Abstract
The rise in the frequency of nosocomial infections is becoming a major problem for public health, in particular in immunocompromised patients. Aspergillus fumigatus is an opportunistic fungus normally present in the environment directly responsible for lethal invasive infections. Recent results suggest that the metabolic pathways related to amino acid metabolism can regulate the fungus-host interaction and that an important role is played by enzymes involved in the catabolism of L-tryptophan. In particular, in A. fumigatus L-tryptophan regulates Aro genes. Among them, AroH encodes a putative pyridoxal 5'-phosphate-dependent aminotransferase. Here we analyzed the biochemical features of recombinant purified AroH by spectroscopic and kinetic analyses corroborated by in silico studies. We found that the protein is dimeric and tightly binds the coenzyme forming a deprotonated internal aldimine in equilibrium with a protonated ketoenamine form. By setting up a new rapid assay method, we measured the kinetic parameters for the overall transamination of substrates and we demonstrated that AroH behaves as an aromatic amino acid aminotransferase, but also accepts L-kynurenine and α-aminoadipate as amino donors. Interestingly, computational approaches showed that the predicted overall fold and active site topology of the protein are similar to those of its yeast ortholog, albeit with some differences in the regions at the entrance of the active site, which could possibly influence substrate specificity. Should targeting fungal metabolic adaptation be of therapeutic value, the results of the present study may pave the way to the design of specific AroH modulators as potential novel agents at the host/fungus interface.
Highlights
Health care-associated, or nosocomial infections is a rising threat in public health
Aspergillus spp. are ubiquitous saprophytic molds that thrive on a diverse array of organic matter. These fungi are inhaled as spores daily without triggering any particular infectious disease, underlining the non-pathogenic potential of the fungus, unless severe immunocompromised hosts are exposed to spores
After addition of 100 μM pyridoxal 5’-phosphate (PLP), the protein solution was subjected to forced dialysis with 100 mM potassium phosphate buffer (KP), pH 7.4 using Amicon Ultra 10 concentrators (Millipore) to remove imidazole and unbound coenzyme
Summary
Health care-associated, or nosocomial infections is a rising threat in public health. The increase of opportunistic or nosocomial fungal infections has been perceived as a consequence of advances and more aggressive treatment modalities as organ transplantation, stem cell transplantation, immunomodulatory molecules or sophisticated chemotherapy. These conditions represent high risk factors for invasive fungal infections. Recent studies highlight the importance of regulatory cells in the defense against the fungus, as fungus-specific T regulatory cells have been described in healthy individuals (Bacher et al, 2016) This implies that resistance to fungal infections requires a very sophisticated but balanced environment where immune metabolism represents an important aspect (Choera et al, 2017). Targeting metabolic reprogramming in the fungus could be exploited for therapeutic intervention
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