The Lipid Raft-Associated Protein Stomatin Is Required for Accumulation of Dectin-1 in the Phagosomal Membrane and for Full Activity of Macrophages against Aspergillus fumigatus.

Alveolar macrophages belong to the first line of defense against inhaled conidia of the human-pathogenic fungus Aspergillus fumigatus. In lung alveoli, they contribute to phagocytosis and elimination of conidia. As a counterdefense, conidia have a gray-green pigment that enables them to survive in phagosomes of macrophages for some time. Previously, we showed that this conidial pigment interferes with the formation of flotillin-dependent lipid raft microdomains in the phagosomal membrane, thereby preventing the formation of functional phagolysosomes. Besides flotillins, stomatin is a major component of lipid rafts and can be targeted to the membrane. However, only limited information on stomatin is available, in particular on its role in defense against pathogens. To determine the function of this integral membrane protein, a stomatin-deficient macrophage line was generated by CRISPR/Cas9 gene editing. Immunofluorescence microscopy and flow cytometry revealed that stomatin contributes to the phagocytosis of conidia and is important for recruitment of the ß-glucan receptor dectin-1 to both the cytoplasmic membrane and phagosomal membrane. In stomatin knockout cells, fusion of phagosomes and lysosomes, recruitment of the vATPase to phagosomes, and tumor necrosis factor alpha (TNF-α) levels were reduced when cells were infected with pigmentless conidia. Thus, our data suggest that stomatin is involved in maturation of phagosomes via fostering fusion of phagosomes with lysosomes. IMPORTANCE Stomatin is an integral membrane protein that contributes to the uptake of microbes, e.g., spores of the human-pathogenic fungus Aspergillus fumigatus. By generation of a stomatin-deficient macrophage line by advanced genetic engineering, we found that stomatin is involved in the recruitment of the ß-glucan receptor dectin-1 to the phagosomal membrane of macrophages. Furthermore, stomatin is involved in maturation of phagosomes via fostering fusion of phagosomes with lysosomes. The data provide new insights on the important role of stomatin in the immune response against human-pathogenic fungi.

Targeting of phagolysosomes containing conidia of the fungus Aspergillus fumigatus with polymeric particles.

Conidia of the airborne human-pathogenic fungus Aspergillus fumigatus are inhaled by humans. In the lung, they are phagocytosed by alveolar macrophages and intracellularly processed. In macrophages, however, conidia can interfere with the maturation of phagolysosomes to avoid their elimination. To investigate whether polymeric particles (PPs) can reach this intracellular pathogen in macrophages, we formulated dye-labeled PPs with a size allowing for their phagocytosis. PPs were efficiently taken up by RAW 264.7 macrophages and were found in phagolysosomes. When macrophages were infected with conidia prior to the addition of PPs, we found that they co-localized in the same phagolysosomes. Mechanistically, the fusion of phagolysosomes containing PPs with phagolysosomes containing conidia was observed. Increasing concentrations of PPs increased fusion events, resulting in 14% of phagolysosomes containing both conidia and PPs. We demonstrate that PPs can reach conidia-containing phagolysosomes, making these particles a promising carrier system for antimicrobial drugs to target intracellular pathogens. KEY POINTS: • Polymer particles of a size larger than 500 nm are internalized by macrophages and localized in phagolysosomes. • These particles can be delivered to Aspergillus fumigatus conidia-containing phagolysosomes of macrophages. • Enhanced phagolysosome fusion by the use of vacuolin1 can increase particle delivery.

The leucine biosynthetic pathway is crucial for adaptation to iron starvation and virulence in Aspergillus fumigatus.

In contrast to mammalia, fungi are able to synthesize the branched-chain amino acid leucine de novo. Recently, the transcription factor LeuB has been shown to cross-regulate leucine biosynthesis, nitrogen metabolism and iron homeostasis in Aspergillus fumigatus, the most common human mold pathogen. Moreover, the leucine biosynthetic pathway intermediate α-isopropylmalate (α-IPM) has previously been shown to posttranslationally activate LeuB homologs in S. cerevisiae and A. nidulans. Here, we demonstrate that in A. fumigatus inactivation of both leucine biosynthetic enzymes α-IPM synthase (LeuC), which disrupts α-IPM synthesis, and α-IPM isomerase (LeuA), which causes cellular α-IPM accumulation, results in leucine auxotrophy. However, compared to lack of LeuA, lack of LeuC resulted in increased leucine dependence, a growth defect during iron starvation and decreased expression of LeuB-regulated genes including genes involved in iron acquisition. Lack of either LeuA or LeuC decreased virulence in an insect infection model, and inactivation of LeuC rendered A. fumigatus avirulent in a pulmonary aspergillosis mouse model. Taken together, we demonstrate that the lack of two leucine biosynthetic enzymes, LeuA and LeuC, results in significant phenotypic consequences indicating that the regulator LeuB is activated by α-IPM in A. fumigatus and that the leucine biosynthetic pathway is an attractive target for the development of antifungal drugs.

