Standing among the front defense strategies against pathogens, host phagocytic cells release various oxidants. Therefore, pathogens have to cope with stressful conditions at the site of infection. Peroxiredoxins (Prx) are highly reactive and abundant peroxidases that can support virulence and persistence of pathogens in distinct hosts. Here, we revealed that the opportunistic human pathogen A. fumigatus presents three 1-Cys Prx (Prx6 subfamily), which is unprecedented. We showed that PrxB and PrxC were in mitochondria, while Prx1 was in cytosol. As observed for other Prxs, recombinant Prx1 and PrxC decomposed H₂O₂ at elevated velocities (rate constants in the 10⁷ M⁻¹s⁻¹ range). Deletion mutants for each Prx displayed higher sensitivity to oxidative challenge in comparison with the wild-type strain. Additionally, cytosolic Prx1 was important for A. fumigatus survival upon electron transport dysfunction. Expression of Prxs was dependent on the SakA^HOG1 MAP kinase and the Yap1^YAP1 transcription factor, a global regulator of the oxidative stress response in fungi. Finally, cytosolic Prx1 played a major role in pathogenicity, since it is required for full virulence, using a neutropenic mouse infection model. Our data indicate that the three 1-Cys Prxs act together to maintain the redox balance of A. fumigatus.

Mammalian peroxiredoxin class 6 (Prdx6) are bifunctional enzymes. Non-mammalian Prdx6 enzymes display Cys-based peroxidase activity, but to date their putative phospholipase A₂ (PLA₂ activities) has not been experimentally investigated. Initially, we observed that five non-mammalian Prdx6 enzymes (enzymes from Arabidopsis thaliana (AtPER1), Triticum aestivum (TaPER1), Pseudomonas aeruginosa (PaLsfA) and Aspergillus fumigatus (AfPrx1 and AfPrxC)) present features compatible with PLA₂ activities in mammalian Prdx6 by amino acid sequences alignment and tertiary structure modeling. Employing unilamellar liposomes with tracer amounts of [³H]-1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and thin layer chromatography, all the tested non-mammalian Prdx6 enzymes displayed PLA₂ activities, with values ranging from 3.4 to 6.1 nmol/min/mg protein. It was previously shown that Thr177 phosphorylation of human Prdx6 increases its PLA₂ activity, especially at neutral pH. Therefore, we investigated if human Erk2 kinase could also phosphorylate homologous Thr residues in non-mammalian Prdx6 proteins. We observed phosphorylation of the conserved Thr in three out of the five non-mammalian Prdx enzymes by mass spectrometry. In the case of the mitochondrial Prdx6 from A. fumigatus (AfPrxC), we also observed phosphorylation by western blot, and as a consequence, the PLA₂ activity was increased in acidic and neutral conditions by the human Erk2 kinase treatment. The possible physiological meanings of these PLA₂ activities described open new fields for future research.

Industrial scale manufacture of pharmaceutical grade hyaluronic acid increasingly uses a recombinant source of hyaluronidase to achieve size-specific polymers, necessary for both cosmetic and medical purposes. A turbidimetric assay is currently recommended by the United States Pharmacopeia (USP) to measure specific hyaluronidase enzyme activity. However, this monograph lacks critical detail of experimental methodology, uses difficult to source reagents and is technically challenging to perform. Herein, a simplified assay to measure the specific activity of hyaluronidase with improved reliability, consistency and flexibility is presented. Results demonstrated that the source and concentration of substrate significantly influenced turbidity formation, which occurred only under acidic conditions and when measured by absorbance at 400 nm rather than 640 nm. Replacement of horse serum as the binding protein agent with bovine serum albumin enhanced accuracy and precision, consistently meeting acceptance criteria. The improved protocol also introduced flexible timing, enabling reliable measurements within a 30 mins to 1 h window, compared to a fixed 30-minute time point. Inference by different compounds at concentrations normally used in standard buffers and fermentation media was not significant. However, Tris-HCl and Na₂HPO₄ significantly reduced the specific activity of hyaluronidase and are, therefore, not recommended in growth media for yeast cultivation, buffers used for enzyme purification, and in reagents necessary for monitoring production of size-specific hyaluronic acid polymers. A robust assay that can be validated is an important part of any enzyme dependent manufacturing process and based on our empirical experimentation, hereby propose revision of the existing USP monograph.