Aspergillus fumigatus is a mold that causes an array of diseases, the most severe form being invasive aspergillosis which occurs in highly immunocompromised individuals. We demonstrated that tissue-resident alveolar macrophages are critical for maintaining host resistance against Aspergillus fumigatus due to their role in the early type I/III interferon response. Specifically, CD11c-Cre x Mavs fl/flmice have a decreased interferon response and survival outcomes when challenged with A. fumigatus, which can be functionally restored via adoptive transfer of wild-type SiglecF +alveolar macrophages. Why alveolar macrophages are potent inducers of the type I/III interferon response after Aspergillus fumigatus challenge remains elusive. Here, we demonstrate that TGF-beta metabolically programs alveolar macrophages to be potent interferon inducers via enhanced dependency on oxidative phosphorylation and peroxisome biogenesis. The type I/III interferon responses were partially related to the inability of alveolar macrophages to recognize mycoviral double-stranded RNA (dsRNA). Aspergillus fumigatus strains infected with polymycovirus 1 (PmV1) are shown to induce a robust type I/III interferon responses both in vitro and in vivo. Critically, Mda5/MAVS-dependent interferon response can still be induced in a fungal dsRNA independent manner, which warrant further explorations. In this study we identified key metabolic traits of alveolar macrophages which enable robust Mda5/MAVS signaling necessary for initiating the Aspergillus fumigatus induced interferon response, which maintains host resistance against this ubiquitous mold, through both fungal dsRNA-dependent and -independent mechanisms.

Conclusions:The presence of NTM in respiratory cultures of PwCF does not appear to decrease likelihood of transplantation referral, although a positive NTM culture was associated with lower likelihood of listing for and performance of lung transplantation.This is particularly notable for those PwCF who have had Mabsc in cultures.A limitation of this analysis is that we do not know the specific factors that decreased the probability of listing and transplantation.Additional sensitivity analyses will be conducted to include modeling to assess the effect of persistence and temporality of culture positivity as it is related to transplantation status and multivariate logistic regression evaluating interactions of other key clinical features that could affect transplant status.Whether the presence of NTM should be a factor in transplantation decisions should be determined; assessment of post-transplantation outcomes is planned.

amounts, pyoverdine chelated Fe(III) from ferric-cefiderocol, indicating that siderophores might prevent cefiderocol uptake by displacing Fe(III).Conclusions: These data show that P. aeruginosa cefiderocol crossprotection is beta-lactamase independent; bacterial siderophores and cefiderocol compete for iron, and because of their greater Fe(III)-affinity, pyoverdine and enterobactin limit cefiderocol uptake by susceptible siblings.Understanding what drives the cooperative behavior might improve the use of cefiderocol and delay the spread of cefiderocol resistance.

airway.These manifestations were not seen in the 2-month-old rats.Analysis of mucin content in the BALF of all animals indicated a potential correlation between high Muc5ac protein levels and high markers of lung damage as assessed using μCT.No correlation was observed for Muc5b.Bacterial burden of P. aeruginosa was equal in the lungs of 2-and 6-monthold rats, but S. aureus was only retained in the 6-month-old group.Conclusions: In adult CF rats, chronic co-infection with P. aeruginosa and S. aureus led to structural lung damage and decline, reflecting what has been reported clinically for PwCF.This decline was not observed in younger rats lacking the complete CF airway phenotype, indicating the importance of the CF environment in disease progression.In adult rats, S. aureus infection was also sustained over the course of the study, supporting clinical evidence that, despite having an antagonistic relationship in vitro, S. aureus escapes killing by P. aeruginosa in the context of CF.

Background: Although Aspergillus fumigatus is the most common filamentous fungus isolated from people with cystic fibrosis (CF), a standardized microbiology laboratory protocol for fungus culture of CF sputum remains to be developed.Sputum expectoration and collection in clinic, necessary for fungus culture, has decreased because of widespread

Abstract Type I and III interferons act as important activators of antifungal neutrophil response in the lungs. The RIG-I like receptor (RLR) family, including RIG-I and Mda5, are cytosolic RNA sensors that signal through the MAVS adaptor in order to activate interferon responses against viruses. Whether the RLR family has broader effects on host immunity against other pathogen families remains to be fully explored. Herein we demonstrate that Mda5/MAVS signaling was essential for host resistance against pulmonary Aspergillus fumigatus challenge through the regulation of antifungal leukocyte responses. Aspergillus fumigatus induction of type I interferons was partially dependent on Mda5/MAVS signaling, while type III interferon expression was entirely dependent on Mda5/MAVS signaling. Activation of the interferon pathway was driven by prolonged exposure to fungal spores, rather than germ tubes. Ultimately, interferon signaling drove the expression of CXCL9 and CXCL10, SNPs in the latter are associated with invasive aspergillosis in human patients. Our data suggest a broader role of the RLR family in the regulation of innate immunity to include invasive fungal infections.

