Biology subjects

Blango, M. G.

Publications and source records attributed to Blango, M. G..

12 recordsLinked to original sources

bioRxiv2026

Human neutrophils release extracellular RNA that modulates Aspergillus fumigatus gene expression across kingdoms.

Extracellular RNAs (exRNAs) are emerging as potent bidirectional interkingdom effectors of host-pathogen interactions, particularly in plant and insect systems. Here, using small RNA-sequencing and genetic manipulation, we provide evidence consistent with a function of the human neutrophil exRNA-ome in modulating gene expression across kingdoms in the fungal pathogen Aspergillus fumigatus. The neutrophil extracellular space contains a diverse repertoire of RNA, which was corroborated using a droplet-digital stem-loop RT-PCR protocol to measure differential abundance of several tRNA-derived RNAs between two extracellular fractions. A closer inspection of the extracellular miRNA pool revealed enrichment of antimicrobial Let-7 family sRNAs and immunomodulatory miR-223, among others. Secretion of these sRNAs was largely impervious to infection status, suggesting that neutrophils produce and maintain an antimicrobial exRNA environment against A. fumigatus that requires further targeting. Cross-kingdom miRNA target prediction and exRNA delivery experiments revealed that putative fungal targets are modulated independently of the fungal argonaute proteins and in a donor-dependent manner. Although heterogeneous, for donors that exhibited a silencing effect, mutated target sequences in the fungus did reverse silencing. We hypothesize that the exRNA landscape produced by neutrophils contributes to their multifaceted role in antifungal defense.

microbiology↗

bioRxiv2026

Characterization of tRNA ligase function in pathogenic fungi and trypanosomes reveals the ligase domain as a promising drug target

The majority of eukaryotes encode some intron-containing pre-tRNAs. Splicing of these pre-tRNAs requires a dedicated tRNA splicing machinery. The fungal and trypanosome tRNA ligase, Trl1, and the human RNA ligase, RTCB, catalyze an essential step in tRNA splicing. However, Trl1 and RTCB are nonhomologous and biochemically and structurally distinct from each other. Therefore, Trl1 could serve as a broad-spectrum antifungal and anti-trypanosomal target. While the functions and requirements of the three catalytic Trl1 domains have been extensively characterized in the model yeast Saccharomyces cerevisiae, the roles of Trl1 orthologs in pathogenic fungi remain unexplored. Here, we validate Trl1 as one of the few promising novel drug targets for the development of antifungal therapeutics. Functional analyses of the three Trl1 domains show that only the "sealing" domain is essential for growth and viability in Candida albicans and Aspergillus fumigatus. In contrast, the two "healing" domains are dispensable in these pathogenic fungi, suggesting the presence of redundant healing enzymes, unlike in S. cerevisiae. These findings indicate that only the sealing domain is a good drug target. Our analysis also shows that the Mucor enzyme, which only contains the sealing domain, is essential. Using a Caenorhabditis elegans infection model of C. albicans, we further demonstrated that inhibiting Trl1 expression protects worms during an established infection. In contrast to these fungal pathogens, we show that all three domains of Trl1 are essential in Trypanosoma brucei. Our findings show that the essentiality of the Trl1 sealing is conserved in important human pathogens and provides an impetus for future drug development. SIGNIFICANCEFungal infections are an important cause of human disease and death and difficult to treat and there is an urgent need to develop additional drugs. Based on studies in yeast, one promising target for antifungal drug development is the tRNA splicing pathway. Human tRNA ligase is fundamentally distinct from the fungal one. To investigate the possibility of developing tRNA ligase-targeting drugs, we investigated the function of the catalytic domains of fungal tRNA ligase in different fungal pathogens. Surprisingly, only the first domain is essential in these pathogens and yeast is not a good model fungus. In contrast, all three domains of Trypanosome tRNA ligase are essential. These findings provide an impetus for future drug development.

molecular biology↗

bioRxiv2026

The Small RNA MicC is a Multifunctional Regulator of Extraintestinal Pathogenic Escherichia coli Fitness across Multiple Host Niches

