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Exploitation of the RNA interference pathway of the human fungal pathogen Aspergillus fumigatus

ABSTRACT: RNA-based therapeutics are an emerging class of drugs that are changing the way we treat infectious diseases. The versatility of this class is clearly on display in the rapid design and adaptation of the mRNA vaccines to SARS-CoV-2. Despite the success of these therapeutics against viruses, research into RNA-based therapeutics against human fungal pathogens, a major source of human morbidity and mortality, is lacking. Here, we provide proof-of-principle that RNA-based therapeutics hold potential against human fungal pathogens like Aspergillus fumigatus. We provide an improved mechanistic description of the RNA interference pathway of this important human pathogen by describing the genetic variation in RNAi-related genes using a large collection of environmental and clinical genomes, the proteins regulated by the system using advanced proteomics analysis, and the RNAi components essential for hairpin-induced silencing. We then exploit this pathway using a heterologously expressed hairpin RNA construct to silence the pabA gene of A. fumigatus to inhibit growth. The data presented here provide a foundation for a mechanistic description of novel RNA regulatory pathways in A. fumigatus and provide a first step towards the development of RNA-based therapeutics against this important human fungal pathogen.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Neosartorya Fumigata (aspergillus Fumigatus)

TISSUE(S): Spore

SUBMITTER: Thomas Krüger

LAB HEAD: Axel Brakhage

PROVIDER: PXD033984 | Pride | 2023-02-01

REPOSITORIES: Pride

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			Action	DRS
	1-WT-a.raw	Raw
	1-WT-b.raw	Raw
	1-WT-c.raw	Raw
	1-agoAB-a.raw	Raw
	1-agoAB-b.raw	Raw

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Functional analysis of the Aspergillus fumigatus kinome reveals a DYRK kinase involved in septal plugging is a novel antifungal drug target

Project description:More than 10 million people suffer from lung diseases caused by the pathogenic fungus Aspergillus fumigatus. The azole class of antifungals represent first line therapeutics for most of these infections however resistance is rising. Identification of novel antifungal targets that, when inhibited, synergise with the azoles will aid the development of agents that can improve therapeutic outcomes and supress the emergence of resistance. As part of the A. fumigatus genome-wide knockout program (COFUN), we have completed the generation of a library that consists of 120 genetically barcoded null mutants in genes that encode the protein kinase cohort of A. fumigatus. We have employed a competitive fitness profiling approach (Bar-Seq), to identify targets which when deleted result in hypersensitivity to the azoles and fitness defects in a murine host. The most promising candidate from our screen is a previously uncharacterised DYRK kinase orthologous to Yak1 of Candida albicans, a TOR signalling pathway kinase involved in modulation of stress responsive transcriptional regulators. Here we show that the orthologue YakA has been repurposed in A. fumigatus to regulate blocking of the septal pore upon exposure to stress via phosphorylation of the Woronin body tethering protein Lah. Loss of YakA function reduces the ability of A. fumigatus to penetrate solid media and impacts growth in murine lung tissue. We also show that 1-ethoxycarbonyl-beta-carboline (1-ECBC), a compound previously shown to inhibit Yak1 in C. albicans prevents stress mediated septal spore blocking and synergises with the azoles to inhibit A. fumigatus growth.

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A phylogenomic approach to explore the Aspergillus fumigatus conidia surforme and its role in pathogenesis

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Differentiated PLB-985 cells as a model system for Aspergillus fumigatus infection

Project description:Fungal infections remain a tremendous source of global morbidity and mortality, with more than 1 billion individuals affected by fungal infections each year. Invasive infections are particularly dangerous and kill numbers on par with tuberculosis or malaria. Aspergillus fumigatus is a major source of invasive fungal infections in humans, and is particularly dangerous to the immunocompromised. In many cases, research into invasive fungal infections is limited by a lack of model systems. In this study we hypothesized that cultured, differentiated human PLB-985 neutrophil-like cells could serve as model system to study the host-pathogenesis of A. fumigatus. We tested this hypothesis by defining the phagocytosis and intracellular trafficking of A. fumigatus conidia by PLB-985 cells, the production of neutrophil extracellular traps, and by characterizing extracellular vesicles produced in response to infection. As part of this analysis, we performed a detailed LC-MS/MS-based proteomic comparison of extracellular vesicles isolated by two different methods, centrifugation-based isolation or size-exclusion chromatography. We find numerous similarities between primary neutrophils and PLB-985 cells that suggest differentiated PLB-985 cells can serve as a tractable in vitro model system for further study of many aspects of A. fumigatus pathogenesis.

