Biology subjects

Kelani, A. A.

Publications and source records attributed to Kelani, A. A..

4 recordsLinked to original sources

bioRxiv2024

Elucidation of the Aspergillus fumigatus tRNA-derived RNA repertoire from conidia and mycelium.

Aspergillus fumigatus is a ubiquitous filamentous fungus and dangerous human pathogen that produces a limited pool of small RNAs, consisting in large part of tRNA-derived RNAs (tDRs). Here, we improve our understanding of the small RNAs produced in conidia and mycelium of A. fumigatus using small RNA-sequencing of strains lacking RNA interference (RNAi) machinery and a cutting-edge tDR-sequencing approach. We find little evidence of small RNAs dependent on the canonical RNAi machinery under laboratory growth conditions, but reveal tDRs to be differentially abundant across fungal morphotypes, with specific fragments proving dominant in each assessed condition (e.g., Asp(GTC)-5tRH in conidia; His(GTG)-5tRH in mycelium). Consistent with the literature, we observed distinct patterns of tDRs from nuclear- and mitochondria-derived tDRs, which was confirmed for wild-type fungus with tDR-seq. By inducing canonical RNAi with overexpression of an inverted-repeat transgene, we determined A. fumigatus to produce predominantly 20-nt, 5 uridine-containing small RNAs from the transgene, a population reliant on Argonaute and Dicer-like proteins. Surprisingly, we found that overexpression of this double-stranded RNA (dsRNA) limited growth of both wild-type and RNAi-deficient strains, with a strain lacking the two A. fumigatus RNA-dependent RNA polymerase orthologs particularly vulnerable. Dicer-like- and Argonaute-double knockout strains with few detectable transgene-derived small RNAs were also susceptible to growth inhibition, suggesting excessive dsRNA limits growth in A. fumigatus. Ultimately, we have provided an improved description of small RNA biogenesis in A. fumigatus and uncovered an intriguing link between dsRNA metabolism and filamentous growth. IMPORTANCEThe mechanisms of small RNA biogenesis in fungi are diverse and multifactorial. Here, using a panel of RNA interference gene deletion strains and multiple small RNA sequencing approaches, we reveal limited input of the canonical RNAi machinery on the small RNA landscape of the human fungal pathogen, Aspergillus fumigatus under normal growth conditions. We identify specific patterns of tRNA-derived RNAs unique to fungal morphotypes, which may hold promise for future diagnostic efforts. We then probed the mechanism of double-stranded RNA processing by the organism after artificial induction, revealing a potential link between proper double-stranded RNA processing and fungal growth arrest, improving our understanding of the evolution of double-stranded RNA regulation in a ubiquitous mold.

microbiology↗

bioRxiv2024

Mod5 mediates a molecular trade-off between optimal gene expression and antifungal resistance.

Increasing antifungal drug resistance is a major concern associated with human fungal pathogens like Aspergillus fumigatus. Genetic mutation and epimutation mechanisms clearly drive resistance, yet the epitranscriptome remains relatively untested. Here, deletion of the A. fumigatus tRNA-modifying isopentenyl transferase ortholog, Mod5, led to altered stress response and unexpected resistance against the antifungal drug 5-fluorocytosine (5-FC). After confirming the canonical isopentenylation activity of Mod5 by LC-MS/MS and Nano-tRNAseq, we performed simultaneous profiling of transcriptomes and proteomes to reveal a comparable overall response to 5-FC stress; however, a premature activation of cross-pathway control (CPC) genes in the knockout was further increased after antifungal treatment. We identified several orthologues of the A. nidulans Major Facilitator Superfamily (MFS) transporter nmeA as specific CPC-client genes in A. fumigatus. Overexpression of Mod5-target tRNATyrG{Psi}A in the {Delta}mod5 strain rescued select phenotypes but failed to reverse 5-FC resistance, whereas deletion of nmeA largely, but incompletely, reverted the resistance phenotype, implying additional relevant exporters. In conclusion, 5-FC resistance in the absence of Mod5 and i6A likely originates from multifaceted transcriptional and translational changes that skew the fungus towards premature CPC-dependent activation of antifungal toxic-intermediate exporter nmeA, offering a potential mechanism reliant on RNA modification to facilitate transient antifungal resistance.

microbiology↗

bioRxiv2023

RNA-based sensitive fungal pathogen detection

Detecting fungal pathogens, a major cause of severe systemic infections, remains challenging due to the difficulty and time-consuming nature of diagnostic methods. This delay in identification hinders targeted treatment decisions and may lead to unnecessary use of broad-spectrum antibiotics. To expedite treatment initiation, one promising approach is to directly detect pathogen nucleic acids such as DNA, which is often preferred to RNA because of its inherent stability. However, a higher number of RNA molecules per cell makes RNA a more promising diagnostic target which is particularly prominent for highly expressed genes such as rRNA. Here, we investigated the utility of a minimal input-specialized reverse transcription protocol to increase diagnostic sensitivity. This proof-of-concept study demonstrates that fungal rRNA detection by the minimal input protocol is drastically more sensitive compared to detection of genomic DNA even with high levels of human RNA background. This approach can detect several of the most relevant human pathogenic fungal genera, such as Aspergillus, Candida, and Fusarium and thus represents a powerful, cheap, and easily adaptable addition to currently available diagnostic assays.

microbiology↗

bioRxiv2022

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

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 collection of 217 environmental and 83 clinical genomes, where we found that RNAi components are conserved even in clinical strains. Using endogenously expressed inverted-repeat transgenes complementary to a conditionally essential gene (pabA) or a nonessential gene (pksP), we determined that a subset of the RNAi componentry is active in inverted-repeat transgene silencing in conidia and mycelium. Analysis of mRNA-seq data from RNAi double-knockout strains linked the A. fumigatus dicer-like enzymes (DclA/B) and RNA-dependent RNA polymerases (RrpA/B) 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.

microbiology↗