Mouse Model Of Invasive Aspergillosis Research Articles

Sphingosine as a New Antifungal Agent against Candida and Aspergillus spp.

This study investigated whether sphingosine is effective as prophylaxis against Aspergillus spp. and Candida spp. In vitro experiments showed that sphingosine is very efficacious against A. fumigatus and Nakeomyces glabrataa (formerly named C. glabrata). A mouse model of invasive aspergillosis showed that sphingosine exerts a prophylactic effect and that sphingosine-treated animals exhibit a strong survival advantage after infection. Furthermore, mechanistic studies showed that treatment with sphingosine leads to the early depolarization of the mitochondrial membrane potential (Δψm) and the generation of mitochondrial reactive oxygen species and to a release of cytochrome C within minutes, thereby presumably initiating apoptosis. Because of its very good tolerability and ease of application, inhaled sphingosine should be further developed as a possible prophylactic agent against pulmonary aspergillosis among severely immunocompromised patients.

Overexpression of the Aspergillus fumigatus Small GTPase, RsrA, Promotes Polarity Establishment during Germination.

Cell polarization comprises highly controlled processes and occurs in most eukaryotic organisms. In yeast, the processes of budding, mating and filamentation require coordinated mechanisms leading to polarized growth. Filamentous fungi, such as Aspergillus fumigatus, are an extreme example of cell polarization, essential for both vegetative and pathogenic growth. A major regulator of polarized growth in yeast is the small GTPase Rsr1, which is essential for bud-site selection. Here, we show that deletion of the putative A. fumigatus ortholog, rsrA, causes only a modest reduction of growth rate and delay in germ tube emergence. In contrast, overexpression of rsrA results in a morphogenesis defect, characterized by a significant delay in polarity establishment followed by the establishment of multiple growth axes. This aberrant phenotype is reversed when rsrA expression levels are decreased, suggesting that correct regulation of RsrA activity is crucial for accurate patterning of polarity establishment. Despite this finding, deletion or overexpression of rsrA resulted in no changes of A. fumigatus virulence attributes in a mouse model of invasive aspergillosis. Additional mutational analyses revealed that RsrA cooperates genetically with the small GTPase, RasA, to support A. fumigatus viability.

Structural basis for the core-mannan biosynthesis of cell wall fungal-type galactomannan in Aspergillus fumigatus

Fungal cell walls and their biosynthetic enzymes are potential targets for novel antifungal agents. Recently, two mannosyltransferases, namely core-mannan synthases A (CmsA/Ktr4) and B (CmsB/Ktr7), were found to play roles in the core-mannan biosynthesis of fungal-type galactomannan. CmsA/Ktr4 is an α-(1→2)-mannosyltransferase responsible for α-(1→2)-mannan biosynthesis in fungal-type galactomannan, which covers the cell surface of Aspergillus fumigatus Strains with disrupted cmsA/ktr4 have been shown to exhibit strongly suppressed hyphal elongation and conidiation alongside reduced virulence in a mouse model of invasive aspergillosis, indicating that CmsA/Ktr4 is a potential novel antifungal candidate. In this study we present the 3D structures of the soluble catalytic domain of CmsA/Ktr4, as determined by X-ray crystallography at a resolution of 1.95 Å, as well as the enzyme and Mn2+/GDP complex to 1.90 Å resolution. The CmsA/Ktr4 protein not only contains a highly conserved binding pocket for the donor substrate, GDP-mannose, but also has a unique broad cleft structure formed by its N- and C-terminal regions and is expected to recognize the acceptor substrate, a mannan chain. Based on these crystal structures, we also present a 3D structural model of the enzyme-substrate complex generated using docking and molecular dynamics simulations with α-Man-(1→6)-α-Man-(1→2)-α-Man-OMe as the model structure for the acceptor substrate. This predicted enzyme-substrate complex structure is also supported by findings from single amino acid substitution CmsA/Ktr4 mutants expressed in ΔcmsA/ktr4 A. fumigatus cells. Taken together, these results provide basic information for developing specific α-mannan biosynthesis inhibitors for use as pharmaceuticals and/or pesticides.

