Fungal infections remain a threat due to the lack of broad spectrum fungal vaccines and protective antigens. Recent studies showed that attenuated Blastomyces dermatitidis confers protection via T cell recognition of an unknown, but conserved antigen. Using transgenic CD4+ T cells recognizing this antigen, we identify an amino acid determinant within the chaperone calnexin that is conserved across diverse fungal ascomycetes. Calnexin, typically an ER protein, also localizes to the surface of yeast, hyphae and spores. T cell epitope mapping unveiled a 13-residue sequence conserved across Ascomycota. Infection with divergent ascomycetes including dimorphic fungi, opportunistic molds, and the agent causing white nose syndrome in bats induces expansion of calnexin-specific CD4+ T cells. Vaccine delivery of calnexin in glucan particles induces fungal antigen-specific CD4+ T cell expansion and resistance to lethal challenge with multiple fungal pathogens. Thus, the immunogeneticity and conservation of calnexin make this fungal protein a promising vaccine target.

Pythiosis is a life-threatening infectious disease caused by the oomycete Pythium insidiosum. Direct exposure to Py. insidiosum zoospores can initiate infections of the eye, limb, gastrointestinal tract, or skin/subcutaneous tissue. Treatments for pythiosis have mostly relied on surgery. Antifungal drugs are generally ineffective against Py. insidiosum. However, one patient with an invasive Py. insidiosum infection recovered completely following treatment with terbinafine and itraconazole. Additionally, the drug target sterol biosynthetic enzymes have been identified in the oomycete Aphanomyces euteiches. It remains an open question whether Py. insidiosum is susceptible to the antifungal drugs and harbors any of the known drug target enzymes. Here, we determined the in vitro susceptibilities of terbinafine and itraconazole against 30 isolates of Py. insidiosum. We also analyzed endogenous sterols and searched for genes encoding the sterol biosynthetic enzymes in the genomes of Py. insidiosum and related oomycetes. The susceptibility assay showed that the growth of each of the Py. insidiosum isolates was inhibited by the antifungal agents, but only at difficult-to-achieve concentrations, which explains the clinical resistance of the drugs in the treatment of pythiosis patients. Genome searches of Py. insidiosum and related oomycetes demonstrated that these organisms contained an incomplete set of sterol biosynthetic enzymes. Gas chromatographic mass spectrometry did not detect any sterol end products in Py. insidiosum. In conclusion, Py. insidiosum possesses an incomplete sterol biosynthetic pathway. Resistance to antifungal drugs targeting enzymes in the ergosterol biosynthetic pathway in Py. insidiosum was due to modifications or losses of some of the genes encoding the drug target enzymes.

Chromoblastomycosis is a chronic skin infection caused by the pigmented saprophytic mould Fonsecaea pedrosoi. Chronicity of infection can be broken by a coordinated innate recognition of the spores by pattern recognition receptors. While Mincle signaling via theKeywords: Chromoblastomycosis r C-type lectin r Fungi r Dectin-2 r Mincle r T-cell differentiation r Th17 cell Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionChromoblastomycosis is a chronic progressive fungal infection of mammalian skin and subcutaneous tissue that is caused by which gradually enlarge and become verrucous nodules and psoriasis like plaques that may extend as satellites along the lymphatics or disseminate through scratching [5]. Chromoblastomycosis treatment is difficult and most therapeutic attempts provide only a modest rate of success [6,7].Little is known about the protective host defense mechanisms against chromoblastomycosis. Innate immunity mediated by neutrophils and macrophages is thought to be principally responsible for host protection [2,3]. However, the chronic nature of infection with F. pedrosoi may be due to an inappropriate innate immune response [8]. F. pedrosoi is recognized by the C-type lectin receptors (CLRs) Dectin-1 and Mincle, yet fails to induce the production of proinflammatory TNF-α by macrophages and DCs. Inflammatory responses to F. pedrosoi and clearance of infection can be reinstated by exogenous TLR costimulation [8]. Imiquimod (TLR7 ligand) and LPS (TLR4 ligand) application augments TNF-α production and accelerates healing in murine models [8] TLR costimulation does not augment Ag-specific T-cell development in F. pedrosoi-infected miceWe have previously shown that F. pedrosoi spores alone failed to induce innate inflammatory responses by macrophages. The failure of innate recognition could be reinstated by TLR costimulation [8]. To explore whether Ag-specific T-cell responses can be augmented by TLR costimulation we injected F. pedrosoi spore-infected mice with LPS as described [8]. Surprisingly, LPS (TLR4 agonist) and Imiquimod (TLR7 agonist) treatment did not increase the activation (CD44), expansion, and differentiation of Ag-specific 1807 cells as indicated by the numbers and frequencies of activated (CD44 + ) and cytokine producing (IFN-γ and IL-17) 1807 cells (Fig. 1A-E). Thus, unlike innate inflammatory responses, the generation of adaptive cell-mediated immune responses could not be augmented by costimulation of the TLR pathway. sought to investigate whether surface expressed CLRs are involved in the innate recognition of the fungal spores. We cocultured live F. pedrosoi spores with CLR transformed B3Z T-cell hybridoma cells expressing an NFAT-lacZ β-galatosidase reporter of ITAM signaling [15]. In response to spore stimulation, lacZ activity was increased in reporter cells expressing Dectin-1 and Dectin-2 and to a lesser extent MCL (Dectin-3, Clecsf8, or Clec4d) and Mincle, but not in cells expressing FcRγ only (Fig....

Pythium insidiosum is an oomycete that causes a life-threatening infectious disease called pythiosis in humans and animals living in tropical and subtropical countries. Here, we report the first draft genome sequence of P. insidiosum. The genome of P. insidiosum is 53.2 Mb and contains 14,962 open reading frames.