Small non-coding RNAs (sRNA) modulate diverse bacterial functions ranging from carbon metabolism to virulence gene expression. Previous research showed that the sRNA chaperone Hfq is critical for the fitness of Extraintestinal Pathogenic Escherichia coli (ExPEC), a major cause of both bloodstream and urinary tract infections (UTI). Using the reference ExPEC strain UTI89, we created deletion mutants to probe the effects of seven conserved Hfq-dependent sRNAs (DsrA, RprA, OxyS, RyhB, MicF, MicC, Spf) on resistance to oxidative stress. All of the sRNA mutants grew normally in replete lysogeny broth, but the spf and micC mutants exhibited additive effects upon challenge with reactive oxygen species generated by methyl viologen. In a murine UTI model, the spf mutant resembled the wild-type strain, whereas UTI89{Delta}micC was unable to effectively colonize the bladder despite behaving like wild type within the kidneys. This correlated with a greatly reduced ability of the micC mutant to survive within bladder epithelial cells and paralleled UTI89{Delta}micC defects in gut colonization, virulence in a sepsis model, and complement resistance. Although MicC downregulated expression of its only known target, OmpC, aberrant modulation of this porin did not entirely account for the decreased stress resistance of UTI89{Delta}micC. Rather, RNA-Seq, sRNA target predictions, and in vitro phenotypic assays revealed that MicC can impact multiple pathways linked to niche establishment, including motility, chemotaxis, and various metabolic processes. These data are consistent with MicC serving as a multifunctional regulator of ExPEC stress responses and niche-specific fitness through OmpC-dependent and - independent mechanisms. IMPORTANCEPathogenic strains of Escherichia coli are exceptionally common causes of diarrheal disease, urinary tract infection, sepsis, and meningitis. The ability of these pathogens to cause such a wide range of maladies is in part attributable to their ability to quickly adapt to and thrive within disparate and often hostile environments, including the gut, bladder, kidneys, and bloodstream. Adaptation to new environments requires rapid and precise changes in gene expression. To accomplish this feat, E. coli utilizes a suite of regulatory RNA called small RNA (sRNA). In this paper, we identified the sRNA MicC as a critical facilitator of E. coli fitness and virulence within diverse host environments via effects on the expression of multiple genes involved in bacterial motility, energy acquisition, and various other pathways. Delineating how sRNAs like MicC impact disease processes will aid the development of novel therapeutics to better combat E. coli infections.

microbiology↗

bioRxiv2025

Comprehensive mapping of the 5' and 3' untranslated regions of Aspergillus fumigatus reveals new insights into gene regulation

In the twenty years since the first genome sequencing of Aspergillus fumigatus, the field has seen an explosion in both the number of sequenced genomes and our molecular understanding of this ubiquitous human fungal pathogen. Despite an improved knowledge of the A. fumigatus genome, we still know little about the transcriptome, with key regulatory sequences like the untranslated regions of mRNA based only on in silico predictions and bulk-RNA-seq. Here, we provide an improved description of 5' and 3' untranslated regions of A. fumigatus poly(A)-enriched RNA through experimental mapping of transcription start sites and polyadenylation sites using 5' and 3' End-Seq. We assigned high-quality 5' ends to 2,747 genes (average length 126 nt), 3' ends to 7,079 genes (average length 268 nt), and improved our understanding of the regulatory landscape of A. fumigatus gene expression. We leveraged the refined 5' UTRs to identify upstream open reading frames and binding sites for important RNA binding proteins like the translational regulator Ssd1 and the 3' UTRs to define binding sites for PUF proteins known to contribute to mRNA localization and regulation. Although a single isoform typically dominated expression, we observed 148 instances of alternative start sites and 1,675 alternative stop sites. Interestingly, we detected multiple examples of premature transcriptional termination, including the first evidence for promoter-proximal premature transcriptional termination in a member of the Eurotiomycetes. Ultimately, we provide a resource to the Aspergillus community and an accurate starting point for unravelling the complexities of gene regulation in an important human pathogen.