Molecular mechanism of Aspergillus fumigatus biofilm disruption by fungal and bacterial glycoside hydrolases

During infection, the fungal pathogen Aspergillus fumigatus forms biofilms that enhance its resistance to antimicrobials and host defenses. An integral component of the biofilm matrix is galactosaminogalactan (GAG), a cationic polymer of α-1,4-linked galactose and partially deacetylated N-acetylgalactosamine (GalNAc). Recent studies have shown that recombinant hydrolase domains from Sph3, an A. fumigatus glycoside hydrolase involved in GAG synthesis, and PelA, a multifunctional protein from Pseudomonas aeruginosa involved in Pel polysaccharide biosynthesis, can degrade GAG, disrupt A. fumigatus biofilms, and attenuate fungal virulence in a mouse model of invasive aspergillosis. The molecular mechanisms by which these enzymes disrupt biofilms have not been defined. We hypothesized that the hydrolase domains of Sph3 and PelA (Sph3h and PelAh, respectively) share structural and functional similarities given their ability to degrade GAG and disrupt A. fumigatus biofilms. MALDI-TOF enzymatic fingerprinting and NMR experiments revealed that both proteins are retaining endo-α-1,4-N-acetylgalactosaminidases with a minimal substrate size of seven residues. The crystal structure of PelAh was solved to 1.54 Å and structure alignment to Sph3h revealed that the enzymes share similar catalytic site residues. However, differences in the substrate-binding clefts result in distinct enzyme-substrate interactions. PelAh hydrolyzed partially deacetylated substrates better than Sph3h, a finding that agrees well with PelAh's highly electronegative binding cleft versus the neutral surface present in Sph3h Our insight into PelAh's structure and function necessitate the creation of a new glycoside hydrolase family, GH166, whose structural and mechanistic features, along with those of GH135 (Sph3), are reported here.

Contribution of ATPase copper transporters in animal but not plant virulence of the crossover pathogen Aspergillus flavus

ABSTRACT The ubiquitous fungus Aspergillus flavus is notorious for contaminating many important crops and food-stuffs with the carcinogenic mycotoxin, aflatoxin. This fungus is also the second most frequent Aspergillus pathogen after A. fumigatus infecting immunosuppressed patients. In many human fungal pathogens including A. fumigatus, the ability to defend from toxic levels of copper (Cu) is essential in pathogenesis. In A. fumigatus, the Cu-fist DNA binding protein, AceA, and the Cu ATPase transporter, CrpA, play critical roles in Cu defense. Here, we show that A. flavus tolerates higher concentrations of Cu than A. fumigatus and other Aspergillus spp. associated with the presence of two homologs of A. fumigatus CrpA termed CrpA and CrpB. Both crpA and crpB are transcriptionally induced by increasing Cu concentrations via AceA activity. Deletion of crpA or crpB alone did not alter high Cu tolerance, suggesting they are redundant. Deletion of both genes resulted in extreme Cu sensitivity that was greater than that following deletion of the regulatory transcription factor aceA. The ΔcrpAΔcrpB and ΔaceA strains were also sensitive to ROI stress. Compared to wild type, these mutants were impaired in the ability to colonize maize seed treated with Cu fungicide but showed no difference in virulence on non-treated seed. A mouse model of invasive aspergillosis showed ΔcrpAΔcrpB and to a lesser degree ΔaceA to be significantly reduced in virulence, following the greater sensitivity of ΔcrpAΔcrpB to Cu than ΔaceA.

The Aspergillus fumigatus Sialidase (Kdnase) Contributes to Cell Wall Integrity and Virulence in Amphotericin B-Treated Mice.