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Augmented enterocyte damage during Candida albicans and Proteus mirabilis coinfection

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Project description:Increasing antifungal drug resistance is a major concern associated with human fungal infections. Genetic mutation and epimutation mechanisms are clearly linked to resistance in some fungi, but few studies have investigated RNA modifications (the epitranscriptome) as a mediator of resistance. Here, deletion of the Aspergillus fumigatus tRNA-modifying isopentenyl transferase ortholog, Mod5, led to altered growth and stress response, but also unexpected resistance against the antifungal drug 5-fluorocytosine (5-FC). Simultaneous profiling of the transcriptome and proteome of 5-FC-stressed wild-type and ∆mod5 strains revealed a similar general adaptation to stress; however, the knockout displayed elevated expression of cross-pathway control (CPC) genes. Quantification of CPC transcription factor cpcA and client gene argB showed premature CPC activation in ∆mod5, which was further increased upon treatment with 5-FC. By associating codon usage patterns with proteomics abundances, we observed the number of tRNATyrGΨA-decoded codons to negatively correlate with protein abundance in the knockout, indicative of modification-tuneable transcripts. Subsequent overexpression of tRNATyrGΨA in the ∆mod5 strain rescued select phenotypes but, surprisingly, failed to reverse 5-FC resistance. Investigation of the purported tRNA gene-mediated silencing function of Mod5 uncovered a negative correlation between tRNA proximity and gene induction under normal growth, suggesting that tRNA gene-mediated silencing is active in A. fumigatus. In conclusion, 5-FC resistance in the absence of the bifunctional Mod5 protein likely originates from multifaceted transcriptional and translational changes that skew the fungus towards starvation responses over optimal growth, potentially offering a mechanism reliant on RNA modification and tRNA gene-mediated silencing capable of facilitating transient antifungal resistance.

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The Role of NAD+-dependent lysine deacetylases (Sirtuin) and their Inhibitors in Aspergillus fumigatus acetylome and virulence

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2024-06-20 | PXD044964 | Pride

Functional characterization of the RNA interference pathway in A. fumigatus reveals its potential for antifungal therapy

Project description:The RNA interference (RNAi) pathway has evolved numerous functionalities in eukaryotes, with many on display in Kingdom Fungi. RNAi can regulate gene expression, facilitate drug resistance, or even be altogether lost to improve growth potential in some fungal pathogens. In the WHO fungal priority pathogen, Aspergillus fumigatus, the RNAi system is known to be intact and functional. To extend our limited understanding of A. fumigatus RNAi, we first investigated the genetic variation in RNAi-associated genes in a large collection of environmental and clinical genomes, where we found that RNAi is evolutionarily conserved even in clinical strains. Using endogenously expressed inverted-repeat transgenes complementary to both a conditionally essential gene and a nonessential gene, we determined that a subset of the RNAi componentry is active in inverted-repeat transgene silencing in conidia and mycelium. A multi-condition mRNA-seq analysis linked the A. fumigatus dicer-like enzymes and RNA-dependent RNA polymerases to regulation of conidial ribosome biogenesis genes; however, surprisingly few endogenous small RNAs were identified in conidia that could explain this broad change. Although RNAi was not clearly linked to growth or stress response defects in the RNAi knockouts, serial passaging of RNAi knockout strains for six generations resulted in lineages with diminished spore production over time, indicating that loss of RNAi can exert a fitness cost on the fungus. Cumulatively, A. fumigatus RNAi appears to play an active role in defense against double-stranded RNA species alongside a previously unappreciated housekeeping function in regulation of conidial ribosomal biogenesis genes.

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