Cathelicidin-inspired antimicrobial peptides as novel antifungal compounds.

Fungal infections in humans are increasing worldwide and are currently mostly treated with a relative limited set of antifungals. Resistance to antifungals is increasing, for example, in Aspergillus fumigatus and Candida auris, and expected to increase for many medically relevant fungal species in the near future. We have developed and patented a set of cathelicidin-inspired antimicrobial peptides termed 'PepBiotics'. These peptides were initially selected for their bactericidal activity against clinically relevant Pseudomonas aeruginosa and Staphylococcus aureus isolates derived from patients with cystic fibrosis and are active against a wide range of bacteria (ESKAPE pathogens). We now report results from studies that were designed to investigate the antifungal activity of PepBiotics against a set of medically relevant species encompassing species of Aspergillus, Candida, Cryptococcus, Fusarium, Malassezia, and Talaromyces. We characterized a subset of PepBiotics and show that these peptides strongly affected metabolic activity and/or growth of a set of medically relevant fungal species, including azole-resistant A. fumigatus isolates. PepBiotics showed a strong inhibitory activity against a large variety of filamentous fungi and yeasts species at low concentrations (≤1 µM) and were fungicidal for at least a subset of these fungal species. Interestingly, the concentration of PepBiotics required to interfere with growth or metabolic activity varied between different fungal species or even between isolates of the same fungal species. This study shows that PepBiotics display strong potential for use as novel antifungal compounds to fight a large variety of clinically relevant fungal species.

Expression profile analysis reveals that Aspergillus fumigatus but not Aspergillus niger makes type II epithelial lung cells less immunological alert.

BACKGROUND: Aspergillus fumigatus is the main causative agent of aspergillosis. Infections rarely occur in immunocompetent individuals, indicating efficient clearance of conidia by pulmonary defense mechanisms. Other aspergilli like Aspergillus niger also cause infections but to a much lesser extent. Our previous studies showed that A. fumigatus and A. niger have different behavior in the presence of type II alveolar A549 epithelial cells. A. fumigatus conidia are more efficiently internalized by these cells and germination is delayed when compared to A. niger. In addition, hyphae that have escaped the epithelial cells grow parallel to the epithelium, while A. niger grows away from this cell layer. RESULTS: Here it is shown that global gene expression of A. fumigatus and A. niger is markedly different upon contact with A549 cells. A total of 545 and 473 genes of A. fumigatus and A. niger, respectively, were differentially expressed when compared to growth in the absence of A549 cells. Notably, only 53 genes (approximately 10%) were shared in these gene sets. The different response was also illustrated by the fact that only 4 out of 75 GO terms were shared that were enriched in the differentially expressed gene sets. The orthologues of A. fumigatus genes involved in hypoxia regulation and heat shock were also up-regulated in A. niger, whereas thioredoxin reductase and allergen genes were found up-regulated in A. fumigatus but down-regulated in A. niger. Infection with A. fumigatus resulted in only 62 up and 47 down-regulated genes in A549. These numbers were 17 and 34 in the case of A. niger. GO terms related with immune response were down-regulated upon exposure to A. fumigatus but not in the case of A. niger. This indicates that A. fumigatus reprograms A549 to be less immunologically alert. CONCLUSIONS: Our dual transcriptomic analysis supports earlier observations of a marked difference in life style between A. fumigatus and A. niger when grown in the presence of type II epithelial cells. The results indicate important differences in gene expression, amongst others down regulation of immune response genes in lung epithelial cells by A. fumigatus but not by A niger.

Comparative genotyping and phenotyping of Aspergillus fumigatus isolates from humans, dogs and the environment.