Aspergillus fumigatus is a filamentous fungus that can cause a life-threatening invasive pulmonary aspergillosis (IPA) in immunocompromised individuals. We previously characterized an exo-sialidase from A. fumigatus that prefers the sialic acid substrate, 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (Kdn); hence it is a Kdnase. Sialidases are known virulence factors in other pathogens; therefore, the goal of our study was to evaluate the importance of Kdnase in A. fumigatus. A kdnase knockout strain (Δkdnase) was unable to grow on medium containing Kdn and displayed reduced growth and abnormal morphology. Δkdnase was more sensitive than wild type to hyperosmotic conditions and the antifungal agent, amphotericin B. In contrast, Δkdnase had increased resistance to nikkomycin, Congo Red and Calcofluor White indicating activation of compensatory cell wall chitin deposition. Increased cell wall thickness and chitin content in Δkdnase were confirmed by electron and immunofluorescence microscopy. In a neutropenic mouse model of invasive aspergillosis, the Δkdnase strain had attenuated virulence and a significantly lower lung fungal burden but only in animals that received liposomal amphotericin B after spore exposure. Macrophage numbers were almost twofold higher in lung sections from mice that received the Δkdnase strain, possibly related to higher survival of macrophages that internalized the Δkdnase conidia. Thus, A. fumigatus Kdnase is important for fungal cell wall integrity and virulence, and because Kdnase is not present in the host, it may represent a potential target for the development of novel antifungal agents.

Host immune status-specific production of gliotoxin and bis-methyl-gliotoxin during invasive aspergillosis in mice

Delayed diagnosis in invasive aspergillosis (IA) contributes to its high mortality. Gliotoxin (GT) and bis-methyl-gliotoxin (bmGT) are secondary metabolites produced by Aspergillus during invasive, hyphal growth and may prove diagnostically useful. Because IA pathophysiology and GT’s role in virulence vary depending on the underlying host immune status, we hypothesized that GT and bmGT production in vivo may differ in three mouse models of IA that mimic human disease. We defined temporal kinetics of GT and bmGT in serum, bronchoalveolar lavage fluid (BALF) and lungs of A. fumigatus-infected chronic granulomatous disease (CGD), hydrocortisone-treated, and neutropenic mice. We harvested lungs for assessment of fungal burden, histology and GT/bmGT biosynthetic genes’ mRNA induction. GT levels were higher in neutropenic versus CGD or steroid-treated lungs. bmGT was persistently detected only in CGD lungs. GT, but not bmGT, was detected in 71% of sera and 50% of BALF of neutropenic mice; neither was detected in serum/BALF of CGD or steroid-treated mice. Enrichment of GT in Aspergillus-infected neutropenic lung correlated with fungal burden and hyphal length but not induction of GT biosynthetic genes. In summary, GT is detectable in mouse lungs, serum and BALF during neutropenic IA, suggesting that GT may be useful to diagnose IA in neutropenic patients.

Animal Models for Studying Triazole Resistance in Aspergillus fumigatus

Infections caused by triazole-resistant Aspergillus fumigatus are associated with a higher probability of treatment failure and mortality. Because clinical experience in managing these infections is still limited, mouse models of invasive aspergillosis fulfill a critical void for studying treatment regimens designed to overcome resistance. The type of immunosuppression, the route of infection, the timing of antifungal administration, and the end points used to assess antifungal activity affect the interpretation of data from these models. Nevertheless, these models provide important insights that help guide treatment decisions in patients with triazole-resistant invasive aspergillosis. Animal models confirmed that a high triazole minimal inhibitory concentration corresponded with triazole treatment failure and that the efficacy of other classes of drugs, such as the polyenes and echinocandins, was not affected by the presence of triazole resistance mutations. Furthermore, the feasibility of triazole dose escalation, combination therapy, and prophylaxis were explored as strategies to overcome resistance.

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.

