Fluorescent antimicrobial peptides are promising structures for in situ, real-time imaging of fungal infection. Here we report a fluorogenic probe to image Aspergillus fumigatus directly in human pulmonary tissue. We have developed a fluorogenic Trp-BODIPY amino acid with a spacer-free C-C linkage between Trp and a BODIPY fluorogen, which shows remarkable fluorescence enhancement in hydrophobic microenvironments. The incorporation of our fluorogenic amino acid in short antimicrobial peptides does not impair their selectivity for fungal cells, and enables rapid and direct fungal imaging without any washing steps. We have optimized the stability of our probes in human samples to perform multi-photon imaging of A. fumigatus in ex vivo human tissue. The incorporation of our unique BODIPY fluorogen in biologically relevant peptides will accelerate the development of novel imaging probes with high sensitivity and specificity.

To sustain iron homeostasis, microorganisms have evolved fine-tuned mechanisms for uptake, storage and detoxification of the essential metal iron. In the human pathogen Aspergillus fumigatus, the fungal-specific bZIP-type transcription factor HapX coordinates adaption to both iron starvation and iron excess and is thereby crucial for virulence. Previous studies indicated that a HapX homodimer interacts with the CCAAT-binding complex (CBC) to cooperatively bind bipartite DNA motifs; however, the mode of HapX-DNA recognition had not been resolved. Here, combination of in vivo (genetics and ChIP-seq), in vitro (surface plasmon resonance) and phylogenetic analyses identified an astonishing plasticity of CBC:HapX:DNA interaction. DNA motifs recognized by the CBC:HapX protein complex comprise a bipartite DNA binding site 5′-CSAATN12RWT-3′ and an additional 5′-TKAN-3′ motif positioned 11–23 bp downstream of the CCAAT motif, i.e. occasionally overlapping the 3′-end of the bipartite binding site. Phylogenetic comparison taking advantage of 20 resolved Aspergillus species genomes revealed that DNA recognition by the CBC:HapX complex shows promoter-specific cross-species conservation rather than regulon-specific conservation. Moreover, we show that CBC:HapX interaction is absolutely required for all known functions of HapX. The plasticity of the CBC:HapX:DNA interaction permits fine tuning of CBC:HapX binding specificities that could support adaptation of pathogens to their host niches.

Filamentous fungi differentiate along complex developmental programs directed by abiotic and biotic signals. Currently, intrinsic signals that govern fungal development remain largely unknown. Here we show that an endogenously produced and secreted fungal oxylipin, 5,8-diHODE, induces fungal cellular differentiation, including lateral branching in pathogenic Aspergillus fumigatus and Aspergillus flavus, and appressorium formation in the rice blast pathogen Magnaporthe grisea. The Aspergillus branching response is specific to a subset of oxylipins and is signaled through G-protein coupled receptors. RNA-Seq profiling shows differential expression of many transcription factors in response to 5,8-diHODE. Screening of null mutants of 33 of those transcription factors identifies three transcriptional regulators that appear to mediate the Aspergillus branching response; one of the mutants is locked in a hypo-branching phenotype, while the other two mutants display a hyper-branching phenotype. Our work reveals an endogenous signal that triggers crucial developmental processes in filamentous fungi, and opens new avenues for research on the morphogenesis of filamentous fungi.

Highlights In vitro assembled CRISPR/Cas9-gRNA complexes can mediate selection free transformation. Single gRNAs can be used to target mutagenesis effectively. Targeted point mutations can be achieved utilising single strand DNA oligonucleotides as repair template.