Biology subjects

Bruno, V. M.

Publications and source records attributed to Bruno, V. M..

9 recordsLinked to original sources

bioRxiv2025

The HosA histone deacetylase regulates stress resistance, host cell interactions, and virulence in Aspergillus fumigatus

The capacity of Aspergillus fumigatus to cause invasive pulmonary aspergillosis depends on its ability to adapt to dynamic and stressful microenvironments within the host. Epigenetic regulation, including histone deacetylation, plays a critical role in fungal adaptation to stress. Here, we investigated the role of the class I histone deacetylase (HDAC) HosA in A. fumigatus stress resistance, host cell interactions, and virulence. A {Delta}hosA mutant had increased susceptibility to intracellular oxidant stress induced by menadione. It also had impaired capacity to invade and damage two pulmonary epithelial cell lines in vitro. In a corticosteroid-immunosuppressed mouse model of invasive aspergillosis, mice infected with the {Delta}hosA mutant survived significantly longer than those infected with the wild-type strain, despite having similar pulmonary fungal burden. The {Delta}hosA mutant also induced a weaker inflammatory response than the wild-type strain. Transcriptomic analysis revealed that HosA regulates genes involved in secondary metabolite biosynthesis and energy metabolism, functioning as both an activator and repressor of distinct gene sets. Collectively, these results indicate that HosA is a key epigenetic regulator that governs A. fumigatus interactions with host cells and virulence during invasive pulmonary aspergillosis. ImportanceEpigenetic modifications in A. fumigatus can be induced by environmental changes and stresses such as those induced by interaction with host cells. HosA, a class I histone deacetylase, has been shown to play a key role in regulating secondary metabolism in several Aspergillus species, but its function in A. fumigatus was previously unknown. We found that deletion of hosA increased susceptibility to intracellular, but not extracellular, oxidative stress. The {Delta}hosA mutant also exhibited significantly reduced pulmonary epithelial cell invasion and host cell damage, as well as attenuated virulence in immunosuppressed mice. Together, these findings indicate that HosA functions as a key epigenetic regulator that governs stress resistance, secondary metabolism, and fungal-host interactions. Defining the functions of HosA could provide critical insight into the epigenetic mechanisms that control fungal pathogenicity and may reveal a potential therapeutic target for invasive aspergillosis.

microbiology↗

bioRxiv2024

Epidermal Growth Factor Receptor Signaling Governs the Host Inflammatory Response to Invasive Aspergillosis

The epidermal growth factor receptor (EGFR) has been identified as an epithelial cell receptor for Mucorales fungi and Candida albicans. Blocking EGFR with small molecule inhibitors reduces disease severity in mouse models of mucormycosis and oropharyngeal candidiasis. In contrast, cases of invasive aspergillosis have been reported in cancer patients who were treated with EGFR inhibitors, suggesting that EGFR signaling may play a protective role in the host defense against this infection. Here, we analyzed transcriptomic data from the lungs of mice with invasive aspergillosis and found evidence that Aspergillus fumigatus infection activates multiple genes that are predicted to function in the EGFR signaling pathway. We also found that A. fumigatus infection activates EGFR in both a human small airway epithelial (HSAE) cell line and in the lungs of immunosuppressed mice. EGFR signaling in HSAE cells is required for maximal endocytosis of A. fumigatus and for fungal-induced proinflammatory cytokine and chemokine production. In a corticosteroid immunosuppressed mouse model of invasive pulmonary aspergillosis, inhibition of EGFR with gefitinib decreased whole lung chemokine levels and reduced accumulation of phagocytes in the lung, leading to a decrease in fungal killing, an increase in pulmonary fungal burden, and accelerated mortality. Thus, EGFR signaling is required for pulmonary epithelial cells to orchestrate the host innate immune defense against invasive aspergillosis in immunosuppressed hosts. ImportanceWhen A. fumigatus infects the lungs, it invades epithelial cells that line the airways. During this process, the fungus interacts with epithelial cell receptors. This interaction stimulates epithelial cells to endocytose the fungus. It also induces these cells to secret proinflammatory cytokines and chemokines that recruit phagocytes to the site of infection where they can kill the fungus. Here, we show that in small airway epithelial cells, the epidermal growth factor receptor (EGFR) acts a sensor for A. fumigatus that triggers the production of chemokines in response to fungal infection. In corticosteroid-immunosuppressed mice, blocking EGFR with the kinase inhibitor, gefitinib reduces chemokine production in the lungs. This leads to decreased accumulation of neutrophils and dendritic cell in the lungs, reduced A. fumigatus killing, and increased mortality. These results provide a potential explanation as to why some cancer patients who are treated with EGFR inhibitors develop invasive aspergillosis.

