The formation of cryptococcus neoformans biofilms contributes to the pyroptosis of cerebral microvascular endothelial cells Free

BackgroundCryptococcus neoformans primarily affects non-central nervous systems in immunocompetent individuals, causing pulmonary cryptococcosi. In immunocompromised patients, particularly those with AIDS, it can spread to the brain, leading to fatal cryptococcal meningitis. Biofilms, structural assemblies of microorganisms embedded in a polysaccharide matrix, are common in pathogenic fungi and exhibit resistance to antifungal agents and host defenses. Clinically significant, approximately 65% of infections involve biofilm formation on tissue surfaces, organ surfaces. Cryptococcus neoformans biofilms induce inflammatory cytokines IL-1β and activate caspase-1 in immune cells via NLRP3 signaling. Disruption of the PMT4 gene, which regulates biofilm formation in Cryptococcus neoformans, leads to substantial biofilm degradation, thereby reducing antifungal resistance and improving patient outcomes. Previous research showed Cryptococcus neoformans induces pyroptosis in cerebral endothelial cells, and this study investigates the virulence factors and biofilm's role in this pathway.

MethodsCryptococcus neoformans was resuscitated in culture medium and subcultured for stabilization. Cells were suspended in medium at a concentration of 10^7/ml. Cryptococcus neoformans (10^7/ml) was inoculated into 6-well plates, and the control group was cultured in medium at 37°C for 48 hours without agitation. Following the completion of the adhesion phase, the wells were gently washed five times with PBS to remove unattached cells. At this stage, the remaining cells adhering to the plate base were considered as the biofilm. The number of residual Cryptococcus neoformans was counted by the cell counting plate. Brain microvascular endothelial cells were treated with Cryptococcus neoformans biofilms and free Cryptococcus neoformans for 12 hours. Conduct CCK8 detection for cell viability and viable cells number examined using bisBenzimide H 33342 trihydrochloride. Supernatants were collected for ELISA analysis, while cells were lysed with RIPA buffer for protein extraction and subsequent Western blotting. Image J software was utilized for analysis, and SPSS (IBM SPSS Statistics 26) was employed for statistical evaluations, with data analyzed using one-way ANOVA.

Results Live cell staining was performed and examined using bisBenzimide H 33342 trihydrochloride. The results showed that there was no statistically significant difference in the number of viable cells between the Cryptococcus neoformans treatment group alone and the control group, while the number of viable cells in the Cryptococcus neoformans biofilm group decreased significantly compared with the blank control group(P<0.01). Interestingly, the results of the CCK8 cell viability assay revealed that the cell survival rate of the free Cryptococcus neoformans group was significantly higher than that of the Cryptococcus neoformans biofilm group (P<0.05). Following biofilm treatment, the expression levels of pyroptosis-related proteins were significantly upregulated, including NLRP3 (P<0.05), NT-GSDMD/GSDMD (P<0.01), and P20/Caspase1 (P<0.01). Notably, the inflammatory cytokines associated with pyroptosis, IL-18 (P<0.001) and IL-1β (P<0.001), exhibited marked increases in their expression levels.

Conclusion In this study, we designed an in vitro infection model utilizing Cryptococcus neoformans biofilms and employed free-state Cryptococcus neoformans as a control for comparison. The hypothesis was examined comprehensively at both cytokine and protein expression levels. Our findings demonstrated that the Cryptococcus neoformans biofilm significantly enhanced the expression of NLRP3, Caspase 1, and NT-GSDMD, accompanied by a substantial upregulation of pro-inflammatory cytokines IL-18 and IL-1β. These molecular changes were associated with pyroptosis in cerebral microvascular endothelial cells. These findings indicate that Cryptococcus neoformans biofilm is a key factor in pyroptosis of cerebral microvascular endothelial cells, revealing the mechanism and offering new strategies for preventing and treating cryptococcal meningitis. These findings underscore the importance of targeting biofilm-associated pathways as a potential therapeutic strategy against this devastating disease. (Acknowledgements: This study was supported by Grant from School of Public Health of Southern Medical University, China, Grant No.GW202431; Corresponding author: Hong Cao, gzhcao@smu.edu.cn).