A fungal lytic polysaccharide monooxygenase is required for cell wall integrity, thermotolerance, and virulence of the fungal human pathogen Cryptococcus neoformans.

Fungi often adapt to environmental stress by altering their size, shape, or rate of cell division. These morphological changes require reorganization of the cell wall, a structural feature external to the cell membrane composed of highly interconnected polysaccharides and glycoproteins. Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that are typically secreted into the extracellular space to catalyze initial oxidative steps in the degradation of complex biopolymers such as chitin and cellulose. However, their roles in modifying endogenous microbial carbohydrates are poorly characterized. The CEL1 gene in the human fungal pathogen Cryptococcus neoformans (Cn) is predicted by sequence homology to encode an LPMO of the AA9 enzyme family. The CEL1 gene is induced by host physiological pH and temperature, and it is primarily localized to the fungal cell wall. Targeted mutation of the CEL1 gene revealed that it is required for the expression of stress response phenotypes, including thermotolerance, cell wall integrity, and efficient cell cycle progression. Accordingly, a cel1Δ deletion mutant was avirulent in two models of C. neoformans infection. Therefore, in contrast to LPMO activity in other microorganisms that primarily targets exogenous polysaccharides, these data suggest that CnCel1 promotes intrinsic fungal cell wall remodeling events required for efficient adaptation to the host environment.

Knowing friend from foe.

How does a protein at the cell wall determine if a newly encountered fungus is safe to fuse with?

Lytic polysaccharide monooxygenases and other histidine-brace copper proteins: structure, oxygen activation and biotechnological applications.

Lytic polysaccharide monooxygenases (LPMOs) are mononuclear copper enzymes that catalyse the oxidative cleavage of glycosidic bonds. They are characterised by two histidine residues that coordinate copper in a configuration termed the Cu-histidine brace. Although first identified in bacteria and fungi, LPMOs have since been found in all biological kingdoms. LPMOs are now included in commercial enzyme cocktails used in industrial biorefineries. This has led to increased process yield due to the synergistic action of LPMOs with glycoside hydrolases. However, the introduction of LPMOs makes control of the enzymatic step in industrial stirred-tank reactors more challenging, and the operational stability of the enzymes is reduced. It is clear that much is still to be learned about the interaction between LPMOs and their complex natural and industrial environments, and fundamental scientific studies are required towards this end. Several atomic-resolution structures have been solved providing detailed information on the Cu-coordination sphere and the interaction with the polysaccharide substrate. However, the molecular mechanisms of LPMOs are still the subject of intense investigation; the key question being how the proteinaceous environment controls the copper cofactor towards the activation of the O-O bond in O2 and cleavage of the glycosidic bonds in polysaccharides. The need for biochemical characterisation of each putative LPMO is discussed based on recent reports showing that not all proteins with a Cu-histidine brace are enzymes.

Secondary metabolite production by cereal-associated penicillia during cultivation on cereal grains.

Cereals are vulnerable substrates for fungal growth and subsequent mycotoxin contamination. One of the major fungal genera to colonize the ecosystem of stored grain is Penicillium, especially species in the series of Viridicata and Verrucosa. Culturing these species on grains, we hoped to induce the production of relevant secondary metabolites produced by these fungi in the early stage of cereal breakdown. In a multivariate setup six different cereal grains (wheat, rye, barley, oat, rice, and maize), one kind of white beans, and two standard fungal media, Yeast Extract Sucrose agar (YES agar) and Czapek Yeast Autolysate agar (CYA agar), were inoculated with the ten most important cereal-associated species from Penicillium (P. aurantiogriseum, P. cyclopium, P. freii, P. melanoconidium, P. neoechinulatum, P. polonicum, P. tricolor, P. viridicatum, P. hordei, and P. verrucosum). P. nordicum is a meat-associated species, which was included due to its chemical association with P. verrucosum, in addition to see if a substrate change would alter the profile of known chemistry. We found that cereals function very well as substrates for secondary metabolite production, but did not present significantly different secondary metabolite profiles, concerning known chemistry, as compared to standard laboratory agar media. However, white beans altered the semi-quantitative secondary metabolite profiles for several species. Correlations between substrates and certain metabolites were observed, as illuminated by principal component analysis. Many bioactive secondary metabolites were observed for the first time in the analyzed fungal species, including ergot type alkaloids in P. hordei.

Production of the Fusarium Mycotoxin Moniliformin by Penicillium melanoconidium.

Moniliformin is a mycotoxin produced by several cereal associated Fusaria. Here, we show for the first time that moniliformin can be produced by the cereal fungus, Penicillium melanoconidium (4 out of 4 strains), but not in the related species in the Viridicata series. Moniliformin was detected in 10 out of 11 media: two agars and several cereal and bean types. Moniliformin was identified by a novel mixed-mode anionic exchange reversed phase chromatographic method which was coupled to both tandem mass spectrometry (MS) and high resolution MS. Mixed-mode chromatography showed superior peak shape compared to that of HILIC and less matrix interference compared to that of reversed phase chromatography, but during a large series of analyses, the column was fouled by matrix interferences. Wheat and beans were artificially infected by P. melanoconidium containing up to 64 and 11 mg/kg moniliformin, respectively, while penicillic acid, roquefortine C, and penitrem A levels in wheat were up to 1095, 38, and 119 mg/kg, respectively.