microbiology↗

bioRxiv2023

Tick extracellular vesicles impair epidermal homeostasis through immune-epithelial networks during hematophagy

Wound healing has been extensively studied through the lens of inflammatory disorders and cancer, but limited attention has been given to hematophagy and arthropod-borne diseases. Hematophagous ectoparasites, including ticks, subvert the wound healing response to maintain prolonged attachment and facilitate blood-feeding. Here, we unveil a strategy by which extracellular vesicles (EVs) ensure blood-feeding and arthropod survival in three medically relevant tick species. Through single cell RNA sequencing and murine genetics, we demonstrate that wildtype animals infested with EV-deficient Ixodes scapularis display a unique epidermal sub-population with a mesenchymal-like transcriptional program and an overrepresentation of pathways connected to wound healing. Furthermore, tick EVs inhibit proliferation and diminish the capacity of wound closure in keratinocytes. This occurrence was linked to phosphoinositide 3-kinase activity, keratinocyte growth factor 1 (KGF-1) and transforming growth factor {beta} (TGF-{beta}) levels. Collectively, we uncovered a strategy employed by a blood-feeding arthropod that disrupts the circuitry in cutaneous wound healing, contributing to ectoparasite fitness.

microbiology↗

bioRxiv2023

Common Analysis of Direct RNA SequencinG CUrrently Leads to Misidentification of 5-Methylcytosine Modifications at GCU Motifs

RNA modifications, such as methylation, can be detected with Oxford Nanopore Technologies direct RNA sequencing. One commonly used tool for detecting 5-methylcytosine (m5C) modifications is Tombo, which uses an "Alternative Model" to detect putative modifications from a single sample. We examined direct RNA sequencing data from diverse taxa including virus, bacteria, fungi, and animals. The algorithm consistently identified a 5-methylcytosine at the central position of a GCU motif. However, it also identified a 5-methylcytosine in the same motif in fully unmodified in vitro transcribed RNA, suggesting that this a frequent false prediction. In the absence of further validation, several published predictions of 5-methylcytosine in human coronavirus and human cerebral organoid RNA in a GCU context should be reconsidered. IMPORTANCEThe detection of chemical modifications to RNA is a rapidly expanding field within epigenetics. Nanopore sequencing technology provides an attractive means of detecting these modifications directly on the RNA, but accurate modification predictions are dependent upon the software developed to interpret the sequencing results. One of these tools, Tombo, allows users to detect modifications using sequencing results from a single RNA sample. However, we find that this method falsely predicts modifications in a specific sequence context across a variety of RNA samples, including RNA that lacks modifications. Results from previous publications include predictions in human coronaviruses with this sequence context and should be reconsidered. Our results highlight the importance of using RNA modification detection tools with caution in the absence of a control RNA sample for comparison.

genomics↗

bioRxiv2021

Mucoricin is a Ricin-Like Toxin that is Critical for the Pathogenesis of Mucormycosis

Fungi of the order Mucorales cause mucormycosis, a lethal infection with an incompletely understood pathogenesis. We now demonstrate that Mucorales fungi produce a toxin that plays a central role in virulence. Polyclonal antibodies against this toxin inhibit its ability to damage human cells in vitro, and prevent hypovolemic shock, organ necrosis, and death in mice with mucormycosis. RNAi inhibition of the toxin in Rhizopus delemar, compromises the ability of the fungus to damage host cells and attenuates virulence in mice. This 17 kDa toxin has structural and functional features of the plant toxin, ricin, including the ability to inhibit protein synthesis by its N-glycosylase activity, the existence of a motif that mediates vascular leak, and a lectin sequence. Antibodies against the toxin inhibit R. delemar- or toxin-mediated vascular permeability in vitro and cross-react with ricin. A monoclonal anti-ricin B chain antibody binds to the toxin and also inhibits its ability to cause vascular permeability. Therefore, we propose the name "mucoricin" for this toxin. Not only is mucoricin important in the pathogenesis of mucormycosis but our data suggest that a ricin- like toxin is produced by organisms beyond the plant and bacterial kingdoms. Importantly, mucoricin should be a promising therapeutic target.

microbiology↗

bioRxiv2020

Determining Aspergillus fumigatus transcription factor expression and function during invasion of the mammalian lung

