Biology subjects

Verbon, A.

Publications and source records attributed to Verbon, A..

7 recordsLinked to original sources

bioRxiv2025

Genome-wide mapping of the Galleria mellonella larvae transcription start sites during fungal infection and treatment

Using Low Quantity single strand CAGE (LQ-ssCAGE), we mapped the transcription start sites (TSS). We annotated the 5 end of the invertebrate Galleria mellonella, an upcoming and booming experimental model in infectious disease and immunology research. However, the current genome annotation of this model organism lacks annotation of the 5 end and TSS information. G. mellonella larva was infected with the fungal pathogen Madurella mycetomatis to map TSS under healthy and infection conditions. Larvae were first treated with itraconazole or ravuconazole, and then RNA-seq and LQ-ssCAGE libraries were prepared and sequenced 4, 30, and 52 hours following infection. The LQ-ssCAGE data was processed to identify CAGE transcription start site (CTSS), uni-, and bi-directional clusters. LQ-ssCAGE enabled us to precisely identify 39,410 TSS and 249 active enhancers; we assigned genomic features to the resulting TSSs and enhancers. The majority of the TSS peaks are annotated as promoter regions, while the enhancers were annotated as intergenic and genic. Furthermore, we confirmed the quality of TSS calling by promoter shapes and GC bias. Furthermore, we identified a set of super-enhancers and predicted de-novo motifs. The raw and processed data was deposited to NCBI GEO GSE282923. CTSS, TSS peaks, and enhancers coordinated are available through the ZENBU Genome browser. In this study, we reported the first atlas of TSS and active enhancers of G. mellonella.

genomics↗

bioRxiv2022

Iron regulatory pathways differentially expressed during Madurella mycetomatis grain development in Galleria mellonella

BackgroundMycetoma is a neglected, chronic granulomatous infection of the subcutaneous tissue, most often caused by the fungal pathogen Madurella mycetomatis. Characteristic of the infection is the formation of grains. However, knowledge of the function and formation of the grain is limited. To map the processes leading to M. mycetomatis grain formation, we used a Galleria mellonella larvae infection model and time-course transcriptomic profiling. MethodsGalleria mellonella larvae were infected with M. mycetomatis genome strain mm55. At 4h, 24h, 72h and 168h post-inoculation, RNA was extracted from larval content. Two types of sequencing libraries were prepared for time-course transcriptomic profiling and analysis. FindingsIn the infected G. mellonella, 88.0% of the RNA sequence reads mapped to G. mellonella, while only 0.01% mapped to M. mycetomatis. Differential Gene Expression analysis revealed that 3,498 G. mellonella and 136 M. mycetomatis genes were differentially expressed during infection. Most of the enriched GO terms of both host and pathogen are linked to energy pathways, nucleobase metabolic process as well as cation and iron transport. Genes related to iron transport were highly expressed by both G. mellonella (transferrin and ferritin) and M. mycetomatis (SidA, SidD and SidI). A protein-protein interaction network analysis of D. melanogaster homologous genes in M. mycetomatis revealed the expression of the entire siderophore biosynthesis pathway throughout infection. InterpretationThe identification of the importance of iron acquisition during grain formation can be exploited as a potential novel diagnostic and therapeutic strategy for mycetoma. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSMycetoma is a chronic, neglected tropical infectious disease, characterised by a large subcutaneous mass and the formation of black grains in the affected tissue. Treatment for mycetoma is disappointing as in 25-50% of the patients recurrences are noted and up to 15% of patients will have to undergo amputation. The main reason behind this poor treatment response is the formation of protective structures by the pathogen upon entering the human body. These structures are called grains and provide a strong barrier for antifungal agents. Although grains are the hallmark of mycetoma, it is currently not known how these grains are formed. To improve the current therapy, it is important to gain insights in grain formation. Added value of this studyWe unravel the processes leading to grain formation and development in an invertebrate model of Madurella mycetomatis grain. We were able to build a model of grain formation and demonstrated that iron sequestering plays an important role in this process. Our findings were an important milestone in understanding the pathogenesis of mycetoma which has been a mystery for decades. Implications of all the available evidenceThe findings, will provide leads for future drug development of mycetoma treatment and therefore, improve patients live and end the need for amputations.