Triacetylfusarinine C: A urine biomarker for diagnosis of invasive aspergillosis.

OBJECTIVES: Early diagnosis of invasive aspergillosis (IA) remains challenging, with available diagnostics being limited by inadequate sensitivities and specificities. Triacetylfusarinine C, a fungal siderophore that has been shown to accumulate in urine in animal models, is a potential new biomarker for diagnosis of IA. METHODS: We developed a method allowing absolute and matrix-independent mass spectrometric quantification of TAFC. Urine TAFC, normalized to creatinine, was determined in 44 samples from 24 patients with underlying hematologic malignancies and probable, possible or no IA according to current EORTC/MSG criteria and compared to other established biomarkers measured in urine and same-day blood samples. RESULTS: TAFC/creatinine sensitivity, specificity, positive and negative likelihood ratio for probable versus no IA (cut-off ≥ 3) were 0.86, 0.88, 6.86, 0.16 per patient. CONCLUSION: For the first time, we provide proof for the occurrence of TAFC in human urine. TAFC/creatinine index determination in urine showed promising results for diagnosis of IA offering the advantages of non-invasive sampling. Sensitivity and specificity were similar as reported for GM determination in serum and bronchoalveolar lavage, the gold standard mycological criterion for IA diagnosis.

The Zn2Cys6-type transcription factor LeuB cross-links regulation of leucine biosynthesis and iron acquisition in Aspergillus fumigatus.

Both branched-chain amino acids (BCAA) and iron are essential nutrients for eukaryotic cells. Previously, the Zn2Cys6-type transcription factor Leu3/LeuB was shown to play a crucial role in regulation of BCAA biosynthesis and nitrogen metabolism in Saccharomyces cerevisiae and Aspergillus nidulans. In this study, we found that the A. fumigatus homolog LeuB is involved in regulation of not only BCAA biosynthesis and nitrogen metabolism but also iron acquisition including siderophore metabolism. Lack of LeuB caused a growth defect, which was cured by supplementation with leucine or iron. Moreover, simultaneous inactivation of LeuB and HapX, a bZIP transcription factor required for adaptation to iron starvation, significantly aggravated the growth defect caused by inactivation of one of these regulators during iron starvation. In agreement with a direct role in regulation of both BCAA and iron metabolism, LeuB was found to bind to phylogenetically conserved motifs in promoters of genes involved in BCAA biosynthesis, nitrogen metabolism, and iron acquisition in vitro and in vivo, and was required for full activation of their expression. Lack of LeuB also caused activation of protease activity and autophagy via leucine depletion. Moreover, LeuB inactivation resulted in virulence attenuation of A. fumigatus in Galleria mellonella. Taken together, this study identified a previously uncharacterized direct cross-regulation of BCCA biosynthesis, nitrogen metabolism and iron homeostasis as well as proteolysis.

Exploiting the Concept of Multivalency with 68Ga- and 89Zr-Labelled Fusarinine C-Minigastrin Bioconjugates for Targeting CCK2R Expression.

Cholecystokinin-2 receptors (CCK2R) are overexpressed in a variety of malignant diseases and therefore have gained certain attention for peptide receptor radionuclide imaging. Among extensive approaches to improve pharmacokinetics and metabolic stability of minigastrin (MG) based radioligands, the concept of multivalency for enhanced tumour targeting has not been investigated extensively. We therefore utilized fusarinine C (FSC) as chelating scaffold for novel mono-, di-, and trimeric bioconjugates for targeting CCK2R expression. FSC-based imaging probes were radiolabelled with positron emitting radionuclides (gallium-68 and zirconium-89) and characterized in vitro (log⁡D, IC50, and cell uptake) and in vivo (metabolic stability in BALB/c mice, biodistribution profile, and microPET/CT imaging in A431-CCK2R/A431-mock tumour xenografted BALB/c nude mice). Improved targeting did not fully correlate with the grade of multimerization. The divalent probe showed higher receptor affinity and increased CCK2R mediated cell uptake while the trimer remained comparable to the monomer. In vivo biodistribution studies 1 h after administration of the 68Ga-labelled radioligands confirmed this trend, but imaging at late time point (24 h) with 89Zr-labelled counterparts showed a clearly enhanced imaging contrast of the trimeric probe compared to the mono- and dimer. Furthermore, in vivo stability studies showed a higher metabolic stability for multimeric probes compared to the monomeric bioconjugate. In summary, we could show that FSC can be utilized as suitable scaffold for novel mono- and multivalent imaging probes for CCK2R-related malignancies with partly improved targeting properties for multivalent conjugates. The increased tumour accumulation of the trimer 24 h postinjection (p.i.) can be explained by slower clearance and increased metabolic stability of multimeric conjugates.