Unsaturated fatty acid perturbation combats emerging triazole antifungal resistance in the human fungal pathogen Aspergillus fumigatus.

Contemporary antifungal therapies utilized to treat filamentous fungal infections are inhibited by intrinsic and emerging drug resistance. Consequently, there is an urgent need to develop novel antifungal compounds that are effective against drug-resistant filamentous fungi. Here, we utilized an Aspergillus fumigatus cell-based high-throughput screen to identify small molecules with antifungal activity that also potentiated triazole activity. The screen identified 16 hits with promising activity against A. fumigatus. A nonspirocyclic piperidine, herein named MBX-7591, exhibited synergy with triazole antifungal drugs and activity against pan-azole-resistant A. fumigatus isolates. MBX-7591 has additional potent activity against Rhizopus species and CO2-dependent activity against Cryptococcus neoformans. Chemical, genetic, and biochemical mode of action analyses revealed that MBX-7591 increases cell membrane saturation by decreasing oleic acid content. MBX-7591 has low toxicity in vivo and shows good efficacy in decreasing fungal burden in a murine model of invasive pulmonary aspergillosis. Taken together, our results suggest MBX-7591 is a promising hit with a novel mode of action for further antifungal drug development to combat the rising incidence of triazole-resistant filamentous fungal infections.IMPORTANCEThe incidence of infections caused by fungi continues to increase with advances in medical therapies. Unfortunately, antifungal drug development has not kept pace with the incidence and importance of fungal infections, with only three major classes of antifungal drugs currently available for use in the clinic. Filamentous fungi, also called molds, are particularly recalcitrant to contemporary antifungal therapies. Here, a recently developed Aspergillus fumigatus cell reporter strain was utilized to conduct a high-throughput screen to identify small molecules with antifungal activity. An emphasis was placed on small molecules that potentiated the activity of contemporary triazole antifungals and led to the discovery of MBX-7591. MBX-7591 potentiates triazole activity against drug-resistant molds such as A. fumigatus and has activity against Mucorales fungi. MBX-7591's mode of action involves inhibiting the conversion of saturated to unsaturated fatty acids, thereby impacting fungal membrane integrity. MBX-7591 is a novel small molecule with antifungal activity poised for lead development.

Intestinal Bacteroides modulates inflammation, systemic cytokines, and microbial ecology via propionate in a mouse model of cystic fibrosis.

Persons with cystic fibrosis (CF), starting in early life, show intestinal microbiome dysbiosis characterized in part by a decreased relative abundance of the genus Bacteroides. Bacteroides is a major producer of the intestinal short chain fatty acid propionate. We demonstrate here that cystic fibrosis transmembrane conductance regulator-defective (CFTR-/-) Caco-2 intestinal epithelial cells are responsive to the anti-inflammatory effects of propionate. Furthermore, Bacteroides isolates inhibit the IL-1ß-induced inflammatory response of CFTR-/- Caco-2 intestinal epithelial cells and do so in a propionate-dependent manner. The introduction of Bacteroides-supplemented stool from infants with cystic fibrosis into the gut of CftrF508del mice results in higher propionate in the stool as well as the reduction in several systemic pro-inflammatory cytokines. Bacteroides supplementation also reduced the fecal relative abundance of Escherichia coli, indicating a potential interaction between these two microbes, consistent with previous clinical studies. For a Bacteroides propionate mutant in the mouse model, pro-inflammatory cytokine KC is higher in the airway and serum compared with the wild-type (WT) strain, with no significant difference in the absolute abundance of these two strains. Taken together, our data indicate the potential multiple roles of Bacteroides-derived propionate in the modulation of systemic and airway inflammation and mediating the intestinal ecology of infants and children with CF. The roles of Bacteroides and the propionate it produces may help explain the observed gut-lung axis in CF and could guide the development of probiotics to mitigate systemic and airway inflammation for persons with CF.IMPORTANCEThe composition of the gut microbiome in persons with CF is correlated with lung health outcomes, a phenomenon referred to as the gut-lung axis. Here, we demonstrate that the intestinal microbe Bacteroides decreases inflammation through the production of the short-chain fatty acid propionate. Supplementing the levels of Bacteroides in an animal model of CF is associated with reduced systemic inflammation and reduction in the relative abundance of the opportunistically pathogenic group Escherichia/Shigella in the gut. Taken together, these data demonstrate a key role for Bacteroides and microbially produced propionate in modulating inflammation, gut microbial ecology, and the gut-lung axis in cystic fibrosis. These data support the role of Bacteroides as a potential probiotic in CF.