Pharmacokinetic-Pharmacodynamic Target Attainment Analysis to Support VL-2397 Dose Selection for a Phase 2 Trial in Patients with Invasive Aspergillosis

BackgroundVL-2397 is a novel antifungal agent in clinical development for treatment of invasive aspergillosis (IA). The analysis objectives were to: 1) develop a population PK model using data from a Phase 1 trial in healthy adult volunteers, 2) define the PK-PD driver of efficacy in a mouse model of IA, and 3) conduct PK-PD target attainment analyses to aid in Phase 2 dose selection for patients with IA.MethodsData from two studies were used: a dose fractionation study in a mouse model of IA and a Phase 1 study in healthy adult volunteers receiving single- and multiple-ascending doses of intravenous (IV) VL-2397 ranging from 3 mg to 1200 mg daily. Mouse lung fungal burden (LFB) measured on Day 4 after infection with Aspergillus fumigatus FP1305 (MIC of 1 µg/mL) was used to assess the predictive value of three measures of VL-2397 exposure: total-drug AUC0-24:MIC (tAUC0-24:MIC), total-drug Cmax:MIC, and total-drug %T>MIC; total drug was used because serum protein binding is similar in mice and humans. A population PK model for VL-2397 in humans was developed in NONMEM v7.1.2 and formed the basis of Monte Carlo simulations. The model was also fit to mouse PK data to estimate the VL-2397 exposure in mice.ResultsVL-2397 concentration-time data in humans revealed nonlinear PK behavior consistent with highly-bound compounds. A robust fit to the data was obtained using a two-compartment model with linear elimination and concentration-dependent binding in both central and peripheral compartments. The results of the dose fractionation study suggested that tAUC0-24:MIC was the most predictive (r2 = 0.713) driver of efficacy, with a value of 21.8 on Day 1 associated with a 2-log reduction in LFB on Day 4. Comparisons of the simulated VL-2397 tAUC0-24:MIC on Day 1 in humans receiving daily dosing regimens of 300 mg, 600 mg, and 900 mg of VL-2397 to the 2-log target (figure) suggested that a dose of 600 mg Q24 would provide robust (99.9%) PK-PD target attainment up to an A. fumigatus MIC of 2 µg/mL.ConclusionThe PK-PD driver of efficacy for VL-2397 was tAUC0-24:MIC and a value of 21.8 was associated with a 2-log reduction in LFB in a mouse model of IA. PK-PD target attainment assessments indicate that a VL-2397 dose of 600 mg IV Q24 will provide adequate target attainment up to an MIC of 2 µg/mL.Disclosures C. M. Rubino, Vical Incorporated: Consultant, Consulting fee; L. R. Smith, Vical Incorporated: Employee, Salary and Stock Options; M. P. Mammen, Vical Incorporated: Employee, Salary and Stock Options; A. M. Hopkins, Vical Incorporated: Employee, Salary and Stock Options; E. A. Lakota, Vical Incorporated: Consultant, Consulting fee; S. M. Sullivan, Vical Incorporated: Employee, Salary and Stock Options

A computational model of invasive aspergillosis in the lung and the role of iron.

BackgroundInvasive aspergillosis is a severe infection of immunocompromised hosts, caused by the inhalation of the spores of the ubiquitous environmental molds of the Aspergillus genus. The innate immune response in this infection entails a series of complex and inter-related interactions between multiple recruited and resident cell populations with each other and with the fungal cell; in particular, iron is critical for fungal growth.ResultsA computational model of invasive aspergillosis is presented here; the model can be used as a rational hypothesis-generating tool to investigate host responses to this infection. Using a combination of laboratory data and published literature, an in silico model of a section of lung tissue was generated that includes an alveolar duct, adjacent capillaries, and surrounding lung parenchyma. The three-dimensional agent-based model integrates temporal events in fungal cells, epithelial cells, monocytes, and neutrophils after inhalation of spores with cellular dynamics at the tissue level, comprising part of the innate immune response. Iron levels in the blood and tissue play a key role in the fungus’ ability to grow, and the model includes iron recruitment and consumption by the different types of cells included. Parameter sensitivity analysis suggests the model is robust with respect to unvalidated parameters, and thus is a viable tool for an in silico investigation of invasive aspergillosis.ConclusionsUsing laboratory data from a mouse model of invasive aspergillosis in the context of transient neutropenia as validation, the model predicted qualitatively similar time course changes in fungal burden, monocyte and neutrophil populations, and tissue iron levels. This model lays the groundwork for a multi-scale dynamic mathematical model of the immune response to Aspergillus species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-016-0275-2) contains supplementary material, which is available to authorized users.

