Aspergillus fumigatus is the most important airborne fungal pathogen of immunosuppressed humans. A. fumigatus is able to produce dihydroxynaphthalene melanin, which is predominantly present in the conidia. Its biosynthesis is an important virulence determinant. Here, we show that A. fumigatus is able to produce an alternative melanin, i.e., pyomelanin, by a different pathway, starting from L-tyrosine. Proteome analysis indicated that the L-tyrosine degradation enzymes are synthesized when the fungus is grown with L-tyrosine in the medium. To investigate the pathway in detail, we deleted the genes encoding essential enzymes for pigment production, homogentisate dioxygenase (hmgA) and 4-hydroxyphenylpyruvate dioxygenase (hppD). Comparative Fourier transform infrared spectroscopy of synthetic pyomelanin and pigment extracted from A. fumigatus cultures confirmed the identity of the observed pigment as pyomelanin. In the hmgA deletion strain, HmgA activity was abolished and the accumulation of homogentisic acid provoked an increased pigment formation. In contrast, homogentisic acid and pyomelanin were not observed with an hppD deletion mutant. Germlings of the hppD deletion mutant showed an increased sensitivity to reactive oxygen intermediates. The transcription of both studied genes was induced by L-tyrosine. These results confirmed the function of the deleted genes and the predicted pathway in A. fumigatus. Homogentisic acid is the major intermediate, and the L-tyrosine degradation pathway leading to pyomelanin is similar to that in humans leading to alkaptomelanin.

Aspergillus fumigatus is the most important air-borne fungal pathogen of humans. The interaction of the pathogen with the host's immune system represents a key process to understand pathogenicity. For elimination of invading microorganisms, they need to be efficiently phagocytosed and located in acidified phagolysosomes. However, as shown previously, A. fumigatus is able to manipulate the formation of functional phagolysosomes. Here, we demonstrate that in contrast to pigmentless pksP mutant conidia of A. fumigatus, the gray-green wild-type conidia inhibit the acidification of phagolysosomes of alveolar macrophages, monocyte-derived macrophages, and human neutrophil granulocytes. Therefore, this inhibition is independent of the cell type and applies to the major immune effector cells required for defense against A. fumigatus. Studies with melanin ghosts indicate that the inhibitory effect of wild-type conidia is due to their dihydroxynaphthalene (DHN)-melanin covering the conidia, whereas the hydrophobin RodA rodlet layer plays no role in this process. This is also supported by the observation that pksP conidia still exhibit the RodA hydrophobin layer, as shown by scanning electron microscopy. Mutants defective in different steps of the DHN-melanin biosynthesis showed stronger inhibition than pksP mutant conidia but lower inhibition than wild-type conidia. Moreover, A. fumigatus and A. flavus led to a stronger inhibition of phagolysosomal acidification than A. nidulans and A. terreus. These data indicate that a certain type of DHN-melanin that is different in the various Aspergillus species, is required for maximal inhibition of phagolysosomal acidification. Finally, we identified the vacuolar ATPase (vATPase) as potential target for A. fumigatus based on the finding that addition of bafilomycin which inhibits vATPase, led to complete inhibition of the acidification whereas the fusion of phagosomes containing wild-type conidia and lysosomes was not affected.

The opportunistic human pathogenic fungus Aspergillus fumigatus is able to produce the dark brown pigment pyomelanin by degradation of L-tyrosine. Pyomelanin was shown to protect the fungus against reactive oxygen intermediates as well as cell wall disturbing compounds and is therefore assumed to protect against immune effector cells during the infection process. Several genes for tyrosine degradation and pyomelanin formation are organized in a cluster in the genome of A. fumigatus. Here, we aimed at further analyzing tyrosine degradation and a possible role of pyomelanin in virulence. For this purpose, the function of two not yet characterized genes of the cluster, i.e., hmgX and hmgR, was analyzed. Generation of corresponding gene deletion mutants and reconstituted strains revealed that hmgX and hmgR are essential for tyrosine degradation. Both mutants, ΔhmgX and ΔhmgR, were not able to use tyrosine as sole carbon or nitrogen source and revealed impaired pyomelanin production. HmgR harbors a Zn(II)2Cys6-DNA binding domain. Analyses of the steady state mRNA levels revealed that HmgR acts as a transcriptional activator for the genes of the tyrosine degradation cluster. Consistently, an HmgR-eGFP fusion protein was localized in the nucleus of A. fumigatus cells. By contrast, HmgX was found to be localized in the cytoplasm and does not contribute to regulation of gene transcription. HPLC analyses showed that HmgX is crucial for the conversion of p-hydroxyphenylpyruvate to homogentisic acid, the main intermediate in pyomelanin formation. Thus, HmgX is supposed to function as an accessory factor to mediate specific activity of HppD. Remarkably, the ability to degrade tyrosine and to form pyomelanin is dispensable for virulence of A. fumigatus in a murine infection model.

Recalls of some batches of metformin have occurred due to the detection of N-nitrosodimethylamine (NDMA) in amounts above the acceptable intake (AI) of 96 ng per day. Prior to the recalls, an international regulatory laboratory network had been monitoring drugs for nitrosamine impurities with each laboratory independently developing and validating multiple analytical procedures to detect and measure nitrosamines in metformin drugs used in their jurisdictions. Here, we provide an overview of the analysis of metformin active pharmaceutical ingredients (APIs) and drug products with 1090 samples (875 finished dosage forms (FDFs) and 215 API samples) tested beginning in November of 2019 through July of 2020. Samples were obtained internationally by a variety of approaches, including purchased, received from firms via information requests or selected by regional regulatory authorities (either at wholesalers or during GMP inspections). Only one nitrosamine (NDMA) was detected and was only present in some batches of metformin products. For API samples, 213 out of 215 lots tested had no measurable level of NDMA. For FDF samples tested, the number of batches with NDMA above the AI amount for patient safety was 17.8% (156/875). Based on these data, although the presence of NDMA was of concern, 82.2% of the samples of metformin drug products tested met quality and safety standards for patients. Regulatory agencies continue to collaborate extensively and work with marketing authorization holders to understand root causes of nitrosamine formation and agree on corrective actions to mitigate the presence of NDMA in future metformin batches. Graphical abstract