Bik promotes proteasomal degradation to control low-grade inflammation.

Although chronic low-grade inflammation does not cause immediate clinical symptoms, over the longer term, it can enhance other insults or age-dependent damage to organ systems and thereby contribute to age-related disorders, such as respiratory disorders, heart disease, metabolic disorders, autoimmunity, and cancer. However, the molecular mechanisms governing low-level inflammation are largely unknown. We discovered that Bcl-2-interacting killer (Bik) deficiency causes low-level inflammation even at baseline and the development of spontaneous emphysema in female but not male mice. Similarly, a single nucleotide polymorphism that reduced Bik levels was associated with increased inflammation and enhanced decline in lung function in humans. Transgenic expression of Bik in the airways of Bik-deficient mice inhibited allergen- or LPS-induced lung inflammation and reversed emphysema in female mice. Bik deficiency increased nuclear but not cytosolic p65 levels because Bik, by modifying the BH4 domain of Bcl-2, interacted with regulatory particle non-ATPase 1 (RPN1) and RPN2 and enhanced proteasomal degradation of nuclear proteins. Bik deficiency increased inflammation primarily in females because Bcl-2 and Bik levels were reduced in lung tissues and airway cells of female compared with male mice. Therefore, controlling low-grade inflammation by modifying the unappreciated role of Bik and Bcl-2 in facilitating proteasomal degradation of nuclear proteins may be crucial in treating chronic age-related diseases.

The cystic fibrosis treatment Trikafta affects the growth, viability, and cell wall of Aspergillus fumigatus biofilms.

IMPORTANCE: PwCF commonly test positive for pathogenic fungi, and more than 90% of the cystic fibrosis patient population is approved for the modulator treatment, Trikafta. Therefore, it is critical to understand how fungal communities, specifically A. fumigatus, respond to Trikafta exposure. Therefore, we sought to determine whether Trikafta impacted the biology of A. fumigatus biofilms. Our data demonstrate that Trikafta reduces biomass in several laboratory strains as well as clinical strains isolated from the expectorated sputum of pwCF. Furthermore, Trikafta reduces fungal viability and the capacity of biofilms to recover following treatment. Of particular importance, Trikafta affects how A. fumigatus biofilms respond to cell wall stressors, suggesting that Trikafta modulates components of the cell wall. Since the cell wall directly affects how a host immune system will respond to and effectively neutralize pathogens, our work, demonstrating that Trikafta impacts the A. fumigatus cell wall, is potentially highly relevant to fungal-induced disease pathogenesis.

Postinfluenza Environment Reduces Aspergillus fumigatus Conidium Clearance and Facilitates Invasive Aspergillosis In Vivo.

Aspergillus fumigatus is a human fungal pathogen that is most often avirulent in immunecompetent individuals because the innate immune system is efficient at eliminating fungal conidia. However, recent clinical observations have shown that severe influenza A virus (IAV) infection can lead to secondary A. fumigatus infections with high mortality. Little is currently known about how IAV infection alters the innate antifungal immune response. Here, we established a murine model of IAV-induced A. fumigatus (IAV-Af) superinfection by inoculating mice with IAV followed 6 days later by A. fumigatus conidia challenge. We observed increased mortality in the IAV-Af-superinfected mice compared to mice challenged with either IAV or A. fumigatus alone. A. fumigatus conidia were able to germinate and establish a biofilm in the lungs of the IAV-Af superinfection group, which was not seen following fungal challenge alone. While we did not observe any differences in inflammatory cell recruitment in the IAV-Af superinfection group compared to single-infection controls, we observed defects in Aspergillus conidial uptake and killing by both neutrophils and monocytes after IAV infection. pHrodo Green zymosan bioparticle (pHrodo-zymosan) and CM-H2DCFDA [5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate] staining, indicators of phagolysosome maturation and reactive oxygen species (ROS) production, respectively, revealed that the fungal killing defect was due in part to reduced phagolysosome maturation. Collectively, our data demonstrate that the ability of neutrophils and monocytes to kill and clear Aspergillus conidia is strongly reduced in the pulmonary environment of an IAV-infected lung, which leads to invasive pulmonary aspergillosis and increased overall mortality in our mouse model, recapitulating what is observed clinically in humans. IMPORTANCE Influenza A virus (IAV) is a common respiratory virus that causes seasonal illness in humans, but can cause pandemics and severe infection in certain patients. Since the emergence of the 2009 H1N1 pandemic strains, there has been an increase in clinical reports of IAV-infected patients in the intensive care unit (ICU) developing secondary pulmonary aspergillosis. These cases of flu-Aspergillus superinfections are associated with worse clinical outcomes than secondary bacterial infections in the setting of IAV. To date, we have a limited understanding of the cause(s) of secondary fungal infections in immunocompetent hosts. IAV-induced modulation of cytokine production and innate immune cellular function generates a unique immune environment in the lung, which could make the host vulnerable to a secondary fungal infection. Our work shows that defects in phagolysosome maturation in neutrophils and monocytes after IAV infection impair the ability of these cells to kill A. fumigatus, thus leading to increased fungal germination and growth and subsequent invasive aspergillosis. Our work lays a foundation for future mechanistic studies examining the exact immune modulatory events occurring in the respiratory tract after viral infection leading to secondary fungal infections.