Efficacy of oleylphosphocholine (OlPC) in vitro and in a mouse model of invasive aspergillosis.

Invasive aspergillosis (IA) has become increasingly common and is characterised by high morbidity and mortality. Upcoming resistance threatens treatment with azoles and highlights the continuous need for novel therapeutics. This laboratory study investigated the in vitro and in vivo potential of the alkylphospholipid oleylphosphocholine (OlPC) against Aspergillus. In vitro activities of OlPC, miltefosine, posaconazole and voriconazole were determined for Aspergillus fumigatus, A. niger, A. terreus and A. flavus. In vivo efficacy of OlPC was evaluated in a systemic A. fumigatus mouse model, adopting a short-term and long-term oral or intraperitoneal dosing regimen. OlPC showed good in vitro activity against A. fumigatus (IC50 = 1.04 μmol l(-1)). Intraperitoneal administration of 50 mg kg(-1) day(-1) OlPC significantly reduced the fungal organ burdens at 4 days post-infection (dpi). Although 5- and 10-day OlPC treatment improved survival, organ burdens were not affected at 10 and 15 dpi. While this study showed excellent in vitro activity of OlPC against Aspergillus spp., its therapeutic efficacy in an acute mouse model for IA was less convincing. Given the limited therapeutic options in the current antifungal market for invasive infections, OlPC activity should be assessed in a less stringent in vivo model, potentially in combination treatment with other already marketed antifungal drugs.

A Polysaccharide Virulence Factor from Aspergillus fumigatus Elicits Anti-inflammatory Effects through Induction of Interleukin-1 Receptor Antagonist

The galactosaminogalactan (GAG) is a cell wall component of Aspergillus fumigatus that has potent anti-inflammatory effects in mice. However, the mechanisms responsible for the anti-inflammatory property of GAG remain to be elucidated. In the present study we used in vitro PBMC stimulation assays to demonstrate, that GAG inhibits proinflammatory T-helper (Th)1 and Th17 cytokine production in human PBMCs by inducing Interleukin-1 receptor antagonist (IL-1Ra), a potent anti-inflammatory cytokine that blocks IL-1 signalling. GAG cannot suppress human T-helper cytokine production in the presence of neutralizing antibodies against IL-1Ra. In a mouse model of invasive aspergillosis, GAG induces IL-1Ra in vivo, and the increased susceptibility to invasive aspergillosis in the presence of GAG in wild type mice is not observed in mice deficient for IL-1Ra. Additionally, we demonstrate that the capacity of GAG to induce IL-1Ra could also be used for treatment of inflammatory diseases, as GAG was able to reduce severity of an experimental model of allergic aspergillosis, and in a murine DSS-induced colitis model. In the setting of invasive aspergillosis, GAG has a significant immunomodulatory function by inducing IL-1Ra and notably IL-1Ra knockout mice are completely protected to invasive pulmonary aspergillosis. This opens new treatment strategies that target IL-1Ra in the setting of acute invasive fungal infection. However, the observation that GAG can also protect mice from allergy and colitis makes GAG or a derivative structure of GAG a potential treatment compound for IL-1 driven inflammatory diseases.

