Insertion Pool Sequencing Identifies Essential Fungal Virulence Factors in vivo
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB23309 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR2190340_1.fastq.gz | Fastqsanger.gz | |||
| ERR2190340_2.fastq.gz | Fastqsanger.gz | |||
| ERR2190341_1.fastq.gz | Fastqsanger.gz | |||
| ERR2190341_2.fastq.gz | Fastqsanger.gz | |||
| ERR2190342_1.fastq.gz | Fastqsanger.gz |
Items per page: 5 1 - 5 of 24 |
Similar Datasets
Project description:Large-scale insertional mutagenesis screens can be powerful genome-wide tools if they are streamlined with efficient downstream analysis, which is a serious bottleneck in complex biological systems. A major impediment to the success of next-generation sequencing (NGS)-based screens for virulence factors is that the genetic material of pathogens is often underrepresented within the eukaryotic host, making detection extremely challenging. We therefore established insertion Pool-Sequencing (iPool-Seq) on maize infected with the biotrophic fungus U. maydis. iPool-Seq features tagmentation, unique molecular barcodes, and affinity purification of pathogen insertion mutant DNA from in vivo-infected tissues. In a proof of concept using iPool-Seq, we identified 28 virulence factors, including 23 that were previously uncharacterized, from an initial pool of 195 candidate effector mutants. Because of its sensitivity and quantitative nature, iPool-Seq can be applied to any insertional mutagenesis library and is especially suitable for genetically complex setups like pooled infections of eukaryotic hosts.
| S-EPMC5912717 | biostudies-literature
Project description:Fungal acetylome comparative analysis identifies an essential role of acetylation in human fungal pathogen virulence
| PRJNA476875 | ENA
Project description:acylation also called S-palmitoylation is an important reversible protein post-translational modification in organisms. However, its function remains unknown in pathogenic fungi. Here, we found treatment of rice false fungus Ustilaginoidea virens with S-palmitoylation inhibitor 2-BP resulted in a significant decrease in fungal virulence and growth rate. Deletion of the S-acyltransferase proteins from U. virens indicated that two S-acyltransferases UvPfa3 and UvPfa4 were required for fungal virulence. Comprehensive identification of S-palmitoylation sites and proteins in U. virens revealed a total of 4089 S-palmitoylation sites were identified within 2192 proteins and S-palmitoylation proteins were involved in diverse biological processes. Interestingly, S-palmitoylation proteins were significantly enriched in mitogen-activated protein kinase (MAPK) pathway and MAP kinase UvSlt2 was confirmed to be a S-palmitoylation protein which was palmitoylated by UvPfa4. Mutations of S-palmitoylation sites in UvSlt2 resulted in significantly reduced fungal virulence and decreased kinase enzymatic activity and phosphorylation level. Molecular dynamics simulations demonstrated mutation of S-palmitoylation sites in UvSlt2 caused decreased hydrophobic solvent-accessible surface area, and thereby affected binding between the UvSlt2 enzyme and substrates. Taken together, S-palmitoylation promotes U. virens virulence through palmitoylating MAP kinase UvSlt2 by palmitoyltransferase UvPfa4 and enhancing enzymatic activity and phosphorylation level of the kinase, thereby increasing hydrophobic solvent-accessible surface area and binding activity between the UvSlt2 enzyme and its substrates. Our studies provide a framework for dissecting the biological functions of S-palmitoylation, and reveal an important role for S-palmitoylation in regulating virulence of pathogen.
2024-10-14 | PXD054433 | Pride
Project description:acylation also called S-palmitoylation is an important reversible protein post-translational modification in organisms. However, its function remains unknown in pathogenic fungi. Here, we found treatment of rice false fungus Ustilaginoidea virens with S-palmitoylation inhibitor 2-BP resulted in a significant decrease in fungal virulence and growth rate. Deletion of the S-acyltransferase proteins from U. virens indicated that two S-acyltransferases UvPfa3 and UvPfa4 were required for fungal virulence. Comprehensive identification of S-palmitoylation sites and proteins in U. virens revealed a total of 4089 S-palmitoylation sites were identified within 2192 proteins and S-palmitoylation proteins were involved in diverse biological processes. Interestingly, S-palmitoylation proteins were significantly enriched in mitogen-activated protein kinase (MAPK) pathway and MAP kinase UvSlt2 was confirmed to be a S-palmitoylation protein which was palmitoylated by UvPfa4. Mutations of S-palmitoylation sites in UvSlt2 resulted in significantly reduced fungal virulence and decreased kinase enzymatic activity and phosphorylation level. Molecular dynamics simulations demonstrated mutation of S-palmitoylation sites in UvSlt2 caused decreased hydrophobic solvent-accessible surface area, and thereby affected binding between the UvSlt2 enzyme and substrates. Taken together, S-palmitoylation promotes U. virens virulence through palmitoylating MAP kinase UvSlt2 by palmitoyltransferase UvPfa4 and enhancing enzymatic activity and phosphorylation level of the kinase, thereby increasing hydrophobic solvent-accessible surface area and binding activity between the UvSlt2 enzyme and its substrates. Our studies provide a framework for dissecting the biological functions of S-palmitoylation, and reveal an important role for S-palmitoylation in regulating virulence of pathogen.