microbiology↗

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↗

bioRxiv2021

PLB-985 neutrophil-like cells release antifungal extracellular vesicles against the human-pathogenic fungus Aspergillus fumigatus

Fungal infections remain a major global concern. Emerging fungal pathogens and increasing rates of resistance mean that additional research efforts and resources must be allocated to advancing our understanding of fungal pathogenesis and developing new therapeutic interventions. Neutrophilic granulocytes are a major cell type involved in protection against the important fungal pathogen Aspergillus fumigatus, where they employ numerous defense mechanisms, including production of antimicrobial extracellular vesicles. A major draw-back to work with neutrophils is the lack of a suitable cell line system for the study of fungal pathogenesis. To address this problem, we assessed the feasibility of using differentiated PLB-985 neutrophil-like cells as an in vitro model to study A. fumigatus infection. We find that dimethylformamide-differentiated PLB-985 cells provide a useful recapitulation of many aspects of A. fumigatus interactions with primary human polymorphonuclear leukocytes. We show that differentiated PLB-985 cells phagocytose fungal conidia and acidify conidia-containing phagolysosomes similar to primary neutrophils, release neutrophil extracellular traps, and also produce antifungal extracellular vesicles in response to infection. In addition, we provide an improved method for the isolation of extracellular vesicles produced during infection by employing a size-exclusion chromatography-based approach. Advanced LC-MS/MS proteomics revealed an enrichment of extracellular vesicle marker proteins and a decrease of cytoplasmic proteins in extracellular vesicles isolated using this improved method. Ultimately, we find that differentiated PLB-985 cells can serve as a genetically tractable model to study many aspects of A. fumigatus pathogenesis. IMPORTANCEPolymorphonuclear leukocytes are an important defense against human fungal pathogens, yet our model systems to study this group of cells remains very limited in scope. In this study, we established that differentiated PLB-985 cells can serve as a model to recapitulate several important aspects of human polymorphonuclear leukocyte interactions with the important human fungal pathogen Aspergillus fumigatus. The proposed addition of a cultured neutrophil-like cell line to the experimental toolbox to study fungal pathogenesis will allow for a more mechanistic description of neutrophil antifungal biology. In addition, the easier handling of the cell line compared to primary human neutrophils allowed us to use PLB-985 cells to provide an improved method for isolation of neutrophil-derived extracellular vesicles using size-exclusion chromatography. Together, these results provide significant tools and a baseline knowledge for the future study of neutrophil-derived extracellular vesicles in the laboratory.

microbiology↗

bioRxiv2021

Balanced Input from the tRNA Prenyltransferase MiaA Controls the Stress Resistance and Virulence Potential of Extraintestinal Pathogenic Escherichia coli

An ability to adapt to rapidly changing and often hostile environments is key to the success of many bacterial pathogens. In Escherichia coli, the highly conserved enzymes MiaA and MiaB mediate the sequential prenylation and methylthiolation of adenosine-37 within tRNAs that decode UNN codons. Here, we show that MiaA, but not MiaB, is critical to the fitness and virulence of extraintestinal pathogenic E. coli (ExPEC), a major cause of urinary tract and bloodstream infections. Deletion of miaA has pleiotropic effects, rendering ExPEC especially sensitive to stressors like nitrogen and oxygen radicals and osmotic shock. We find that stress can stimulate striking changes in miaA expression, which in turn can increase translational frameshifting and markedly alter the bacterial proteome. Cumulatively, these data indicate that ExPEC, and likely other organisms, can vary MiaA levels as a means to fine-tune translation and the spectrum of expressed proteins in response to changing environmental challenges.

microbiology↗

bioRxiv2020

Biotinylated surfome profiling identifies potential biomarkers for diagnosis and therapy of Aspergillus fumigatus infection