Casein kinase II activates Bik to induce death of hyperplastic mucous cells in a cell cycle-dependent manner.

Extensive inflammation causes epithelial cell hyperplasia in the airways and Bcl-2-interacting killer (Bik) reduces epithelial cell and mucous cell hyperplasia without affecting resting cells to restore homeostasis. These observations suggest that Bik induces apoptosis in a cell cycle-specific manner, but the mechanisms are not understood. Mice were exposed to an allergen for 3, 14, or 30 days and Bik expression was induced in airway epithelia of transgenic mice. Bik reduced epithelial and mucous cell hyperplasia when mice were exposed to an allergen for 3 or 14 days, but not when exposure lasted for 30 days, and Ki67-positivity was reduced. In culture, Bik expression killed proliferating cells but not quiescent cells. To capture the stage of the cell cycle when Bik induces cell death, airway cells that express fluorescent ubiquitin cell cycle indicators were generated that fluoresce red or green during the G0/G1 and S/G2/M phases of the cells cycle, respectively. Regardless of the cell cycle stage, Bik expression eliminated green-fluorescent cells. Also, Bik, when tagged with a blue-fluorescent protein, was only detected in green cells. Bik phosphorylation mutants at threonine 33 or serine 35 demonstrated that phosphorylation activated Bik to induce death even in quiescent cells. Immunoprecipitation and proteomic approaches identified casein kinase IIα to be responsible for phosphorylating and activating Bik to kill cells in S/G2/M. As casein kinase 2 alpha (CKIIα) is expressed only during the G2/M phase, we conclude that Bik activation in airway epithelial cells selectively targets hyperplastic epithelial cells, while leaving resting airway cells unaffected.

Aspergillus fumigatus Strain-Specific Conidia Lung Persistence Causes an Allergic Broncho-Pulmonary Aspergillosis-Like Disease Phenotype.

Aspergillus fumigatus is a filamentous fungus which can cause multiple diseases in humans. Allergic broncho-pulmonary aspergillosis (ABPA) is a disease diagnosed primarily in cystic fibrosis patients caused by a severe allergic response often to long-term A. fumigatus colonization in the lungs. Mice develop an allergic response to repeated inhalation of A. fumigatus spores; however, no strains have been identified that can survive long-term in the mouse lung and cause ABPA-like disease. We characterized A. fumigatus strain W72310, which was isolated from the expectorated sputum of an ABPA patient, by whole-genome sequencing and in vitro and in vivo viability assays in comparison to a common reference strain, CEA10. W72310 was resistant to leukocyte-mediated killing and persisted in the mouse lung longer than CEA10, a phenotype that correlated with greater resistance to oxidative stressors, hydrogen peroxide, and menadione, in vitro In animals both sensitized and challenged with W72310, conidia, but not hyphae, were viable in the lungs for up to 21 days in association with eosinophilic airway inflammation, airway leakage, serum IgE, and mucus production. W72310-sensitized mice that were recall challenged with conidia had increased inflammation, Th1 and Th2 cytokines, and airway leakage compared to controls. Collectively, our studies demonstrate that a unique strain of A. fumigatus resistant to leukocyte killing can persist in the mouse lung in conidial form and elicit features of ABPA-like disease.IMPORTANCE Allergic broncho-pulmonary aspergillosis (ABPA) patients often present with long-term colonization of Aspergillus fumigatus Current understanding of ABPA pathogenesis has been complicated by a lack of long-term in vivo fungal persistence models. We have identified a clinical isolate of A. fumigatus, W72310, which persists in the murine lung and causes an ABPA-like disease phenotype. Surprisingly, while viable, W72310 showed little to no growth beyond the conidial stage in the lung. This indicates that it is possible that A. fumigatus can cause allergic disease in the lung without any significant hyphal growth. The identification of this strain of A. fumigatus can be used not only to better understand disease pathogenesis of ABPA and potential antifungal treatments but also to identify features of fungal strains that drive long-term fungal persistence in the lung. Consequently, these observations are a step toward helping resolve the long-standing question of when to utilize antifungal therapies in patients with ABPA and fungal allergic-type diseases.