Characterization of Specific Immune Responses to Different Aspergillus Antigens during the Course of Invasive Aspergillosis in Hematologic Patients

Several studies in mouse model of invasive aspergillosis (IA) and in healthy donors have shown that different Aspergillus antigens may stimulate different adaptive immune responses. However, the occurrence of Aspergillus-specific T cells have not yet been reported in patients with the disease. In patients with IA, we have investigated during the infection: a) whether and how specific T-cell responses to different Aspergillus antigens occur and develop; b) which antigens elicit the highest frequencies of protective immune responses and, c) whether such protective T cells could be expanded ex-vivo. Forty hematologic patients have been studied, including 22 patients with IA and 18 controls. Specific T cells producing IL-10, IFN-γ, IL-4 and IL-17A have been characterized through enzyme linked immunospot and cytokine secretion assays on 88 peripheral blood (PB) samples, by using the following recombinant antigens: GEL1p, CRF1p, PEP1p, SOD1p, α1–3glucan, β1–3glucan, galactomannan. Specific T cells were expanded through short term culture. Aspergillus-specific T cells producing non-protective interleukin-10 (IL-10) and protective interferon-gamma (IFN-γ) have been detected to all the antigens only in IA patients. Lower numbers of specific T cells producing IL-4 and IL-17A have also been shown. Protective T cells targeted predominantly Aspergillus cell wall antigens, tended to increase during the IA course and to be associated with a better clinical outcome. Aspergillus-specific T cells could be successfully generated from the PB of 8 out of 8 patients with IA and included cytotoxic subsets able to lyse Aspergillus hyphae. Aspergillus specific T-cell responses contribute to the clearance of the pathogen in immunosuppressed patients with IA and Aspergillus cell wall antigens are those mainly targeted by protective immune responses. Cytotoxic specific T cells can be expanded from immunosuppressed patients even during the infection by using the above mentioned antigens. These findings may be exploited for immunotherapeutic purposes in patients with IA.

A new and clinically relevant murine model of solid-organ transplant aspergillosis.

SUMMARYInvasive fungal infections (IFIs) are a major cause of death in organ transplant patients. The murine hydrocortisone-mediated immunosuppression model of pulmonary aspergillosis is commonly used to characterise IFIs in these patients. However, this model does not take into account the effects of calcineurin inhibitors on transplant immunity to IFIs or the fungal calcineurin pathway, which is required for both virulence and antifungal drug resistance. To address these two issues, a new and clinically relevant transplant immunosuppression model of tacrolimus (FK506) and hydrocortisone-associated pulmonary aspergillosis was developed. We first characterised IFIs in 406 patients with a lung transplant. This showed that all of the patients with pulmonary aspergillosis were immunosuppressed with calcineurin inhibitors and steroids. Murine pharmacokinetic studies demonstrated that an ideal dose of 1 mg/kg/day of FK506 intraperitoneally produced blood trough levels in the human therapeutic range (5–12 ng/ml). There was increased mortality from pulmonary aspergillosis in a transplant-relevant immunosuppression model using both FK506 and hydrocortisone as compared with immunosuppression using hydrocortisone only. Lung histopathology showed neutrophil invasion and tracheobronchitis that was associated with reduced lung tumour necrosis factor-α (TNFα), JE (homologue of human MCP-1) and KC (homologue of human IL-8) at 24 hours, but increased lung TNFα, JE and KC at 48 hours when fungal burden was high. Furthermore, FK506 directly impaired fungal killing in alveolar macrophages in vitro, with FK506-mediated inhibition of the radial growth of Aspergillus fumigatus in vitro occurring at the low concentration of 5 ng/ml. Taken together, these findings show that the immunosuppressive activity of FK506 outweighs its antifungal activity in vivo. These observations demonstrate that FK506 impairs innate immune responses and leads to an incremental increase in susceptibility to IFIs when it is combined with steroids. This new and clinically relevant mouse model of invasive aspergillosis is a valuable addition to the further study of both fungal immunity and antifungal therapy in organ transplantation.