Aspergillus fumigatus is one of the most common airborne fungi capable of causing invasive mycoses in immunocompromised patients and allergic diseases in susceptible individuals. In both cases, fungal surface proteins mediate the first contact with the human immune system to evade immune responses or to induce hypersensitivity. Several methods have been established to study the surface proteome (surfome) of A. fumigatus, like trypsin shaving, glucanase treatment, or formic acid extraction. Biotinylation coupled with LC-MS/MS identification of peptides is a particularly efficient method to identify the surface exposed regions of proteins that potentially mediate interaction with the host. After biotinylation of surface proteins during spore germination, we detected 314 different surface proteins, including several well-known proteins like RodA, CcpA, and DppV, as well as several allergens, heat shock proteins (HSPs), and previously undescribed surface proteins. Using immunofluorescence microscopy, we confirmed the surface localization of three HSPs, which may have moonlighting functions. Collectively, our study generated a comprehensive data set of the A. fumigatus surfome, which complements already existing A. fumigatus surface proteome data and allows us to propose a common core set of A. fumigatus surface proteins. In addition, our study uncovers the surface-exposed regions of many proteins on the surface of spores or hyphae. These surface exposed regions are candidates for direct interaction with host cells and may represent antigenic epitopes that either induce protective immune responses or mediate immune evasion. Thus, the comprehensive datasets provided and compiled here represent reasonable immunotherapy and diagnostic targets for future investigations. HIGHLIGHTSO_LISurface protein biotinylation coupled with LC-MS/MS analysis provides a comprehensive dataset of the A. fumigatus surface proteome. C_LIO_LI314 different A. fumigatus proteins (including immunoreactive proteins, and virulence factors) with surface exposed regions were detected. C_LIO_LISurface localization of three Hsp70 chaperones was confirmed by protein tagging coupled with immunofluorescence. C_LIO_LIBy comparison with other surfome datasets, a core surfome of A. fumigatus was defined, which provides possible biomarkers for diagnosis or therapy. C_LI SIGNIFICANCEAspergillus fumigatus is the most important airborne human pathogenic mold, capable of causing both life-threatening invasive pulmonary aspergillosis in immunocompromised patients and allergic infections in atopic individuals. Despite its obvious medical relevance, timely diagnosis and efficient antifungal treatment of A. fumigatus infection remains a major challenge. Proteins on the surface of conidia (asexually produced spores) and mycelium directly mediate host-pathogen interaction and also may serve as targets for diagnosis and immunotherapy. However, the similarity of protein sequences between A. fumigatus and other organisms, and sometimes even the human host, makes selection of targets for immunological-based studies difficult. Here, using surface protein biotinylation coupled with LC-MS/MS analysis, we identified hundreds of A. fumigatus surface proteins with exposed regions, further defining putative targets for possible diagnostic and immunotherapeutic design.

microbiology↗

bioRxiv2020

The dynamic surface proteomes of allergenic fungal conidia

Fungal spores and hyphal fragments play an important role as allergens in respiratory diseases. In this study, we performed trypsin shaving and secretome analyses to identify the surface-exposed proteins and secreted/shed proteins of Aspergillus fumigatus conidia, respectively. We investigated the surface proteome under different conditions, including temperature variation and germination. We found that the surface proteome of resting A. fumigatus conidia is not static, but instead unexpectedly dynamic, as evidenced by drastically different surface proteomes under different growth conditions. Knockouts of two abundant A. fumigatus surface proteins, ScwA and CweA, were found to function only in fine-tuning the cell wall stress response, implying that the conidial surface is very robust against perturbations. We then compared the surface proteome of A. fumigatus to other allergy-inducing molds, including Alternaria alternata, Penicillium rubens, and Cladosporium herbarum, and performed comparative proteomics on resting and swollen conidia, as well as secreted proteins from germinating conidia. We detected 125 protein ortholog groups, including 80 with putative catalytic activity, in the extracellular region of all four molds, and 42 nonorthologous proteins produced solely by A. fumigatus. Ultimately, this study highlights the dynamic nature of the A. fumigatus conidial surface and provides targets for future diagnostics and immunotherapy.

microbiology↗