2024-10-14 | PXD048216 | Pride
Project description:Lysine acetylation is critical in regulating important biological processes in many organisms, yet little is known about acetylome evolution and its contribution to phenotypic diversity. Here, we compare the acetylomes of baker’s yeast and the three deadliest human fungal pathogens, Cryptococcus neoformans, Candida albicans, and Aspergillus fumigatus. Using mass spectrometry enriched for acetylated peptides together with public data from Saccharomyces cerevisiae, we show that fungal acetylomes are characterized by dramatic evolutionary dynamics and limited conservation in core biological processes. Notably, the levels of protein acetylation in pathogenic fungi correlate with their pathogenicity. Using gene knockouts and pathogenity assays in mice, we identify deacetylases with critical roles in virulence and protein translation elongation. Finally, through mutational analysis of deactylation motifs we find evidence of positive selection at specific acetylation motifs in fungal pathogens. These results shed new light on the pathogenicity regulation mechanisms underlying the evolution of fungal acetylomes.
2019-04-26 | GSE116040 | GEO
Project description:'Mutagenomics' is a combination approach of random mutagenesis, phenotypic screening, and whole-genome re-sequencing to uncover all tagged and untagged mutations linked with phenotypic changes in an organism. In this study, we performed a mutagenomics screen on the wheat pathogenic fungus Zymoseptoria tritici for altered morphogenetic switching and stress sensitivity phenotypes using Agrobacterium-mediated “random” T-DNA mutagenesis (ATMT). Biological screening identified four mutants which were strongly reduced in virulence on wheat. Whole genome re-sequencing defined the positions of the T-DNA insertion events and revealed several unlinked mutations potentially also affecting gene functions. Remarkably, two independent reduced virulence mutant strains, with similarly altered stress sensitivities and aberrant hyphal growth phenotypes, were found to have distinct loss of function mutations in the ZtSSK2 MAPKKK gene. One mutant strain had a direct T-DNA insertion affecting the N-terminus of the predicted protein, whilst the other possessed an unlinked frameshift mutation towards the C-terminus. We used genetic complementation to restore both strains' wild-type (WT) function (virulence, morphogenesis, and stress response). We demonstrated that ZtSSK2 has a non-redundant function with ZtSTE11 in virulence through the biochemical activation of the stress-activated HOG1 MAPK pathway. Moreover, we present data suggesting that SSK2 has a unique role in activating this pathway in response to specific stresses. Finally, dual RNAseq-based transcriptome profiling of WT and SSK2 mutant strains revealed many HOG1-dependent transcriptional changes in the fungus during early infection and suggested that the host response does not discriminate between WT and mutant strains during this early phase. Together these data define new genes implicated in the virulence of the pathogen and emphasise the importance of a whole genome sequencing step in mutagenomic discovery pipelines.
2023-02-25 | GSE225623 | GEO
Project description:Interventions: Gold Standard:Histopathological diagnosis;Index test:AmoyDx Essential NGS Panel (Reversible Terminator Sequencing)
Primary outcome(s): EGFR 20 insertion mutation;consistency
Study Design: Factorial
| 2699936 | ecrin-mdr-crc
Project description:An important, but rarely performed, test of Koch’s molecular postulates involves evaluating the capacity of candidate virulence genes to confer pathogenicity in otherwise non-virulent species. Unbiased genomic surveys of avirulent natural isolates might reveal rare variants possessing specific virulence features, which might prove useful in testing their functional sufficiency. Using a custom pan-genome array, we analyzed a panel of avirulent Burkholderia thailandensis (Bt) isolates related to Burkholderia pseudomallei (Bp), the causative agent of the often fatal human and animal disease melioidosis. We report the discovery of variant Bt isolates exhibiting isolated acquisition of a capsular polysaccharide biosynthesis gene cluster (BpCPS), long regarded as an critical species-specific virulence factor essential for Bp mammalian virulence. BpCPS-expressing Bt strains exhibited certain pathogen-related phenotypes including resistance to human complement binding, but did not exhibit enhanced virulence when assessed in two different in vivo animal infection models. Phylogenetic analysis revealed that the BpCPS-expressing Bt strains likely reside within an evolutionary subgroup distinct from the majority of previously-described Bt strains. Our findings suggest that BpCPS acquisition alone is unlikely to fully explain the ability of Bp to colonize humans and animals, highlighting the importance of other collaborating factors in the pathogenesis of mammalian melioidosis. Genomic DNA of several Bt strains were hybridized against a common reference strain (Bt E264), to see gain/loss
2010-08-14 | E-GEOD-18766 | biostudies-arrayexpress
Project description:Rapid adaptation to grow within the physiological conditions found in the host environment is an essential but poorly understood virulence requirement for systemic pathogens such as Streptococcus pneumoniae. We have now demonstrated an essential role for the one-carbon metabolism pathway and a conditional role depending on strain background for proline biosynthesis for S. pneumoniae growth in serum or CSF and therefore for systemic virulence. RNAseq data demonstrated that loss of one carbon metabolism or proline biosynthesis both have profound but differing effects on S. pneumoniae metabolism in human serum, identifying the metabolic processes dependent on each pathway during systemic infection. These data provide a more detailed understanding of the adaptations required by systemic bacterial pathogens in order to cause infection, and demonstrate that the requirement for some of these adaptations vary between strains from the same species and could therefore underpin strain variations in virulence potential.
2023-09-10 | E-MTAB-13289 | biostudies-arrayexpress