To gain a better understanding of the transcriptional response of Aspergillus fumigatus during invasive pulmonary infection, we used a NanoString nCounter to assess the transcript levels of 467 A. fumigatus genes during growth in the lungs of immunosuppressed mice. These genes included ones known to respond to diverse environmental conditions and those encoding most transcription factors in the A. fumigatus genome. We found that invasive growth in vivo induces a unique transcriptional profile as the organism responds to nutrient limitation and attack by host phagocytes. This in vivo transcriptional response is largely mimicked by in vitro growth in Aspergillus minimal medium that is deficient in nitrogen, iron, and/or zinc. From the transcriptional profiling data, we selected 9 transcription factor genes that were either highly expressed or strongly up-regulated during in vivo growth. Deletion mutants were constructed for each of these genes and assessed for virulence in mice. Two transcription factor genes were found to be required for maximal virulence. One was rlmA, which governs the ability of the organism to proliferate in the lung. The other was ace1, which regulates of the expression of multiple secondary metabolite gene clusters and mycotoxin genes independently of laeA. Using deletion and overexpression mutants, we determined that the attenuated virulence of the {Delta}ace1 mutant is due to decreased expression aspf1, which specifies a ribotoxin, but is not mediated by reduced expression of the fumigaclavine gene cluster or the fumagillin-pseruotin supercluster. Thus, in vivo transcriptional profiling focused on transcription factors genes provides a facile approach to identifying novel virulence regulators. Author summaryAlthough A. fumigatus causes the majority of cases of invasive aspergillosis, the function of most of the genes in its genome remains unknown. To identify genes encoding transcription factors that may be important for virulence, we used a NanoString nCounter to measure the mRNA levels of A. fumigatus transcription factor genes in the lungs of mice with invasive aspergillosis. The transcriptional profiling data indicate that the organism is exposed to nutrient limitation and stress during growth in the lungs, and that it responds by up-regulating genes that encode mycotoxins and secondary metabolites. In vitro, this response was most closely mimicked by growth in medium that was deficient in nitrogen, iron and/or zinc. Using the transcriptional profiling data, we identified two transcription factors that govern A. fumigatus virulence. These were RlmA, which is governs proliferation in the lung and Ace1, which controls the production of mycotoxins and secondary metabolites.

microbiology↗

bioRxiv2020

Mosaic changes to the global transcriptome in response to inhibiting ribosome formation versus inhibition of ribosome function

Cell fate is susceptible to several internal and external stresses. Stress resulting from mutations in genes for ribosomal proteins and assembly factors leads to many congenital diseases, collectively called ribosomopathies. Even though such mutations all depress the cells protein synthesis capacity, they are manifested in many different phenotypes. This prompted us to use Saccharomyces cerevisiae to explore whether reducing the protein synthesis capacity by different mechanisms result in the same or different changes to the global transcriptome. We have compared the transcriptome after abolishing the assembly of new ribosomes and inhibiting the translocation of ribosomes on the mRNA. Our results show that these alternate obstructions generate different mosaics of expression for several classes of genes, including genes for ribosomal proteins, mitotic cell cycle, cell wall synthesis, and protein transport.

genomics↗

bioRxiv2020

Tornadic shear stress induces a transient, calcineurin-dependent hyper-virulent phenotype in Mucorales molds

Trauma-related necrotizing myocutaneous mucormycosis (NMM) has a high morbidity and mortality in victims of combat-related injuries, geo-meteorological disasters, and severe burns. Inspired by the observation that several recent clusters of NMM have been associated with extreme mechanical forces (e.g. during tornados), we studied the impact of mechanical stress on Mucoralean biology and virulence in a Drosophila melanogaster infection model. In contrast to other experimental procedures to exert mechanical stress, tornadic shear challenge (TSC) by magnetic stirring induced a hyper-virulent phenotype in several clinically relevant Mucorales species but not in Aspergillus or Fusarium. Whereas fungal growth rates, morphogenesis, and susceptibility to noxious environments or phagocytes were not altered by TSC, soluble factors released in the supernatant of shear-challenged R. arrhizus spores rendered static spores hyper-virulent. Consistent with a rapid decay of TSC-induced hyper-virulence, minimal transcriptional changes were revealed by comparative RNA sequencing analysis of static and shear-challenged Rhizopus arrhizus. However, inhibition of the calcineurin/heat shock protein 90 (hsp90) stress response circuitry by cyclosporine A (CsA) and tanespimycin abrogated the increased pathogenicity of R. arrhizus spores following TSC. Similarly, calcineurin loss-of-function mutants of Mucor circinelloides displayed no increased virulence capacity in flies after undergoing TSC. Collectively, these results establish that TSC induces hyper-virulence specifically in Mucorales and point out the calcineurin/hsp90 pathway as a key orchestrator of this phenotype. Our findings invite future studies of topical calcineurin inhibitor treatment of wounds as an adjunct mitigation strategy for NMM following high-energy trauma. SignificanceGiven the limited efficacy of current medical treatments in trauma-related necrotizing mucormycosis, there is a dire need to better understand the Mucoralean pathophysiology in order to develop novel strategies to counteract fungal tissue invasion following severe trauma. Here, we describe that tornadic shear stress challenge transiently induces a hyper-virulent phenotype in various pathogenic Mucorales species but not in other molds known to cause wound infections. Our data support a model whereby shear stress-induced hyper-virulence is primarily driven by soluble factors and orchestrated by the calcineurin/hsp90 pathway. Importantly, pharmacological and genetic inhibition of calcineurin signaling abrogated hyper-virulence in shear stress-challenged Mucorales, encouraging further evaluation of (topical) calcineurin inhibitors to improve therapeutic outcomes of NMM after combat-related blast injuries or violent storms.

