TACKLING BIOFILM-RELATED OPPORTUNISTIC FUNGAL INFECTIONS

More than 1.5 million people/year die for a biofilm-related fungal infection. The ability to develop biofilms is widespread among human opportunistic pathogens, among them fungi. Biofilms are a community of microorganisms with the ability to attach to surfaces biotic or abiotic, surrounded by a self-produced extracellular polymeric matrix. The pathogen clearance and the general overcoming of the infection are complicated by biofilm presence as it confers tolerance to antimicrobial treatments. Compromised or altered immune response and an inflammatory milieu can exacerbate the pathogenesis and prolong the resolution of the infection. Candida albicans, between yeasts, and Aspergillus fumigatus between molds, are pathobionts considered the most common agents of opportunistic fungi mediated. In our study we assessed different strategies to eradicate biofilms and counteract biofilm-related infections, targeting both pathogens and host immune response. We investigated the efficacy of urinary fractions after cranberry extract intake by healthy volunteers against C. albicans biofilm. By mass spectrometry analyses, we identified two metabolites picking in the most active urine fractions, 5-(3′,4′- dihydroxy phenyl)-γ-valerolactone and 4-hydroxybenzoic acid, which revealed a strong inhibitory effect on C. albicans adhesion and biofilm formation. Both compounds were also able to downregulate the expression of key genes involved in early phases of biofilm formation. Due to the spread of the use of catheters or prostheses in the medical field, the possibility to develop biomaterials with biofilm inhibitory activity could represent a good 10 strategy to control nosocomial infections. We thus investigated newly synthesized hydrogels and demonstrated their ability to prevent C. albicans growth. To target both pathogen virulence and host immune response, we conducted two different studies, the first on C. albicans systemic candidiasis and the second on A. fumigatus infection in cystic fibrosis patients. We investigated the possible dual action of pilocarpine, a muscarinic receptor agonist, in vitro and in vivo using the model host Galleria mellonella. Pilocarpine showed a direct effect in inhibiting C. albicans biofilm biomass and metabolic activity, and its administration to infected larvae increased larval survival. Because of the different behavior of pilocarpine compared to acetylcholine in the modulation of larva immune response, we concluded that the antifungal activity of pilocarpine might rely on muscarinic-like receptor activation on C. albicans, whereas the strong immunomodulatory effect of acetylcholine (but not of pilocarpine) might imply the engagement of nicotinic receptors. A similar approach, aimed at targeting both infection and inflammation, was used to investigate A. fumigatus persistence in CF patient lungs. Indeed, CF monocytes are characterized by altered intracellular lipid accumulation, that compromise pathogen clearance, and the infection resolution. We demonstrated that the treatment of patient monocytes with Myriocin, by inhibiting the synthesis of sphingolipids and hampering CF inflammation, ameliorate the A. fumigatus conidia internalization and clearance. On the host side, Myriocin 11 was able to reduce the over expression of genes encoding for proinflammatory cytokines and at the same time to increase the expression of gene encoding for pathogen recognizing receptors, crucial for its clearance. Our results indicate that, despite we are still far from clinical practice, exploring alternative anti-biofilm strategies could pave the way to new target discovery and to integrated approaches able to promote the infection resolution.