Aspartic Proteases and Major Cell Wall Components in Candida albicans Trigger the Release of Neutrophil Extracellular Traps

Abstract

Neutrophils use different mechanisms to cope with pathogens that invade the host organism. The most intriguing of these responses is a release of neutrophil extracellular traps (NETs) composed of decondensed chromatin and granular proteins with antimicrobial activity. An important potential target of NETs is Candida albicans—an opportunistic fungal pathogen that employs morphological and phenotype switches and biofilm formation during contact with neutrophils, accompanied by changes in epitope exposition that mask the pathogen from host recognition. These processes differ depending on infection conditions and are thus influenced by the surrounding environment. In the current study, we compared the NET release by neutrophils upon contact with purified main candidal cell surface components. We show here for the first time that in addition to the main cell wall-building polysaccharides (mannans and β-glucans), secreted aspartic proteases (Saps) trigger NETs with variable intensities. The most efficient NET-releasing response is with Sap4 and Sap6, which are known to be secreted by fungal hyphae. This involves mixed, ROS-dependent and ROS-independent signaling pathways, mainly through interactions with the CD11b receptor. In comparison, upon contact with the cell wall-bound Sap9 and Sap10, neutrophils responded via a ROS-dependent mechanism using CD16 and CD18 receptors for protease recognition. In addition to the Saps tested, the actuation of selected mediating kinases (Src, Syk, PI3K, and ERK) was also investigated. β-Glucans were found to trigger a ROS-dependent process of NET production with engagement of Dectin-1 as well as CD11b and CD18 receptors. Mannans were observed to be recognized by TLRs, CD14, and Dectin-1 receptors and triggered NET release mainly via a ROS-independent pathway. Our results thus strongly suggest that neutrophils activate NET production in response to different candidal components that are presented locally at low concentrations at the initial stages of infection. However, NET release seemed to be blocked by increasing numbers of fungal cells.

Bradykinin B2 receptor and dopamine D2 receptor cooperatively contribute to the regulation of neutrophil adhesion to endothelial cells*

“…Neutrophil adhesion to endothelial cells. Human polymorphonuclear cells (PMN) were isolated as described previously (Zawrotniak et al, 2017) from freshly EDTA-treated whole-blood samples obtained from the Regional Blood Donation Center (Krakow, Poland), which complies with the requisite of confidentiality assurance for human participants. Isolated PMN were suspended in RPMI-1640 medium and loaded with Cell Tracker Red CMTPX in RPMI for 30 min at 37°C, following the manufacturer's instructions, and then placed on a monolayer of endothelial cells stimulated as described below.…”

Extracellular proteinases of Candida species pathogenic yeasts

“…). Recently, we found that SAP genes are overexpressed in yeast during contact with NET‐forming neutrophils and that Saps can trigger NET release …”

“…Recently, we found that SAP genes are overexpressed in yeast during contact with NET-forming neutrophils 87 and that Saps can trigger NET release. 120 Candida albicans extracellular proteinases can induce the expression of cytokines in host immune cells. In macrophages, Sap1-Sap3…”

Interactions of Candida albicans Cells with Aerobic and Anaerobic Bacteria during Formation of Mixed Biofilms in the Oral Cavity

“…Such an argument was also strengthened by the observation that pre-treatment of PMNs with interferon-γ or granulocyte colony-stimulating factor (G-CSF) did not significantly enhance their activity against opsonized or nonopsonized C. albicans biofilms. Moreover, neutrophils contacting the mature biofilm did not produce reactive oxygen species, necessary for triggering of phagocytosis, or one of the pathways of NET production [99]. Nevertheless, the precise mechanism of this phenomenon remained to be clarified.…”