DIGE enables the detection of a putative serum biomarker of fungal origin in a mouse model of invasive aspergillosis

Invasive aspergillosis (IA) is a major threat for immunocompromised patients. Diagnostic difficulties often delay specific treatment initiation, which increases mortality. Finding new biomarkers to improve and speed accurate diagnosis is thus vital. To investigate the ability of proteomic methods for discovering new biomarkers of IA, we used a DIGE approach to perform a proteomic analysis on both bronchoalveolar lavages (BAL) and sera at different time-points of infection in a mouse model of invasive pulmonary aspergillosis. Progression of the infection was monitored using a bioluminescent strain of Aspergillus fumigatus. Sera proteins were enriched using the ProteoMiner kit (Biorad). This method allowed us to identify a fungal protein, the A. fumigatus major allergen Asp f 2, in sera of mice one day after the infection. However, this fungal protein was not detected three days after the infection. Importantly, in BAL, this work provides evidence of an in vivo complement evasion mechanism through the cleavage of C3b into three fragments during aspergillosis. Finally, our results underlining the inflammatory host response to IA in both lung and blood compartments at different times of infection may provide new insights into the pathophysiology of this disease.

Protective T-Cell Responses to Several Recombinant Aspergillus Antigens May Be Detected Since the Onset of the Infection in Patients with Invasive Aspergillosis, and May Be Exploited for Therapeutic Purposes,

Abstract 3229 Introduction:Several studies have reported that different components of fungi of the genera Aspergillus spp may induce protective T-cell responses in either mouse models of invasive aspergillosis (IA) or in human healthy subjects.We evaluated the occurrence of Aspergillus-specific T-cell responses to different Aspergillus recombinant antigens in patients with proven IA, during the course of the IA, to identify the antigens most frequently targeted by protective immune responses. We characterized phenotypically and functionally such specific T cells. Finally, from peripheral blood (PB) samples of the same IA proven patients, also collected during the active infection phase, we sought to expand such Aspergillus-specific T cells. Methods:15 patients with proven IA, according to the EORTC/MSG criteria, have been enrolled into the study. Specific immune responses producing interleukin-10 (IL-10), interferon-gamma (IFN-γ), IL-4 and IL-17A were detected and characterized by enzyme linked immunospot (ELISpot) assay and cytokine secretion assay (CSA), in all the patients, during the entire course of the IA. The recombinant antigens used were GEL1p, CRF1p, PEP1p, SOD1p, α1–3glucan, β1–3glucan, and galactomannan (GM). Cytotoxicity has been investigated by means of the colorimetric assay with (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]2H-tetrazolium-5-carboxyanilide) sodium salt plus coenzyme Q0 (XTT assay). Aspergillus-specific T cells were obtained by culturing PB mononuclear cells with a mixture containing PEP1p, GEL1p, α1–3 glucan and β1–3 glucan. The infection course were divided into 4 phases, defined from t1 to t4, each of fifteen days interval, starting from the radiological diagnosis of IA. Results:Aspergillus-specific T cells producing either IL-10 or IFN-γ were detected to all the antigens, but GM. The number of antigens targeted by IFN-γ producing specific T cells progressively increased along the course of IA, being such protective responses to 3 out of 7 antigens at t1 and to 6 out of 7 antigens at t4. At t1, IFN-γ producing specific T cells were only detected to GEL1p, α1–3 glucan and β1–3 glucan. GEL1p and α1–3 glucan resulted the antigens most constantly targeted by IFN-γ producing specific T cells, persisting the responses to these antigens in all the phases of IA. No Aspergillus-specific T cells producing IL-4 to any antigens were detected by the ELISpot assay. Aspergillus-specific T cells producing IL-17A were detected in only one out of 15 patients, and targeted CRF1p. Specific T cells to GM and specific T cells producing IL-4 to all the antigens could be shown only by CSA, suggesting that they are present only at very low frequencies during the infection. After 13-day cultures, Aspergillus-specific T cells were expanded from five out of five patients. The specific T cells tested for lytic activity included a median of 95.8% CD3+ cells, either CD4+ or CD8+ T cells, and showed a median lytic activity of 9.45% either at 3/1 or at 5/1 effector/target cells ratios. Conclusions:In patients with IA, protective immune responses may be detected since the onset and increase during the infection. At the onset of IA, specific T cells producing IFN-γ target antigens involved in the cell wall biosynthesis of Aspergillus. On the other hand, at the same phase, specific protective immune responses to CRF1p, PEP1p, and SOD1p, which are all putative virulence factors for Aspergillus, are absent. Aspergillus-specific T cells may be expanded by a mixture of antigens, even in the course of IA and are able to directly kill fungal hyphae. The above mentioned antigens and the corresponding protective T cells may be exploited for therapeutic strategies of either vaccine or autologous cytotoxic cell infusions in patients at high risk for IA. Disclosures:Luppi:MSD; GILEAD: Research Funding.