BACKGROUND: Aspergillus fumigatus is a ubiquitous saprotrophic fungus and an opportunistic pathogen of humans and animals. Humans and animals can inhale hundreds of A. fumigatus spores daily. Normally this is harmless for humans, but in case of immunodeficiency, invasive pulmonary aspergillosis (IPA) can develop with a high mortality rate. A. fumigatus also causes non-invasive mycoses like sino-nasal aspergillosis (SNA) in dogs. RESULTS: In this study we compared A. fumigatus isolates from humans with suspected IPA, dogs with SNA, and a set of environmental isolates. Phylogenetic inference based on calmodulin (CaM) and beta-tubulin (benA) sequences did not reveal A. fumigatus sub-groups linked to the origin of the isolates. Genotyping and microsatellite analysis showed that each dog was infected by one A. fumigatus genotype, whereas human patients had mixed infections. Azole resistance was determined by antifungal susceptibility testing and sequencing of the cyp51A gene. A total of 12 out of 29 human isolates and 1 out of 27 environmental isolates were azole resistant. Of the azole resistant strains, 11 human isolates showed TR34/L98H (n = 6) or TR46/Y121F/T289A (n = 5). Phenotypically, isolates from dogs were more variable in growth speed and morphology when compared to those isolated from human and the environment. CONCLUSIONS: 1. A. fumigatus from dogs with SNA are phenotypically very diverse in contrast to their environmental and human counterparts. 2. Phenotypic variability can be induced during the chronic infection process in the sinus of the dogs. The basis of this heterogeneity might be due to genomic differences and/or epigenetic variations. 3. Differences in dogs is a could be a result of within-host adaption and might be triggered by environmental factors in the sinus, however this hypothesis still needs to be tested.

The Transcription Factor Roc1 Is a Key Regulator of Cellulose Degradation in the Wood-Decaying Mushroom Schizophyllum commune.

Wood-decaying fungi of the class Agaricomycetes (phylum Basidiomycota) are saprotrophs that break down lignocellulose and play an important role in nutrient recycling. They secrete a wide range of extracellular plant cell wall degrading enzymes that break down cellulose, hemicellulose, and lignin, the main building blocks of plant biomass. Although the production of these enzymes is regulated mainly at the transcriptional level, no activating regulators have been identified in any wood-decaying fungus in the class Agaricomycetes. We studied the regulation of cellulase expression in the wood-decaying fungus Schizophyllum commune. Comparative genomics and transcriptomics on two wild isolates revealed a Zn2Cys6-type transcription factor gene (roc1) that was highly upregulated during growth on cellulose, compared to glucose. It is only conserved in the class Agaricomycetes. A roc1 knockout strain showed an inability to grow on medium with cellulose as sole carbon source, and growth on cellobiose and xylan (other components of wood) was inhibited. Growth on non-wood-related carbon sources was not inhibited. Cellulase gene expression and enzyme activity were reduced in the Δroc1 strain. ChIP-Seq identified 1474 binding sites of the Roc1 transcription factor. Promoters of genes involved in lignocellulose degradation were enriched with these binding sites, especially those of LPMO (lytic polysaccharide monooxygenase) CAZymes, indicating that Roc1 directly regulates these genes. A conserved motif was identified as the binding site of Roc1, which was confirmed by a functional promoter analysis. Together, Roc1 is a key regulator of cellulose degradation and the first identified in wood-decaying fungi in the phylum Basidiomycota. IMPORTANCE Wood-degrading fungi in the phylum Basidiomycota play a crucial role in nutrient recycling by breaking down all components of wood. Fungi have evolved transcriptional networks that regulate expression of wood-degrading enzymes, allowing them to prioritize one nutrient source over another. However, to date all these transcription factors have been identified in the phylum Ascomycota, which is only distantly related to the phylum Basidiomycota. Here, we identified the transcription factor Roc1 as a key regulator of cellulose degradation in the mushroom-forming and wood-degrading fungus Schizophyllum commune. Roc1 is highly conserved in the phylum Basidiomycota. Using comparative genomics, transcriptomics, ChIP-Seq and promoter analysis we have identified direct targets of Roc1, as well as other aspects of the transcriptional response to cellulose.