microbiology↗

bioRxiv2018

Cost Effective, Experimentally Robust Differential Expression Analysis for Human/Mammalian, Pathogen, and Dual-Species Transcriptomics

As sequencing read length has increased, researchers have quickly adopted longer reads for their experiments. Here, we examine host-pathogen interaction studies to assess if using longer reads is warranted. Six diverse datasets encountered in studies of host-pathogen interactions were used to assess what genomic attributes might affect the outcome of differential gene expression analysis including: gene density, operons, gene length, number of introns/exons, and intron length. Principal components analysis, hierarchical clustering with bootstrap support, and regression analyses of pairwise comparisons were undertaken on the same reads, looking at all combinations of paired and unpaired reads trimmed to 36,54,72, and 101-bp. For E coli, 36-bp single end reads performed as well as any other read length and as well as paired end reads. For all other comparisons, 54-bp and 72-bp reads were typically equivalent and different from 36-bp and 101-bp reads. Read pairing improved the outcome in several, but not all, comparisons in no discernable pattern, such that using paired reads is recommended in most scenarios. No specific genome attribute appeared to influence the data. However, experiments with an a priori expected greater biological complexity had more variable results with all read lengths relative to those with decreased complexity. When combined with cost, 54-bp paired end reads provided the most robust, internally reproducible results across all comparisons. However, using 36-bp single end reads may be desirable for bacterial samples, although possibly only if the transcriptional response is expected a priori to be robust.\n\nDATA SUMMARYO_LIThe human only CSHL Encode data set (1) was downloaded from ftp://hgdownload.cse.ucsc.edu/goldenPath/hgl9/encodeDCC/wgEncodeCshlLongRnaSeq/.\nC_LIO_LIThe data from mice vaginas infected with Candida albicans (2) was downloaded from the SRA (url - https://trace.ncbi.nlm.nih.gov/Traces/sra/?study=SRP057050).\nC_LIO_LIThe data from Aspergillus fumigatus cells in contact with human cells was downloaded from the SRA (url - https://www.ncbi.nlm.nih.gov/bioproject/399754).\nC_LIO_LIThe data from a strand-specific library from a study comparing C. albicans cells in contact with human cells with those in media (3) was downloaded from the SRA (url - https://trace.ncbi.nlm.nih.gov/Traces/sra/?study=SRP011085).\nC_LIO_LIThe data from C. albicans in culture media (3) was downloaded from the SRA (url - https://trace.ncbi.nlm.nih.gov/Traces/sra/?study=SRP011085).\nC_LIO_LIThe data from Escherichia coli grown in different media (4) was downloaded from the SRA (url - https://trace.ncbi.nlm.nih.gov/Traces/sra/?study=SRP056578).\nC_LI\n\nI/We confirm all supporting data, code and protocols have been provided within the article or through supplementary data files. {boxtimes}\n\nIMPACT STATEMENTAs sequencing technologies improve, sequencing costs decrease and read lengths increase. We examine host-pathogen interaction studies to assess if using these longer reads is warranted given their increased cost relative to using the same number of shorter reads. To this end we compared the use of various read lengths and read pairing for six diverse host-pathogen datasets with varying genomic attributes including: gene density, operons, gene length, number of introns/exons, and intron length. We find that in the bacterial sample, 36-bp single end reads performed as well as any other read length and as well as paired end reads. When combined with cost, 54-bp paired end reads provided the most robust, internally reproducible results for all other comparisons. Read pairing improved the outcome in several, but not all, comparisons in no discernable pattern, such that using paired reads is recommended in most scenarios. No specific genome attribute appeared to influence the data.

genomics↗