The biofilm produced by Cryptococcus neoformans protects the fungus from the antifungal and anti-melanin effects of cyclosporine.

Understanding Cryptococcus neoformans pathogenesis requires a detailed analysis of the various virulence factors that contribute to its ability to cause disease. Cyclosporine, calcineurin inhibitor, impairs C. neoformans production of a polysaccharide capsule and secretion of urease, which are critical for cryptococcal pathogenesis. Two particularly important virulence factors are the production of cell wall melanin and formation of biofilm. In this study, we investigated cyclosporine's effects on melanin production and biofilm formation in C. neoformans. Initially, we examined melanin production in planktonic cells treated with cyclosporine using an L-DOPA containing melanin-inducing medium. Visual inspection and optical microscopy revealed a notable reduction in the characteristic dark coloration of cultures treated with cyclosporine, which indicate decreased melanin production in daughter cells compared to mother cells. Spectrophotometric analysis also demonstrated a significantly altered ultraviolet-visible (UV/vis) absorption spectra in cyclosporine-treated yeast cells, indicative of structural changes in melanin. Additionally, cyclosporine-treated cells exhibited reduced conductance (P-value < 0.0001), suggesting altered cellular ionic properties. The impact of cyclosporine on biofilm formation and mature biofilm disruption was also assessed. Despite cyclosporine's efficacy in modifying virulence factors during planktonic growth, cyclosporine did not inhibit biofilm formation or melanization under biofilm growth conditions, nor did it disrupt mature biofilms in terms of biomass or metabolic activity. However, there was a significant reduction in extracellular matrix production in cyclosporine-treated non-melanized biofilms. Our findings underscore the complex interplay between cyclosporine and C. neoformans, highlighting its differential effects on melanization and biofilm dynamics, which provides new insights into the shortcomings of cyclosporin for combatting cryptococcosis and informs pathways for future therapeutic strategies against cryptococcosis.

Andrade IB ，Alves V ，Correa-Junior D ，Avellar-Moura I ，Soares J ，Sousa Araújo GR ，Pontes B ，Nosanchuk JD ，Almeida-Paes R ，Frases S ... -  《-》

Melanin, melanin "ghosts," and melanin composition in Cryptococcus neoformans.

Wang Y ，Aisen P ，Casadevall A 《-》

Anti-Cryptococcal activity of a furanone derivative-antibiofilm and opsonophagocytic potential.

Cryptococcus neoformans, an encapsulated fungal pathogen is evolving as a major threat to immune-compromised patients and rarely to healthy individuals also. The cell wall bound capsular polysaccharide, melanin pigment and biofilm formation are major virulence factors that are known to contribute to cryptococcal meningitis. In the present study, a furanone derivative, (E)-5-benzylidenedihydrofuran-2(3H)-one (compound-6) was evaluated against biofilm of seven different strains of C. neoformans in melanized and non-melanized condition. In addition, the efficacy of compound-6 in activation of TLR-2, opsonophagocytosis, and modulation of cytokine expression during phagocytosis were studied. During the biofilm study, we found that moderate capsule size favored biofilm formation. Interestingly, the minimum biofilm eradication concentration (MBEC0.5) of melanized biofilm was found to be achieved at 1- to 1.7-fold higher MBEC0.5 of non-melanized cells. The maximum eradication of 77% and 69% of non-melanized and melanized biofilm were observed. The capsule size was reduced to half of its size with marked changes in morphology. Furthermore, expression of TLR2, iNOS and pro-inflammatory cytokines such as TNF-α, IL-12, and IFN-γ were also facilitated by compound-6. The correlation analysis showed a positive correlation between phagocytosis and the expression of TLR-2, iNOS, IL-6, IL-12. Collectively, the significant effect of compound-6, anti-melanization activity, antibiofilmand effective immunomodulant could be an interesting dual strategy drug agonist against cryptococcal meningitis.

Rathore SS ，Raman T ，Lalitha C ，Ramar M ，Munusamy A ，Ramakrishnan J ... -  《-》

Susceptibility of Cryptococcus neoformans biofilms to antifungal agents in vitro.

Martinez LR ，Casadevall A 《-》

Cryptococcus neoformans/Cryptococcus gattii species complex melanized by epinephrine: Increased yeast survival after amphotericin B exposure.

Cryptococcus neoformans/Cryptococcus gattii complex species are etiological agents of cryptococcosis, a systemic mycosis that cause respiratory infection and meningoencephalitis. To establish the infection, these yeasts produce virulence factors, such as melanin, which contribute to pathogenicity and antifungal tolerance. The aim of this study was to investigate melanin production by the C. neoformans/C. gattii complex in the presence of different precursors of melanogenesis and evaluate the effect of melanization on the antifungal susceptibility of these species to fluconazole, flucytosine and amphotericin B. Epinephrine, norepinephrine, dopamine and caffeic acid were used as substrates for melanin production, and l-dopa was used as positive control. The susceptibility of melanized strains (n = 6), after exposure to epinephrine or l-dopa, was evaluated by broth microdilution assay, and non-melanized strains were used as control. The antifungal activity of amphotericin B against melanized strains was also investigated by time kill assay. All Cryptococcus spp. strains produced melanin after exposure to the tested substrates. After exposure to epinephrine, minimum inhibitory concentration (MIC) ranges were 1-8 μg/mL for fluconazole, 2-8 μg/mL for flucytosine and 0.125-1 μg/mL for amphotericin B, while, after exposure to l-dopa, MIC ranges were 2-8 μg/mL for fluconazole, 4-8 μg/mL for flucytosine, and 0.125-0.5 μg/mL for amphotericin B. Similar results were observed for non-melanized strains. The production of melanin after exposure to epinephrine was higher than that induced by l-dopa. Melanized cells of both species were more tolerant to amphotericin B than the non-melanized control, emphasizing the importance of melanin production for fungal virulence.

Brilhante RSN ，Rocha MGD ，Oliveira JS ，Pereira-Neto WA ，Guedes GMM ，Cordeiro RA ，Sidrim JJC ，Rocha MFG ，Castelo-Branco DSCM ... -  《-》