ABSTRACT
Pentraxin 3 (PTX3), a long pentraxin and a humoral pattern recognition molecule (PRM), has been demonstrated to be protective against Aspergillus fumigatus, an airborne human fungal pathogen. We explored its mode of interaction with A. fumigatus, and the resulting implications in the host immune response. Here, we demonstrate that PTX3 interacts with A. fumigatus in a morphotype-dependent manner: (a) it recognizes germinating conidia through galactosaminogalactan, a surface exposed cell wall polysaccharide of A. fumigatus, (b) in dormant conidia, surface proteins serve as weak PTX3 ligands, and (c) surfactant protein D (SP-D) and the complement proteins C1q and C3b, the other humoral PRMs, enhance the interaction of PTX3 with dormant conidia. SP-D, C3b or C1q opsonized conidia stimulated human primary immune cells to release pro-inflammatory cytokines and chemokines. However, subsequent binding of PTX3 to SP-D, C1q or C3b opsonized conidia significantly decreased the production of pro-inflammatory cytokines/chemokines. PTX3 opsonized germinating conidia also significantly lowered the production of pro-inflammatory cytokines/chemokines while increasing IL-10 (an anti-inflammatory cytokine) released by immune cells when compared to the unopsonized counterpart. Overall, our study demonstrates that PTX3 recognizes A. fumigatus either directly or by interplaying with other humoral PRMs, thereby restraining detrimental inflammation. Moreover, PTX3 levels were significantly higher in the serum of patients with invasive pulmonary aspergillosis (IPA) and COVID-19-associated pulmonary aspergillosis (CAPA), supporting previous observations in IPA patients, and suggesting that it could be a potential panel-biomarker for these pathological conditions caused by A. fumigatus.
Subject(s)
Aspergillus fumigatus , C-Reactive Protein , Complement C1q , Serum Amyloid P-Component , Spores, Fungal , Aspergillus fumigatus/immunology , Serum Amyloid P-Component/metabolism , Serum Amyloid P-Component/immunology , Humans , Spores, Fungal/immunology , C-Reactive Protein/metabolism , C-Reactive Protein/immunology , Complement C1q/metabolism , Complement C1q/immunology , Pulmonary Surfactant-Associated Protein D/metabolism , Pulmonary Surfactant-Associated Protein D/immunology , Complement C3b/immunology , Complement C3b/metabolism , Cytokines/metabolism , Cytokines/immunology , Interleukin-10/metabolism , Interleukin-10/immunology , Aspergillosis/immunology , Aspergillosis/microbiology , Host-Pathogen Interactions/immunology , Immunity, Humoral , Female , PolysaccharidesABSTRACT
More than 10 million people suffer from lung diseases caused by the pathogenic fungus Aspergillus fumigatus. Azole antifungals represent first-line therapeutics for most of these infections but resistance is rising, therefore the identification of antifungal targets whose inhibition synergises with the azoles could improve therapeutic outcomes. Here, we generate a library of 111 genetically barcoded null mutants of Aspergillus fumigatus in genes encoding protein kinases, and show that loss of function of kinase YakA results in hypersensitivity to the azoles and reduced pathogenicity. YakA is an orthologue of Candida albicans Yak1, a TOR signalling pathway kinase involved in modulation of stress responsive transcriptional regulators. We show that YakA has been repurposed in A. fumigatus to regulate blocking of the septal pore upon exposure to stress. Loss of YakA function reduces the ability of A. fumigatus to penetrate solid media and to grow in mouse lung tissue. We also show that 1-ethoxycarbonyl-beta-carboline (1-ECBC), a compound previously shown to inhibit C. albicans Yak1, prevents stress-mediated septal spore blocking and synergises with the azoles to inhibit A. fumigatus growth.
Subject(s)
Antifungal Agents , Aspergillus fumigatus , Dyrk Kinases , Fungal Proteins , Protein Serine-Threonine Kinases , Protein-Tyrosine Kinases , Aspergillus fumigatus/genetics , Aspergillus fumigatus/drug effects , Aspergillus fumigatus/enzymology , Animals , Antifungal Agents/pharmacology , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Fungal Proteins/genetics , Fungal Proteins/metabolism , Fungal Proteins/antagonists & inhibitors , Mice , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Protein-Tyrosine Kinases/antagonists & inhibitors , Azoles/pharmacology , Aspergillosis/microbiology , Aspergillosis/drug therapy , Lung/microbiology , Spores, Fungal/drug effects , Spores, Fungal/genetics , FemaleABSTRACT
The opportunistic fungal pathogen Candida albicans damages host cells via its peptide toxin, candidalysin. Before secretion, candidalysin is embedded in a precursor protein, Ece1, which consists of a signal peptide, the precursor of candidalysin and seven non-candidalysin Ece1 peptides (NCEPs), and is found to be conserved in clinical isolates. Here we show that the Ece1 polyprotein does not resemble the usual precursor structure of peptide toxins. C. albicans cells are not susceptible to their own toxin, and single NCEPs adjacent to candidalysin are sufficient to prevent host cell toxicity. Using a series of Ece1 mutants, mass spectrometry and anti-candidalysin nanobodies, we show that NCEPs play a role in intracellular Ece1 folding and candidalysin secretion. Removal of single NCEPs or modifications of peptide sequences cause an unfolded protein response (UPR), which in turn inhibits hypha formation and pathogenicity in vitro. Our data indicate that the Ece1 precursor is not required to block premature pore-forming toxicity, but rather to prevent intracellular auto-aggregation of candidalysin sequences.