microbiology↗

bioRxiv2020

Induction of selective cell death in HIV-1-infected cells by DDX3 inhibitors leads to depletion of the inducible reservoir

An innovative approach to eliminate HIV-1-infected cells emerging out of latency, the major hurdle to HIV-1 cure, is to pharmacologically reactivate viral expression and concomitantly trigger intracellular pro-apoptotic pathways in order to selectively induce cell death (ICD) of infected cells, without reliance on the extracellular immune system. In this work we demonstrate the effect of DEAD-box polypeptide 3, X-Linked (DDX3) inhibitors on selectively inducing cell death in latent HIV-1-infected cell lines, primary CD4+ T cells and in CD4+ T cells from cART-suppressed people living with HIV-1 (PLWHIV). RNA sequencing analysis revealed that while overall gene expression was minimally dysregulated, DDX3 inhibition in independent donor CD4+ T cells led to significant downregulation of BIRC5 and HSPB1A, genes critical to cell survival during HIV-1 infection. We used single-cell FISH-Flow technology to characterise latency reversal and the contribution of viral RNA to inducing cell death; pharmacological targeting of DDX3 induced HIV-1 RNA expression, resulting in phosphorylation of IRF3, upregulation of IFN{beta} and selective induction of apoptosis in viral RNA-expressing CD4+ T cells from PLWHIV but not bystander cells. DDX3 inhibitor treatment of CD4+ T cells from PLWHIV in an in vitro culture model over five days resulted in an approximately 50% reduction of the inducible latent HIV-1 reservoir as determined by quantitation of CA HIV-1 RNA, by TILDA, as well as by FISH-Flow technology. Our data support the translation of DDX3 inhibitor class compounds into HIV-1 curative strategies and provide proof of concept for pharmacological reversal of latency coupled to induction of apoptosis towards elimination of the inducible reservoir.

microbiology↗

bioRxiv2020

The development of a novel diagnostic PCR for Madurella mycetomatis using a comparative genome approach

Eumycetoma is a neglected tropical disease characterized by large tumorous lesions. It is most commonly caused by the fungus Madurella mycetomatis which accounts for more than 70% of cases in central Africa. Currently, identification of the causative agent can only be reliably performed by a species-specific PCR. However, we recently demonstrated that our M. mycetomatis specific PCR can cross-react with Madurella pseudomycetomatis. We therefore used a comparative genome approach to develop a new M. mycetomatis specific PCR for species identification. For this we compared the published M. mycetomatis genome to genomes of other organisms in BLASTCLUST to identify unique M. mycetomatis predicted protein coding sequences. Based on 16 of these unique sequences, PCR primers were developed. The specificity of these primers was further evaluated in other eumycetoma causing agents including the Madurella sibling species. Out of the 16 tested sequences, only one was unique for M. mycetomatis and this should be used as a novel diagnostic marker for M. mycetomatis.

microbiology↗

bioRxiv2019

Gliotoxin, identified from a screen of fungal metabolites, disrupts 7SK snRNP, releases P-TEFb and reverses HIV-1 latency

A leading pharmacological strategy towards HIV cure requires "shock" or activation of HIV gene expression in latently infected cells with Latency Reversal Agents (LRAs) followed by their subsequent clearance. In a screen for novel LRAs we used fungal secondary metabolites (extrolites) as a source of bio-active molecules. Using orthogonal mass spectrometry (MS) coupled to latency reversal bioassays, we identified gliotoxin (GTX) as a novel LRA. GTX significantly induced HIV-1 gene expression in latent ex vivo infected primary cells and in CD4+ T cells from all aviremic HIV-1+ participants. RNA sequencing identified 7SK RNA, the scaffold of the P-TEFb inhibitory 7SK snRNP complex to be significantly reduced upon GTX treatment of independent donor CD4+T cells. GTX disrupted 7SK snRNP, releasing active P-TEFb, which then phosphorylated RNA Pol II CTD, inducing HIV transcription. Our data highlight the power of combining a medium throughput bioassay, mycology and orthogonal mass spectrometry to identify novel potentially therapeutic compounds.

