Quantitative Analysis of Candida Cell Wall Components by Flow Cytometrywith Triple-Fluorescence Staining

This work was supported by the European Commission within the FP7 Framework Programme [Fungitect-Grant No 602125]. We also thank Thomas Sauer, Vienna Biocenter Campus (VBC), Austria, for technical support at the FACS facility of the MFPL, Karl Kuchler, MFPL-Department of Medical Biochemistry, Medical University of Vienna, Max F. Perutz Laboratories, Campus Vienna Biocenter, Vienna, Austria and Ernst Thuer, Centre for Genomic Regulation, Barcelona, Spain, for advice on statistical approaches. Neil Gow acknowledges the support of the Wellcome Trust and the MRC Centre for Medical MycologyPeer reviewedPublisher PD

The Candida albicans Histone Acetyltransferase Hat1 Regulates Stress Resistance and Virulence via Distinct Chromatin Assembly Pathways

Human fungal pathogens like Candida albicans respond to host immune surveillance by rapidly adapting their transcriptional programs. Chromatin assembly factors are involved in the regulation of stress genes by modulating the histone density at these loci. Here, we report a novel role for the chromatin assembly-associated histone acetyltransferase complex NuB4 in regulating oxidative stress resistance, antifungal drug tolerance and virulence in C. albicans. Strikingly, depletion of the NuB4 catalytic subunit, the histone acetyltransferase Hat1, markedly increases resistance to oxidative stress and tolerance to azole antifungals. Hydrogen peroxide resistance in cells lacking Hat1 results from higher induction rates of oxidative stress gene expression, accompanied by reduced histone density as well as subsequent increased RNA polymerase recruitment. Furthermore, hat1Delta/Delta cells, despite showing growth defects in vitro, display reduced susceptibility to reactive oxygen-mediated killing by innate immune cells. Thus, clearance from infected mice is delayed although cells lacking Hat1 are severely compromised in killing the host. Interestingly, increased oxidative stress resistance and azole tolerance are phenocopied by the loss of histone chaperone complexes CAF-1 and HIR, respectively, suggesting a central role for NuB4 in the delivery of histones destined for chromatin assembly via distinct pathways. Remarkably, the oxidative stress phenotype of hat1Delta/Delta cells is a species-specific trait only found in C. albicans and members of the CTG clade. The reduced azole susceptibility appears to be conserved in a wider range of fungi. Thus, our work demonstrates how highly conserved chromatin assembly pathways can acquire new functions in pathogenic fungi during coevolution with the host

Scientific Reports / Klebsiella pneumoniae prevents spore germination and hyphal development of Aspergillus species

Different bacteria and fungi live as commensal organisms as part of the human microbiota, but shifts to a pathogenic state potentially leading to septic infections commonly occur in immunocompromised individuals. Several studies have reported synergistic or antagonistic interactions between individual bacteria and fungi which might be of clinical relevance. Here, we present first evidence for the interaction between Klebsiella pneumoniae and several Aspergillus species including A. fumigatus, A. terreus, A. niger and A. flavus which cohabit in the lungs and the intestines. Microbiological and molecular methods were employed to investigate the interaction in vitro, and the results indicate that Klebsiella pneumoniae is able to prevent Aspergillus spp. spore germination and hyphal development. The inhibitory effect is reversible, as demonstrated by growth recovery of Aspergillus spp. upon inhibition or elimination of the bacteria, and is apparently dependent on the physical interaction with metabolically active bacteria. Molecular analysis of Klebsiella-Aspergillus interaction has shown upregulation of Aspergillus cell wall-related genes and downregulation of hyphae-related genes, suggesting that Klebsiella induces cell wall stress response mechanisms and suppresses filamentous growth. Characterization of polymicrobial interactions may provide the basis for improved clinical management of mixed infections by setting the stage for appropriate diagnostics and ultimately for optimized treatment strategies.(VLID)492857

The histone chaperone HIR maintains chromatin states to control nitrogen assimilation and fungal virulence

Adaptation to changing environments and immune evasion is pivotal for fitness of pathogens. Yet, the underlying mechanisms remain largely unknown. Adaptation is governed by dynamic transcriptional re-programming, which is tightly connected to chromatin architecture. Here, we report a pivotal role for the HIR histone chaperone complex in modulating virulence of the human fungal pathogen Candida albicans. Genetic ablation of HIR function alters chromatin accessibility linked to aberrant transcriptional responses to protein as nitrogen source. This accelerates metabolic adaptation and increases the release of extracellular proteases, which enables scavenging of alternative nitrogen sources. Furthermore, HIR controls fungal virulence, as HIR1 deletion leads to differential recognition by immune cells and hypervirulence in a mouse model of systemic infection. This work provides mechanistic insights into chromatin-coupled regulatory mechanisms that fine-tune pathogen gene expression and virulence. Furthermore, the data point toward the requirement of refined screening approaches to exploit chromatin modifications as antifungal strategies

Proline catabolism is a key factor facilitating Candida albicans pathogenicity

Candida albicans, the primary etiology of human mycoses, is well-adapted to catabolize proline to obtain energy to initiate morphological switching (yeast to hyphal) and for growth. We report that put1-/- and put2-/- strains, carrying defective Proline UTilization genes, display remarkable proline sensitivity with put2-/- mutants being hypersensitive due to the accumulation of the toxic intermediate pyrroline-5-carboxylate (P5C), which inhibits mitochondrial respiration. The put1-/- and put2-/- mutations attenuate virulence in Drosophila and murine candidemia models and decrease survival in human neutrophils and whole blood. Using intravital 2-photon microscopy and label-free non-linear imaging, we visualized the initial stages of C. albicans cells infecting a kidney in real-time, directly deep in the tissue of a living mouse, and observed morphological switching of wildtype but not of put2-/- cells. Multiple members of the Candida species complex, including C. auris, are capable of using proline as a sole energy source. Our results indicate that a tailored proline metabolic network tuned to the mammalian host environment is a key feature of opportunistic fungal pathogens