HacA-Independent Functions of the ER Stress Sensor IreA Synergize with the Canonical UPR to Influence Virulence Traits in Aspergillus fumigatus

Endoplasmic reticulum (ER) stress is a condition in which the protein folding capacity of the ER becomes overwhelmed by an increased demand for secretion or by exposure to compounds that disrupt ER homeostasis. In yeast and other fungi, the accumulation of unfolded proteins is detected by the ER-transmembrane sensor IreA/Ire1, which responds by cleaving an intron from the downstream cytoplasmic mRNA HacA/Hac1, allowing for the translation of a transcription factor that coordinates a series of adaptive responses that are collectively known as the unfolded protein response (UPR). Here, we examined the contribution of IreA to growth and virulence in the human fungal pathogen Aspergillus fumigatus. Gene expression profiling revealed that A. fumigatus IreA signals predominantly through the canonical IreA-HacA pathway under conditions of severe ER stress. However, in the absence of ER stress IreA controls dual signaling circuits that are both HacA-dependent and HacA-independent. We found that a ΔireA mutant was avirulent in a mouse model of invasive aspergillosis, which contrasts the partial virulence of a ΔhacA mutant, suggesting that IreA contributes to pathogenesis independently of HacA. In support of this conclusion, we found that the ΔireA mutant had more severe defects in the expression of multiple virulence-related traits relative to ΔhacA, including reduced thermotolerance, decreased nutritional versatility, impaired growth under hypoxia, altered cell wall and membrane composition, and increased susceptibility to azole antifungals. In addition, full or partial virulence could be restored to the ΔireA mutant by complementation with either the induced form of the hacA mRNA, hacA i, or an ireA deletion mutant that was incapable of processing the hacA mRNA, ireA Δ10. Together, these findings demonstrate that IreA has both HacA-dependent and HacA-independent functions that contribute to the expression of traits that are essential for virulence in A. fumigatus.

The virulence of the opportunistic fungal pathogen Aspergillus fumigatus requires cooperation between the endoplasmic reticulum-associated degradation pathway (ERAD) and the unfolded protein response (UPR)

The filamentous fungal pathogen Aspergillus fumigatus secretes hydrolytic enzymes to acquire nutrients from host tissues. The production of these enzymes exerts stress on the endoplasmic reticulum (ER), which is alleviated by two stress responses: the unfolded protein response (UPR), which adjusts the protein folding capacity of the ER, and ER-associated degradation (ERAD), which disposes of proteins that fail to fold correctly. In this study, we examined the contribution of these integrated pathways to the growth and virulence of A. fumigatus, focusing on the ERAD protein DerA and the master regulator of the UPR, HacA. A ΔderA mutant grew normally and showed no increase in sensitivity to ER stress. However, expression of the UPR target gene bipA was constitutively elevated in this strain, suggesting that the UPR was compensating for the absence of DerA function. To test this, the UPR was disrupted by deleting the hacA gene. The combined loss of derA and hacA caused a more severe reduction in hyphal growth, antifungal drug resistance and protease secretion than the loss of either gene alone, suggesting that DerA and HacA cooperate to support these functions. Moreover, the ΔderA/ΔhacA mutant was avirulent in a mouse model of invasive aspergillosis, which contrasted the wild type virulence of ΔderA and the reduced virulence of the ΔhacA mutant. Taken together, these data demonstrate that DerA cooperates with the UPR to support the expression of virulence-related attributes of A. fumigatus.

A Role for the Unfolded Protein Response (UPR) in Virulence and Antifungal Susceptibility in Aspergillus fumigatus

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.