Aspergillus fumigatus is the most commonly encountered mold pathogen of humans, predominantly infecting the respiratory system. Colonization and penetration of the lung alveolar epithelium is a key but poorly understood step in the infection process. This study focused on identifying the transcriptional and cell-signaling responses activated in A549 alveolar carcinoma cells incubated in the presence of A. fumigatus wild-type and ΔPrtT protease-deficient germinating conidia and culture filtrates (CF). Microarray analysis of exposed A549 cells identified distinct classes of genes whose expression is altered in the presence of germinating conidia and CF and suggested the involvement of both NFkB and MAPK signaling pathways in mediating the cellular response. Phosphoprotein analysis of A549 cells confirmed that JNK and ERK1/2 are phosphorylated in response to CF from wild-type A. fumigatus and not phosphorylated in response to CF from the ΔPrtT protease-deficient strain. Inhibition of JNK or ERK1/2 kinase activity substantially decreased CF-induced cell damage, including cell peeling, actin-cytoskeleton damage, and reduction in metabolic activity and necrotic death. These results suggest that inhibition of MAPK-mediated host responses to treatment with A. fumigatus CF decreases cellular damage, a finding with possible clinical implications.

cInvasive mycotic infections have become more common during recent decades, posing an increasing threat to public health. However, despite the growing needs, treatments for invasive fungal infections remain unsatisfactory and are limited to a small number of antifungals. The aim of this study was to identify novel fungal cell wall inhibitors from a library of small chemical compounds using a conditional protein kinase C (PKC)-expressing strain of Aspergillus nidulans sensitive to cell wall-active agents. Eight "hit" compounds affecting cell wall integrity were identified from a screen of 35,000 small chemical compounds. Five shared a common basic molecular structure of 4-chloro-6-arylamino-7-nitro-benzofurazane (CANBEF). The most potent compound, CANBEF-24, was characterized further and was shown to inhibit the growth of pathogenic Aspergillus, Candida, Fusarium, and Rhizopus isolates at micromolar concentrations but not to affect the growth of mammalian cell lines. CANBEF-24 demonstrated strong synergy in combination with caspofungin, an antifungal that inhibits cell wall biosynthesis. Genetic and biochemical analyses with Aspergillus nidulans and Saccharomyces cerevisiae indicated that CANBEFs selectively inhibit fungal rRNA maturation and protein synthesis, suggesting that their effect on the cell wall is indirect. CANBEFs were nontoxic in insect (Galleria mellonella, Drosophila melanogaster) and mouse models of fungal infection. Preliminary evidence showing no therapeutic benefit in these models suggests that further cycles of optimization are needed for the development of this novel class of compounds for systemic use.T he number of life-threatening invasive fungal infections has risen dramatically over the past 30 years (1, 2). The vast majority of these infections are caused by species of the genera Candida, Aspergillus, Cryptococcus, and Coccidioides (3). Invasive aspergillosis has now overtaken candidiasis as the most frequent invasive fungal infection found after death in Europe and the United States (4, 5). Today, as many as 4% of all patients dying in modern tertiary care hospitals have invasive aspergillosis caused by fungal pathogen species that belong to the genus Aspergillus, while as many as 2% of these patients suffer from invasive candidiasis caused by Candida species (4, 6). However, despite the growing needs, treatments for invasive fungal infections remain unsatisfactory.There are three main classes of antifungal drugs in common clinical use for the treatment of systemic mycoses: the polyene amphotericin B, which binds fungal membrane ergosterol, leading to cell lysis; azoles, which inhibit ergosterol biosynthesis (fluconazole, itraconazole, voriconazole [VRC], and posaconazole); and the newly introduced echinocandins, such as caspofungin (CAS), which inhibit fungal glucan biosynthesis. Most of these current systemic antifungal treatments interact unfavorably with other medications, have resistance problems, a narrow spectrum of activity, and limited formulations, and are fungistatic rather...

Further development and in vivo validation of haemofungin is warranted.

This work demonstrates the feasibility of using a conditional Aspergillus mutant to conduct a small-molecule library screen to identify novel 'hit' compounds affecting cell wall integrity.

Combinations of caspofungin (CAS) with amphotericin-B (AMB), voriconazole (VRC), terbinafine (TRB) and tacrolimus (FK-506) were tested in vitro with 10 Fusarium isolates. MIC and minimal effective concentrations (MEC) were investigated in accord with the CLSI methodology. Drug interactions were assessed by the fractional inhibitory concentration index (FICI). Synergy occurred in 10/10, 9/10, 7/10 and 4/10 isolates with CAS/FK-506, CAS/TRB, CAS/AMB and CAS/VRC, respectively. Caspofungin MECs reached clinically attainable concentrations with FK-506 and TRB. Hyphal length and DiBAC staining demonstrated enhanced inhibition and killing with CAS/FK-506 and CAS/TRB. The combination of CAS/TRB and CAS/FK-506 is strongly synergistic in vitro against Fusarium spp. Our finding should be further studied in animal models of invasive infections caused by this fungus.