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The development of vaccines against fungi and other intracellular microbes is impeded in part by a lack of suitable adjuvants. While most current vaccines against infectious diseases preferentially induce production of antibodies, cellular immunity is essential for the resolution of fungal infections. Microbes such as fungi and Mycobacterium tuberculosis require Th17 and Th1 cells for resistance, and engage the C-type lectin receptors including Dectin-2. Herein, we discovered a novel Dectin-2 ligand, the glycoprotein Blastomyces Eng2 (Bl-Eng2). Bl-Eng2 triggers robust signaling in Dectin-2 reporter cells and induces IL-6 in human PBMC and BMDC from wild type but not Dectin-2-/- and Card9-/- mice. The addition of Bl-Eng2 to a pan-fungal subunit vaccine primed large numbers of Ag-specific Th17 and Th1 cells, augmented activation and killing of fungi by myeloid effector cells, and protected mice from lethal fungal challenge, revealing Bl-Eng2’s potency as a vaccine adjuvant. Thus, ligation of Dectin-2 by Bl-Eng-2 could be harnessed as a novel adjuvant strategy to protect against infectious diseases requiring cellular immunity., Author summary Despite several million new systemic fungal infections annually worldwide, there are no commercial vaccines available. The lack of appropriate adjuvants is one major impediment to developing safe and effective vaccines against infections with fungal pathogens. Current vaccines against infectious diseases preferentially induce protective antibodies, driven by adjuvants such as alum. While clonally-derived and adoptively transferred monoclonal antibodies may confer protection against fungi, the contribution of antibody to host defense is likely to be limited. Animal studies and clinical observations implicate cellular immunity as an essential component of the resolution of fungal infections. We found a promising adjuvant that augments cell mediated immune responses and vaccine-induced protection against fungal infection. We anticipate that our discovery will be a useful adjuvant for vaccination with non-replicating and safe subunit vaccines against many microbial pathogens that require protective cell mediated immune responses.

Phagocytosis by innate immune cells is an important process for protection against multiple pathologies and is particularly important for resistance to infection. However, phagocytosis has also been implicated in the progression of some diseases, including the dissemination of the human fungal pathogen, Cryptococcus neoformans. Previously, we identified Dectin-1 as a likely phagocytic receptor for C. neoformans spores through the use of soluble components in receptor-ligand blocking experiments. In this study, we used gain-of-function and loss-of-function assays with intact cells to evaluate the in vivo role of Dectin-1 and other C-type lectins in interactions with C. neoformans spores and discovered stark differences in outcome when compared with previous assays. First, we found that non-phagocytic cells expressing Dectin-1 were unable to bind spores and that highly sensitive reporter cells expressing Dectin-1 were not stimulated by spores. Second, we determined that some phagocytes from Dectin-1-/- mice interacted with spores differently than wild type (WT) cells, but the effects varied among assays and were modest overall. Third, while we detected small but statistically significant reductions in phagocytosis by primary alveolar macrophages from Dectin-1-/- mice compared to WT, we found no differences in survival between WT and Dectin-1-/- mice challenged with spores. Further analyses to assess the roles of other C-type lectins and their adapters revealed very weak stimulation of Dectin-2 reporter cells by spores and modest differences in binding and phagocytosis by Dectin-2-/- bone marrow-derived phagocytes. There were no discernable defects in binding or phagocytosis by phagocytes lacking Mannose Receptor, Mincle, Card-9, or FcRγ. Taken together, these results lead to the conclusion that Dectin-1 and other C-type lectins do not individually play a major roles in phagocytosis and innate defense by phagocytes against C. neoformans spores and highlight challenges in using soluble receptor/ligand blocking experiments to recapitulate biologically relevant interactions.

Many transcriptional regulators play roles in morphogenesis of the human pathogen Candida albicans. Recently, Sfl2, a sequence homolog of C. albicans Sfl1, has been shown to be required for hyphal development. In this report, we show that, like Sfl1, Sfl2 could complement the phenotypes of the Saccharomyces cerevisiae sfl1 mutant, and green fluorescent protein-tagged Sfl2 localized in the nuclei of both yeast and hyphal cells in C. albicans, reflecting its role as a transcriptional regulator. In C. albicans, SFL2 expression was induced at a high growth temperature (37 °C) at both transcriptional and translational levels. The deletion of SFL2 impaired filamentation at a high temperature, whereas the overexpression of SFL2 promoted filamentous growth at a low temperature. Sfl2-activated hyphal development needs the existence of Efg1 and Flo8 under aerobic conditions. Thus, in contrast to Sfl1, which represses filamentation, Sfl2 acts as an activator of filamentous growth in C. albicans. Functional analysis of chimeric Sfl proteins demonstrated that the opposite actions of C. albicans Sfl1 and Sfl2 were mainly mediated by their heat shock factor domains. Furthermore, the deletion of SFL2 attenuated virulence in a mouse model of gastrointestinal colonization and dissemination, indicating that Sfl2 is important for virulence in the gastrointestinal model of candidiasis. Our results provide new insights into Sfl2 functions in C. albicans morphogenesis and pathogenesis.

Candida albicans is the most common human fungal pathogen, yet is a normal commensal resident of the human gut. CO(2) levels in the gut are much higher than in air, and it is known that elevated CO(2) concentration promotes C. albicans cells to undergo a phenotypic switch from white to opaque phase. Wor1, the master regulator of opaque cell formation, is required for both the white to opaque transition and opaque maintenance. To elucidate the regulatory mechanism of Wor1, we set out to identify Wor1-interacting proteins using a yeast two-hybrid screen. A SUMO E3 ligase named Wos1 (Wor1 SUMO-ligase 1) was identified to interact with Wor1 and regulate Wor1 SUMOylation. WOS1 expression is upregulated in response to high CO(2), and the induction by CO(2) is dependent on the transcription factor Flo8. Under high CO(2) conditions, Wos1 is required for the white to opaque switch and acts downstream of Flo8. At atmospheric CO(2) levels, overexpression of Wos1 enhances Wor1 SUMOylation and promotes the white to opaque switch. Wor1 is found to be SUMOylated at lysine 385, and loss of this mark by point mutation leads to a defect in CO(2) -mediated opaque cell induction. Together, our genetic and biological data show that Wos1-mediated Wor1 SUMOylation contributes to the regulation of CO(2) -induced white to opaque switching as well as heritable maintenance of the opaque cell type.