Dimorphic fungi cause several endemic mycoses which range from subclinical respiratory infections to life-threatening systemic disease. Pathogenic-phase cells of Histoplasma, Blastomyces, Paracoccidioides and Coccidioides escape elimination by the innate immune response with control ultimately requiring activation of cell-mediated immunity. Clinical management of disease relies primarily on antifungal compounds; however, dimorphic fungal pathogens create a number of challenges for antifungal drug therapy. In addition to the drug toxicity issues known for current antifungals, barriers to efficient drug treatment of dimorphic fungal infections include natural resistance to the echinocandins, residence of fungal cells within immune cells, the requirement for systemic delivery of drugs, prolonged treatment times, potential for latent infections, and lack of optimized standardized methodology for in vitro testing of drug susceptibilities. This review will highlight recent advances, current therapeutic options, and new compounds on the horizon for treating infections by dimorphic fungal pathogens.

bStandardized methodologies for determining the antifungal susceptibility of fungal pathogens is central to the clinical management of invasive fungal disease. Yeast-form fungi can be tested using broth macrodilution and microdilution assays. Reference procedures exist for Candida species and Cryptococcus yeasts; however, no standardized methods have been developed for testing the antifungal susceptibility of yeast forms of the dimorphic systemic fungal pathogens. For the dimorphic fungal pathogen Histoplasma capsulatum, susceptibility to echinocandins differs for the yeast and the filamentous forms, which highlights the need to employ Histoplasma yeasts, not hyphae, in antifungal susceptibility tests. To address this, we developed and optimized methodology for the 96-well microtiter plate-based measurement of Histoplasma yeast growth in vitro. Using optical density, the assay is quantitative for fungal growth with a dynamic range greater than 30-fold. Concentration and assay reaction time parameters were also optimized for colorimetric (MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction) and fluorescent (resazurin reduction) indicators of fungal vitality. We employed this microtiter-based assay to determine the antifungal susceptibility patterns of multiple clinical isolates of Histoplasma representing different phylogenetic groups. This methodology fulfills a critical need for the ability to monitor the effectiveness of antifungals on Histoplasma yeasts, the morphological form present in mammalian hosts and, thus, the form most relevant to disease.T he increasing incidence of fungal disease necessitates adequate and timely assessment of antifungal susceptibility to guide the selection and implementation of antifungal therapies. Consequently, the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) have established reference test methods for susceptibility to the major classes of antifungals, which include the polyenes, azoles, and the recently developed echinocandins (1, 2). These standards utilize broth macrodilution or microdilution assays for testing yeasts (M27-A3 [3] and E.DEF 7.1 [4]) and filamentous fungi (M38-A2 [5] and E.DEF 9.1 [6]). Procedures, including inoculum preparation, media, assay duration, and endpoint definitions, have now been established for testing some of the more commonly encountered yeast-form pathogenic fungi (i.e., Candida species and Cryptococcus). However, notably missing from the M27-A3 and E.DEF 7.1 standards for testing of yeasts are methods for testing the yeast forms of the dimorphic fungi.The dimorphic fungi that cause systemic disease are characterized by distinct yeast and filamentous morphological states (7,8). Temperature is the principal morphology-determining factor, with yeast forms characterizing infections in mammals (9). Histoplasma capsulatum is the most common clinically encountered dimorphic fungal pathogen in the United States, with an estimated 10,000 to 20,000...

Chitinases enzymatically hydrolyze chitin, a highly abundant and utilized polymer of N-acetyl-glucosamine. Fungi are a rich source of chitinases; however, the phylogenetic and functional diversity of fungal chitinases are not well understood. We surveyed fungal chitinases from 373 publicly available genomes, characterized domain architecture, and conducted phylogenetic analyses of the glycoside hydrolase (GH18) domain. This large-scale analysis does not support the previous division of fungal chitinases into three major clades (A, B, C) as chitinases previously assigned to the “C” clade are not resolved as distinct from the “A” clade. Fungal chitinase diversity was partly shaped by horizontal gene transfer, and at least one clade of bacterial origin occurs among chitinases previously assigned to the “B” clade. Furthermore, chitin-binding domains (including the LysM domain) do not define specific clades, but instead are found more broadly across clades of chitinases. To gain insight into biological function diversity, we characterized all eight chitinases (Cts) from the thermally dimorphic fungus, Histoplasma capsulatum: six A clade, one B clade, and one formerly classified C clade chitinases. Expression analyses showed variable induction of chitinase genes in the presence of chitin but preferential expression of CTS3 in the mycelial stage. Activity assays demonstrated that Cts1 (B-I), Cts2 (A-V), Cts3 (A-V), Cts4 (A-V) have endochitinase activities with varying degrees of chitobiosidase function. Cts6 (C-I) has activity consistent with N-acetyl-glucosaminidase exochitinase function and Cts8 (A-II) has chitobiase activity. These results suggest chitinase activity is variable even within subclades and that predictions of functionality require more sophisticated models.

Cryptococcosis is a highly lethal infection with limited drug choices, most of which are highly toxic or complicated by emerging antifungal resistance. There is a great need for new drug targets that are unique to the fungus.

Four new metabolites, 4-epi-citreoviridin (1), auransterol (3), and two analogues (2 and 4) of paxisterol ( 6), together with two known metabolites (15R*,20S*)-dihydroxyepisterol ( 5) and ( 6), were isolated from cultures of the fungal associate, Penicillium aurantiacobrunneum, of the lichen Niebla homalea, endemic to California and Baja California. The structures of all compounds were determined by comprehensive spectroscopic and spectrometric methods, as well as single-crystal X-ray diffraction for the determination of the absolute configuration of 3. Compound 1 showed selective cytotoxicity toward MCF-7 breast and A2780 ovarian cells with IC 50 values of 4.2 and 5.7 μM, respectively.