Expression of Secreted Aspartyl Proteinases in an Experimental Model of Candida albicans-Associated Denture Stomatitis.

PURPOSE: Candida albicans is known to produce secreted aspartyl proteinases (SAPs) to aid adhesion, invasion, and host tissue destruction. SAPs may contribute to denture stomatitis (DS) pathogenesis. The aim of this study was to develop an in vivo experimental model for Candida-associated DS that allows the analysis of SAP2, SAP5, and SAP9 expression by C. albicans from biofilm induced on the denture surface. MATERIALS AND METHODS: Thirty-five male Wistar rats were divided into three groups: control, denture, and denture/Candida group. The last two groups remained with dentures for 2, 4, and 6 days, with or without induced biofilm. SAP expression was concomitant with leukocyte counts as well as clinical and histological changes shown by animal palate. RESULTS: The signs observed at 4 days in the denture/Candida group were clinically closer to the Candida-associated DS, showing a significant increase of neutrophils and decrease of lymphocytes in peripheral blood, presence of inflammation signs on the palate similar to DS Newton type I, and fungal invasion in the epithelial layer. Accordingly, the denture/Candida group at 4 days presented the highest relative expression of all SAPs studied. CONCLUSION: The results showed a coincidence between SAP expression and clinical, microscopic, and blood data. Finally, the molecular findings were consistent with the virulence capacities of C. albicans from biofilm formed on the denture resin, which possibly allowed epithelial invasion by the fungus.

ChIP-seq reveals a role for CrzA in the Aspergillus fumigatus high-osmolarity glycerol response (HOG) signalling pathway.

Aspergillus fumigatus is an opportunistic pathogen and allergen of mammals. Calcium signalling is essential for A. fumigatus pathogenicity and is regulated by the CrzA transcription factor. We used ChIP-seq (Chromatin Immunoprecipitation DNA sequencing) to explore CrzA gene targets in A. fumigatus. In total, 165 potential binding peaks including 102 directly regulated genes were identified, resulting in the prediction of the A[GT][CG]CA[AC][AG] CrzA-binding motif. The 102 CrzA putatively regulated genes exhibited a diverse array of functions. The phkB (Afu3g12530) histidine kinase and the sskB (Afu1g10940) MAP kinase kinase kinase of the HOG (high-osmolarity glycerol response) pathway were regulated by CrzA. Several members of the two-component system (TCS) and the HOG pathway were more sensitive to calcium. CrzA::GFP was translocated to the nucleus upon osmotic stress. CrzA is important for the phosphorylation of the SakA MAPK in response to osmotic shock. The ΔsskB was more sensitive to CaCl2 , NaCl, and paraquat stress, while being avirulent in a murine model of invasive pulmonary aspergillosis. The presence of CaCl2 and osmotic stresses resulted in synergistic inhibition of ΔcrzA and ΔsskB growth. These results suggest there is a genetic interaction between the A. fumigatus calcineurin-CrzA and HOG pathway that is essential for full virulence.

Identification of the cell targets important for propolis-induced cell death in Candida albicans.

Candida albicans is the most common fungal pathogen of humans, forming both commensal and opportunistic pathogenic interactions, causing a variety of skin and soft tissue infections in healthy people. In immunocompromised patients C. albicans can result in invasive, systemic infections that are associated with a high incidence of mortality. Propolis is a complex mixture of several resinous substances which are collected from plants by bees. Here, we demonstrated the fungicidal activity of propolis against all three morphogenetic types of C. albicans and that propolis-induced cell death was mediated via metacaspase and Ras signaling. To identify genes that were involved in propolis tolerance, we screened ~800 C. albicans homozygous deletion mutants for decreased tolerance to propolis. Fifty-one mutant strains were identified as being hypersensitive to propolis including seventeen genes involved in cell adhesion, biofilm formation, filamentous growth, phenotypic switching and pathogenesis (HST7, GIN4, VPS34, HOG1, ISW2, SUV3, MDS3, HDA2, KAR3, YHB1, NUP85, CDC10, MNN9, ACE2, FKH2, and SNF5). We validated these results by showing that propolis inhibited the transition from yeast-like to hyphal growth. Propolis was shown to contain compounds that conferred fluorescent properties to C. albicans cells. Moreover, we have shown that a topical pharmaceutical preparation, based upon propolis, was able to control C. albicans infections in a mouse model for vulvovaginal candidiasis. Our results strongly indicate that propolis could be used as a strategy for controlling candidiasis.

Concanavalin-A induces IL-17 production during the course of Candida albicans infection.

In a previous study, our group verified that 100% of mice survived to a lethal dose of Candida albicans following pretreatment with concanavalin-A (Con-A) for 3 days. This work proposed to investigate whether treatment could mediate an adaptative immune response involving T(H) 17 cells. A significant increase in IL-17 levels at 6 h postinfection was observed and was maintained up to 18 h in the Con-A group, whereas in control mice, a reduction in this cytokine was verified. In addition, T(H) 17 cells develop in the presence of TGF-ß, IL-1 ß, and IL-6 that were increased significantly 2 h postinfection in Con-A-treated mice. Macrophages were involved in the process, engulfing greater numbers of yeast cells, and were activated through TNF-α and interferon-γ produced at significant levels at 2 h postinfection. A significant increase in IL-12 levels was also observed at 2 h postinfection. Thus, activated macrophages were probably more capable of killing and processing Candida antigens, signalizing an adaptative immune response. Macrophages from controls did not prevent yeast-to-hyphae transition and were partially destroyed, as shown in scanning microscopy. These results suggest that treatment with Con-A facilitated the triggering of T(H) 17 and T(H) 1 responses via IL-17 and IFN-γ production, leading to the resolution of C. albicans infection.

Apoptosis of phagocytic cells induced by Candida albicans and production of IL-10.

Macrophages co-incubated with Candida albicans strain CR1 in vitro showed early signs of apoptosis, but evolved to necrosis after 2 h. In this study, we investigated whether strain CR1 caused apoptosis or necrosis of macrophages after its inoculation into mice peritoneal cavity, and whether this correlated with the secretion of IL-10. Peritoneal macrophages from mice that received an inoculum of C. albicans CR1 showed signs of apoptosis and necrosis from 30 min to 2 h afterwards, whereas heat-killed C. albicans did not cause those effects. IL-10 production was low during the first 6 h post-infection, when macrophages predominated in the peritoneal exudate, whereas its higher production after 24 h correlated with an increase of neutrophils in the exudate. Treatment of CR1 with pepstatin (an inhibitor of proteinases) prevented the process of apoptosis and significantly reduced IL-10 production, suggesting that the increased production of IL-10 was caused by processes occurring during the initial phase of infection, such as apoptosis, necrosis and uptake of death cells.