Aspergillus fumigatus strain diversity is reflected in heterogeneous virulence and inflammatory responses. Mechanisms underlying A. fumigatus strain specific immune responses and how this affects disease outcome is unresolved. We observe that the CEA10 strain is able to rapidly efficiently within the airway environment and induces greater lung damage, vascular leakage, and IL-1α release compared to the Af293 strain. Fungal clearance is dependent on IL-1α in mice inoculated with the CEA10 strain, while clearance of AF293 does not require IL-1α. Importantly, both swollen conidia and germlings of the CEA10 strain induce robust IL-1α secretion from macrophages in vitro, whereas only germlings of Af293 induce strong production of IL-1α. Our finding that early fungal germination in the airways drives greater pathology and IL-1α dependent inflammation is further supported by similar results with additional strains. First, serial passage of a non-airway germinating strain (Af293) in low-oxygen conditions results in a strain (EVOL20) that germinates more efficiently in the airways leading to enhanced lung damage, vascular leakage, and IL-1α dependent inflammation. Second, using primary clinical and environmental isolates of A. fumigatus that are able to rapidly germinate in airway conditions follows the same trend. Clinically, our data support the idea that A. fumigatus strain phenotypic variation significantly contributes to disease outcomes. Understanding why different A. fumigatus strains induce distinct immune pathology can reveal novel immunotherapeutic and antifungal targets for the treatment of invasive aspergillosis.

Aspergillus fumigatus is a mold that causes severe pulmonary infections in humans, such as invasive pulmonary Aspergillosis (IPA). Currently, our knowledge of how A. fumigatus growth is controlled in the respiratory tract is limited. Phagocytic alveolar macrophages constitute the first line of defense against inhaled A. fumigatus conidia; subsequently, neutrophils and macrophages are sequentially recruited to the respiratory tract to control fungal growth and germination . But how neutrophils and macrophages are recruited to the respiratory tract after A. fumiguatus infection remains ill defined. During sterile inflammation early neutrophil recruitment is dependent on IL-1α, while late macrophage recruitment is dependent on IL-1β. A. fumigatus infection induced the expression of both IL-1α and IL-1β. However, IL-1R1- and ASC-deficient mice displayed differential susceptibility to IPA. IL-1R1-deficient mice were highly susceptible to A. fumigatus infection as measured by increases in fungal growth, lung tissue damage, and fungal dissemination. In contrast, ASC-deficient mice were only mildly susceptible to A. fumigatus infection. IL-1R1- and ASC-deficient mice displayed a severe defect in neutrophil recruitment during the first 48h and a defect in macrophage recruitment after 48h, respectively. Taken together, our data reveal important non-redundant roles for the IL-1α and IL-1β in controlling A. fumigatus infection in the lung.

Aspergillus fumigatus causes invasive and allergenic disease. Host defense relies on the ability of the respiratory immune system to restrict spore germination into invasive hyphae and to limit fungus-induced or inflammation-induced damage in infected tissues. This review covers the molecular and cellular events that mediate innate and CD4 T-cell responses to A. fumigatus and fungal attributes that counter hostile microenvironments and, in turn, affect host responses.Host recognition of fungal cell wall components is critical for fungal uptake, killing, and the formation of protective innate and CD4 T-cell effector populations. Beyond the known role of neutrophils and macrophages, circulating monocytes, dendritic cells, and natural killer cells contribute to optimal defense against A. fumigatus. Genetic and pharmacologic manipulation of A. fumigatus reveals that hypoxia adaptation, cell wall assembly, and secondary metabolite production in mammalian tissues contribute to fungal pathogenesis and the outcome of infection.Greater understanding of the immune mechanisms that underlie protective responses and fungal pathways that promote microbial adaptation and growth in mammalian tissue provide a conceptual framework for improving current antifungal therapies.

Aspergillus fumigatus is a saprophytic fungus commonly found in soil and compost piles. In immunocompromised patients it takes on a sinister form as a potentially lethal opportunistic human pathogen. We currently have a limited understanding of the in vivo growth mechanisms used by A. fumigatus during invasive pulmonary aspergillosis (IPA). The ability of A. fumigatus to adapt to various microenvironments encountered during growth in the human host may explain why A. fumigatus is the most frequently occurring opportunistic pathogenic mold. The transcriptional and metabolic responses to changing microenvironments found in the mammalian lung require the activation of pathways implicated in resistance to unique stresses. Thus, the production of primary metabolites in vivo may give clues to the critical pathways used by A. fumigatus to cause disease in human hosts. We recently have identified primary metabolites in the mammalian lung typically associated with fungal growth under hypoxic environments suggesting that A. fumigatus may encounter low oxygen tensions during IPA. These and other studies on A. fumigatus metabolism are the focus of this review.

