Cryptococcus neoformans is an environmental fungus that belongs to the phylum Basidiomycetes and is a major pathogen in immunocompromised patients. The ability of C. neoformans to produce melanin pigments represents its second most important virulence factor, after the presence of a polysaccharide capsule. Both the capsule and melanin are closely associated with the fungal cell wall, a complex structure that is essential for maintaining cell morphology and viability under conditions of stress. The amino sugar N-acetylglucosamine (GlcNAc) is a key constituent of the cell-wall chitin and is used for both N-linked glycosylation and GPI anchor synthesis. Recent studies have suggested additional roles for GlcNAc as an activator and mediator of cellular signalling in fungal and plant cells. Furthermore, chitin and chitosan polysaccharides interact with melanin pigments in the cell wall and have been found to be essential for melanization. Despite the importance of melanin, its molecular structure remains unresolved; however, we previously obtained critical insights using advanced nuclear magnetic resonance (NMR) and imaging techniques. In this study, we investigated the effect of GlcNAc supplementation on cryptococcal cell-wall composition and melanization. C. neoformans was able to metabolize GlcNAc as a sole source of carbon and nitrogen, indicating a capacity to use a component of a highly abundant polymer in the biospherenutritionally. C. neoformans cells grown with GlcNAc manifested changes in the chitosan cell-wall content, cell-wall thickness and capsule size. Supplementing cultures with isotopically 15N-labelled GlcNAc demonstrated that the exogenous monomer serves as a building block for chitin/chitosan and is incorporated into the cell wall. The altered chitin-to-chitosan ratio had no negative effects on the mother–daughter cell separation; growth with GlcNAc affected the fungal cell-wall scaffold, resulting in increased melanin deposition and assembly. In summary, GlcNAc supplementation had pleiotropic effects on cell-wall and melanin architectures, and thus established its capacity to perturb these structures, a property that could prove useful for metabolic tracking studies.
Clinicians often have to deal with infections that are difficult to control because they are caused by superbugs resistant to many antibiotics. Alternatives to antibiotic treatment include antimicrobial photodynamic therapy (aPDT). The photodynamic process causes bacterial death, inducing oxidative stress through the photoactivation of photosensitizer molecules in the presence of oxygen. No PDT-resistant bacteria have been selected to date, thus the response to photo-oxidative stress in non-phototrophic bacteria needs further investigation. The opportunistic pathogen Pseudomonas aeruginosa, in particular, has been shown to be more tolerant to PDT than other micro-organisms. In order to find any genetic determinants involved in PDT-tolerance, a panel of transposon mutants of P. aeruginosa PAO1 involved in the quorum sensing signalling system and membrane cytoplasmic transport were photoinactivated as part of this study. Two pseudomonas quinolone signalling (PQS) knock-out mutants, pqsH - and pqsC -, were as PDT-sensitive as the PAO1 wild-type strains. Two PQS hyperproducer variants, pqsA - and rsaL -, were shown to be more tolerant to photo-oxidative stress than the wild-type strain. In the pqsA - mutant, the hyperpigmentation due to the presence of phenazines could protect cells against PDT stress, while in rsaL - no pigmentation was detectable. Furthermore, a mutant impaired in an ATP-binding cassette transport involved in maintaining the asymmetry of the outer membrane was significantly more tolerant to photo-oxidative stress than the wild-type strain. These observations support the involvement of quorum sensing and the importance of the bacterial cell envelope when dealing with photo-oxidative stress induced by photodynamic treatment.