPrxA. In contrast, ascorbate and dihydrolipoic acid supported the peroxidase activity of AfPrxA. Once ascorbate could reduce this protein in vitro (rate constant in the 10 3 M -1 s -1 range), we quantified the intra-cellular erytroascorbate concentration as approximately 200 µM. Therefore, erytroascorbate can act as a reducing agent of AfPrxA in vivo. To investigate the possible involvement of AfPrxA in the virulence of A. fumigatus , we constructed a knockout strain of A. fumigatus (ΔAfPrxA), and a strain expressing an AfPrxA allele without the catalytic cysteine (AfPrxAC28S). Currently, phagocytosis, CFU and TNF-α assays are under evaluation in macrophages (J774) cultures exposed to A. fumigatus strains. Acknowledgements: CEPID Redoxoma; FAPESP; Capes-CNPq.