The number of immunocompromised patients with invasive fungal infections continues to increase and new antifungal therapies are not keeping pace with the growing incidence of these infections and their associated mortality. Calcineurin inhibition is currently used to exert effective immunosuppression following organ transplantation and in treating various other conditions. However, the calcineurin pathway is also intricately involved in the growth and pathogenesis of the three major fungal pathogens of humans, Cryptococcus neoformans, Candida albicans and Aspergillus fumigatus, and the exploitation of fungal calcineurin pathways holds great promise for the future development of novel antifungal agents. This Review summarizes our current understanding of calcineurin biology in these fungal species, and its exciting potential role in treating invasive fungal infections.

SUMMARY Alternaria brassicicola is a necrotrophic pathogen causing black spot disease on virtually all cultivated Brassica crops worldwide. In many plant pathosystems fungal secondary metabolites derived from non-ribosomal peptide synthetases (NPSs) are phytotoxic virulence factors or are antibiotics thought to be important for niche competition with other micro-organisms. However, many of the functions of NPS genes and their products are largely unknown. In this study, we investigated the function of one of the A. brassicicola NPS genes, AbNPS2. The predicted amino acid sequence of AbNPS2 showed high sequence similarity with A. brassicae, AbrePsy1, Cochliobolus heterostrophus, NPS4 and a Stagonospora nodorum NPS. The AbNPS2 open reading frame was predicted to be 22 kb in length and encodes a large protein (7195 amino acids) showing typical NPS modular organization. Gene expression analysis of AbNPS2 in wild-type fungus indicated that it is expressed almost exclusively in conidia and conidiophores, broadly in the reproductive developmental phase. AbNPS2 gene disruption mutants showed abnormal spore cell wall morphology and a decreased hydrophobicity phenotype. Conidia of abnps2 mutants displayed an aberrantly inflated cell wall and an increase in lipid bodies compared with wild-type. Further phenotypic analyses of abnps2 mutants showed decreased spore germination rates both in vitro and in vivo, and a marked reduction in sporulation in vivo compared with wild-type fungus. Moreover, virulence tests on Brassicas with abnps2 mutants revealed a significant reduction in lesion size compared with wild-type but only when aged spores were used in experiments. Collectively, these results indicate that AbNPS2 plays an important role in development and virulence.

ABSTRACT The fungal secondary metabolite gliotoxin produced by Aspergillus fumigatus has been hypothesized to be important in the development of invasive aspergillosis. In this study, we addressed this hypothesis by disrupting a nonribosomal peptide synthetase (NRPS) (encoded by gliP ) predicted to be involved in gliotoxin production. Mutants with a disrupted gliP locus failed to produce gliotoxin, which confirmed the role of the NRPS encoded by gliP in gliotoxin biosynthesis. We found no morphological, developmental, or physiological defects in Δ gliP mutant strains. In addition, disruption of gliP resulted in down regulation of gene expression in the gliotoxin biosynthesis gene cluster, which was restored with addition of exogenous gliotoxin. This interesting result suggests a role for gliotoxin in regulating its own production. Culture filtrates from the Δ gliP mutant were unable to inhibit ionomycin-dependent degranulation of mast cells, suggesting a role for gliotoxin in suppressing mast cell degranulation and possibly in disease development. However, the Δ gliP mutant did not have an impact on survival or tissue burden in a murine inhalational model of invasive aspergillosis. This result suggests that gliotoxin is not required for virulence in an immunosuppressed host with an invasive pulmonary infection.

SUMMARY Alternaria brassicicola is a necrotrophic fungal pathogen that causes black spot disease on members of the Brassicaceae plant family. In order to identify candidate fungal pathogenicity genes and characterize a compatible host response, a suppression subtractive hybridization (SSH) cDNA library enriched for A. brassicicola and Brassica oleracea genes expressed during the interaction was created, along with a fungal cDNA library representing genes expressed during nitrogen starvation (NS). A total of 3749 and 2352 expressed sequence tags (ESTs) were assembled into 2834 and 1264 unisequence sets for the SSH and NS libraries, respectively. We compared two methods to identify the origins (plant vs. fungal) of ESTs in the SSH library using different classification procedures, with and without the availability of a database representing the A. brassicicola whole genome sequence and Brassicaceae-specific genes. BLASTX analyses of the 2834 unisequence set using the GenBank non-redundant database identified 114 fungal genes. Further BLASTN analyses of the genes with unidentifiable origin using a database consisting of the 1264 fungal unisequence set from the nitrogen-starved library identified 94 additional fungal genes. By contrast, BLASTN analyses of the same SSH unisequence set using a partially assembled A. brassicicola whole genome draft sequence identified a total of 310 unisequenes of fungal origin. Our results indicated that even a small number of organism-specific EST sequences can be very helpful to identify pathogen genes in a library derived from infected tissue, partially overcoming the limitation of the public databases for little studied organisms. However, using the whole genome draft sequence of A. brassicicola we were able to identify approximately 30% more fungal genes in the SSH library than without utilizing this resource. The putative role of specific fungal and plant genes identified in this study in a compatible interaction is discussed.