Reducing protein oxidation reverses lung fibrosis.

Idiopathic pulmonary fibrosis is characterized by excessive deposition of collagen in the lung, leading to chronically impaired gas exchange and death1-3. Oxidative stress is believed to be critical in this disease pathogenesis4-6, although the exact mechanisms remain enigmatic. Protein S-glutathionylation (PSSG) is a post-translational modification of proteins that can be reversed by glutaredoxin-1 (GLRX)7. It remains unknown whether GLRX and PSSG play a role in lung fibrosis. Here, we explored the impact of GLRX and PSSG status on the pathogenesis of pulmonary fibrosis, using lung tissues from subjects with idiopathic pulmonary fibrosis, transgenic mouse models and direct administration of recombinant Glrx to airways of mice with existing fibrosis. We demonstrate that GLRX enzymatic activity was strongly decreased in fibrotic lungs, in accordance with increases in PSSG. Mice lacking Glrx were far more susceptible to bleomycin- or adenovirus encoding active transforming growth factor beta-1 (AdTGFB1)-induced pulmonary fibrosis, whereas transgenic overexpression of Glrx in the lung epithelium attenuated fibrosis. We furthermore show that endogenous GLRX was inactivated through an oxidative mechanism and that direct administration of the Glrx protein into airways augmented Glrx activity and reversed increases in collagen in mice with TGFB1- or bleomycin-induced fibrosis, even when administered to fibrotic, aged animals. Collectively, these findings suggest the therapeutic potential of exogenous GLRX in treating lung fibrosis.

Glutathione S-transferase pi modulates NF-κB activation and pro-inflammatory responses in lung epithelial cells.

Nuclear Factor kappa B (NF-κB) is a transcription factor family critical in the activation of pro- inflammatory responses. The NF-κB pathway is regulated by oxidant-induced post-translational modifications. Protein S-glutathionylation, or the conjugation of the antioxidant molecule, glutathione to reactive cysteines inhibits the activity of inhibitory kappa B kinase beta (IKKß), among other NF-κB proteins. Glutathione S-transferase Pi (GSTP) is an enzyme that has been shown to catalyze protein S-glutathionylation (PSSG) under conditions of oxidative stress. The objective of the present study was to determine whether GSTP regulates NF-κB signaling, S-glutathionylation of IKK, and subsequent pro-inflammatory signaling. We demonstrated that, in unstimulated cells, GSTP associated with the inhibitor of NF-κB, IκBα. However, exposure to LPS resulted in a rapid loss of association between IκBα and GSTP, and instead led to a protracted association between IKKß and GSTP. LPS exposure also led to increases in the S-glutathionylation of IKKß. SiRNA-mediated knockdown of GSTP decreased IKKß-SSG, and enhanced NF-κB nuclear translocation, transcriptional activity, and pro-inflammatory cytokine production in response to lipopolysaccharide (LPS). TLK117, an isotype-selective inhibitor of GSTP, also enhanced LPS-induced NF-κB transcriptional activity and pro-inflammatory cytokine production, suggesting that the catalytic activity of GSTP is important in repressing NF-κB activation. Expression of both wild-type and catalytically-inactive Y7F mutant GSTP significantly attenuated LPS- or IKKß-induced production of GM-CSF. These studies indicate a complex role for GSTP in modulating NF-κB, which may involve S-glutathionylation of IKK proteins, and interaction with NF-κB family members. Our findings suggest that targeting GSTP is a potential avenue for regulating the activity of this prominent pro-inflammatory and immunomodulatory transcription factor.