molecular biology↗

bioRxiv2019

Inhibition of fatty acid oxidation as a new target to treat Primary Amoebic Meningoencephalitis by repurposing two well-known drugs

Primary Amoebic Meningoencephalitis (PAM) is a rapidly fatal infection caused by the free-living amoeba Naegleria fowleri. The disease mostly affects healthy children and young adults after contaminated water enters the nose, generally during recreational water activities. The amoeba migrate along the olfactory nerve to the brain, resulting in seizures, coma and eventually death. Previous research has shown that Naegleria gruberi, a close relative of N. fowleri, prefers lipids over glucose as an energy source. Therefore, we tested several inhibitors of fatty acid oxidation alongside the currently used drugs amphotericin B and miltefosine. Our data demonstrate that etomoxir, orlistat, perhexiline, thioridazine and valproic acid inhibited growth of N. gruberi. Furthermore, additive effects were seen when drugs were combined. Both thioridazine and valproic acid inhibit in vitro growth of N. gruberi in concentrations that can be obtained at the site of infection, which is doubtful with the currently used drugs amphotericin B and miltefosine. Both thioridazine and valproic acid have already been used for other diseases. As the development of new drugs and randomized controlled trials for this rare disease is nearly impossible, repurposing drugs is the most promising way to obtain additional drugs to combat PAM. Thioridazine and valproic acid are available drugs without major side-effects and can, therefore, be used as new complementary options in PAM therapy.

microbiology↗

bioRxiv2017

Diagnostic Prediction Tools For Bacteraemia Caused By 3rd Generation Cephalosporin-Resistant Enterobacteriaceae In Suspected Bacterial Infections: A Nested Case-Control Study

ObjectivesCurrent guidelines for empirical antibiotic treatment poorly predict the presence of 3rd generation cephalosporin resistant Enterobacteriaceae (3GC-R EB) as a cause of infection, thereby increasing unnecessary carbapenem use. We aimed to develop diagnostic scoring systems to better predict the presence of 3GC-R EB as a cause of bacteraemia.\n\nMethodsA retrospective nested case-control study was performed that included patients [≥]18 years in whom blood cultures were obtained and intravenous antibiotics were initiated. Each patient with 3GC-R EB bacteraemia was matched to four control infection episodes within the same hospital, based on blood culture date and onset location (community or hospital). Starting from 32 described clinical risk factors at infection onset, selection strategies were used to derive scoring systems for the probability of community- and hospital-onset 3GC-R EB bacteraemia.\n\nResults3GC-R EB bacteraemia occurred in 90 of 22,506 (0.4%) community-onset and in 82 of 8,110 (1.0%) hospital-onset infections, and these cases were matched to 360 community-onset and 328 hospital-onset control episodes, respectively. The derived community-onset and hospital-onset scoring system consisted of 6 and 9 predictors, respectively, with c-statistics of 0.807 (95% confidence interval 0.756-0.855) and 0.842 (0.794-0.887). With selected score cutoffs, the models identified 3GC-R EB bacteraemia with equal sensitivity as existing guidelines, but reduced the proportion of patients classified as at risk for 3GC-R EB bacteraemia (i.e. eligible for empiric carbapenem therapy) with 40% in patients with community-onset and 49% in patients with hospital-onset infection.\n\nConclusionsThese prediction rules for 3GC-R EB bacteraemia may reduce unnecessary empiric carbapenem use.

epidemiology↗