Ablation of Glutaredoxin-1 Modulates House Dust Mite-Induced Allergic Airways Disease in Mice.

Protein S-glutathionylation (PSSG) is an oxidant-induced post-translational modification of protein cysteines that impacts structure and function. The oxidoreductase glutaredoxin-1 (Glrx1) under physiological conditions catalyzes deglutathionylation and restores the protein thiol group. The involvement of Glrx1/PSSG in allergic inflammation induced by asthma-relevant allergens remains unknown. In the present study, we examined the impact of genetic ablation of Glrx1 in the pathogenesis of house dust mite (HDM)-induced allergic airways disease in mice. Wild-type (WT) or Glrx1(-/-) mice were instilled intranasally with HDM on 5 consecutive days for 3 weeks. As expected, overall PSSG was increased in Glrx1(-/-) HDM mice as compared with WT animals. Total cells in bronchoalveolar lavage fluid were similarly increased in HDM-treated WT and Glrx1(-/-) mice. However, in response to HDM, mice lacking Glrx1 demonstrated significantly more neutrophils and macrophages but fewer eosinophils as compared with HDM-exposed WT mice. mRNA expression of the Th2-associated cytokines IL-13 and IL-6, as well as mucin-5AC (Muc5ac), was significantly attenuated in Glrx1(-/-) HDM-treated mice. Conversely, mRNA expression of IFN-γ and IL-17A was increased in Glrx1(-/-) HDM mice compared with WT littermates. Restimulation of single-cell suspensions isolated from lungs or spleens with HDM resulted in enhanced IL-17A and decreased IL-5 production in cells derived from inflamed Glrx1(-/-) mice compared with WT animals. Finally, HDM-induced tissue damping and elastance were significantly attenuated in Glrx1(-/-) mice compared with WT littermates. These results demonstrate that the Glrx1-PSSG axis plays a pivotal role in HDM-induced allergic airways disease in association with enhanced type 2 inflammation and restriction of IFN-γ and IL-17A.

Ablation of the Thiol Transferase Glutaredoxin-1 Augments Protein S-Glutathionylation and Modulates Type 2 Inflammatory Responses and IL-17 in a House Dust Mite Model of Allergic Airway Disease in Mice.

S-glutathionylation has emerged as an oxidant-induced post-translational modification of protein cysteines that affects structure and function. The oxidoreductase glutaredoxin-1 (Glrx1), under physiological conditions, catalyzes deglutathionylation and restores the protein thiol group. The involvement of Grx1/S-glutathionylation in allergic inflammation induced by asthma-relevant allergens remains unknown. In the present study we examined the impact of genetic ablation of Glrx1 for the pathogenesis of house dust mite (HDM)-induced allergic airway disease in mice. Wild-type (WT) or Glrx1(-/-) mice in the BALB/c background were instilled intranasally with 50 µg of HDM 5 consecutive days for 3 weeks and killed 72 hours post final exposure. As expected, overall protein S-glutathionylation was increased in Glrx1(-/-) mice exposed to HDM as compared with WT animals. Total cells in the bronchoalveolar lavage fluid were similarly increased in WT and Glrx1(-/-) HDM-treated mice compared with phosphate-buffered saline-treated control mice. However, in response to HDM, mice lacking Glrx1 demonstrated significantly more neutrophils but fewer eosinophils than HDM-exposed WT mice. mRNA expression of the Th2-associated cytokine IL-13, as well as MUC5ac, was significantly attenuated in Glrx1(-/-) HDM-treated mice compared with WT mice. Conversely, expression of IL-17A was increased in Glrx1(-/-) HDM mice compared with WT mice. Last, HDM-induced tissue damping and elastance were significantly attenuated in Glrx1(-/-) mice compared with WT littermates. These results demonstrate that the Grx1/S-glutathionylation redox status plays a pivotal role in HDM-induced allergic inflammation and airway hyperresponsiveness and suggest a potential role of Glrx1/S-glutathionylation in controlling the nature of the HDM-induced adaptive immune responses by promoting Type-2-driven inflammation and restricting IL-17A.