Coupling tRNAGly gene redundancy with staphylococcal cell wall integrity, antibiotic susceptibility and virulence potential
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PROVIDER: PRJNA1204950 | ENA |
REPOSITORIES: ENA
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Project description:Redundancy in the number of tRNA genes which occur across species is poorly understood and, in many cases, remains essentially unexplored. In Staphylococcus aureus, several tRNAGly genes encode isoacceptors that either participate in protein synthesis or cell wall formation, upon aminoacylation by the sole glycyl-tRNA synthetase (GlyRS). Transcription of GlyRS, is regulated by a glyS T-box riboswitch in the 5’UTR, which responds to all tRNAGly isoacceptors and exhibits species-specific RNA conformations. To address whether disruption of the tRNAGly expression impacts the balance between ribosomal translation and cell wall formation, we used CRISPR/Cas9 editing to ablate one proteinogenic tRNAGly gene copy which is the strongest ligand of the glyS T-box riboswitch. Interestingly, no discernible impact on the growth and general translational activity of S. aureus was observed, suggesting that the remaining tRNAs could make up for this deficiency. However, transcriptomics and proteomics analyses revealed that the edited strain had deficient cell wall integrity, and subsequent experimentation showed increased susceptibility to antibiotics targeting the cell wall and resistance to lysostaphin. Interestingly, the observed alteration in the proteome level were independent of the glycine codon usage. Moreover, the edited strain exhibited reduced biofilm formation but retained its ability to invade human cell cultures. Overall, the present study highlights the important role of tRNA gene copy redundancy in the physiology of an important pathogen like S. aureus and consolidates the regulatory role of tRNAs in metabolic homeostasis.
2025-07-16 | GSE285601 | GEO
Project description:Redundancy in the number of tRNA genes which occur across species is poorly understood and, in many cases, remains essentially unexplored. In Staphylococcus aureus, several tRNAGly genes encode isoacceptors that either participate in protein synthesis or cell wall formation, upon aminoacylation by the sole glycyl-tRNA synthetase (GlyRS). Transcription of GlyRS, is regulated by a glyS T-box riboswitch in the 5’UTR, which responds to all tRNAGly isoacceptors and exhibits species-specific RNA conformations. To address whether disruption of the tRNAGly expression impacts the balance between ribosomal translation and cell wall formation, we used CRISPR/Cas9 editing to ablate one proteinogenic tRNAGly gene copy which is the stronger ligand of the glyS T-box riboswitch. Interestingly, no discernible impact on the growth and translational activity of S. aureus was observed, suggesting that the remaining tRNAs could make up for this deficiency. However, transcriptomics and proteomics analyses revealed that the edited strain had deficient cell wall integrity, and subsequent experimentation showed increased susceptibility to antibiotics targeting the cell wall. Interestingly, the observed alteration in the proteome level were independent of the glycine codon usage. Moreover, the edited strain exhibited reduced biofilm formation but retained its ability to invade human cell cultures. Overall, the present study highlights the important role of tRNA gene copy redundancy in the physiology of an important pathogen like S. aureus and consolidates the regulatory role of tRNAs in metabolic homeostasis.
2025-07-21 | PXD059048 | Pride
Project description:Chitin is an important fungal cell wall component that is cross-linked to β-glucan for structural integrity. Acquisition of chitin to glucan cross-links has previously been shown to be performed by transglycosylation enzymes in S. cerevisiae, called Congo Red hypersensitive (Crh) enzymes. Here, we characterized the impact of deleting all seven gene members of the crh gene family (crhA-G) in Aspergillus niger on cell wall integrity, cell wall composition and genome-wide gene expression. In this study, we show that the seven-fold knockout strain shows slightly compact plate growth, but no increased sensitivity to cell wall perturbing compounds. Additionally, we found that the cell wall composition of this knockout strain was virtually identical to that of the wild type. In congruence with these data, genome-wide expression analysis revealed very limited changes in gene expression and no signs of activation of the cell wall integrity response pathway. However, deleting the entire crh gene family in cell wall mutants that are deficient in either galactofuranose (ΔugmA) or α-glucan (ΔagsE and ΔagsA, ΔagsE), resulted in a synthetic growth defect and an increased sensitivity towards Congo Red compared the parental strains. Altogether, these results indicate that loss of the crh gene family in A. niger does not trigger the cell wall integrity response in itself, but does play an important role in ensuring cell wall integrity in mutant strains with reduced galactofuranose or α-glucan.
2019-12-21 | GSE142461 | GEO
Project description:Synchronizing production of antibacterial compounds and integration of DNA released by dead cells, competence is one of the most efficient bacterial evolutionary and adaptative strategies. In most streptococci, this tactic is orchestrated by the ComRS system, a pheromone communication device providing competence bimodal initiation and sharp time window of activation. Understanding how this developmental process integrates multiple inputs to fine-tune the adequate response is a long-standing question. Actually, essential genes involved in the regulation of ComRS have been challenging to study. In this work, we built a conditional mutant library using the CRISPR-interference technology and developed three complementary screens to investigate competence genetic regulation in the human commensal Streptococus salivarius. We highlighted that competence increases upon cell-wall impairment, suggesting a connection between cell envelope stress and competence activation. Notably, we report the key role played by StkP, a serine-threonine kinase known to regulate cell-wall assembly. We showed that StkP controls competence by a mechanism responding to peptidoglycan fragments. Together, those data suggest a key cell-wall sensing mechanism coupling competence to cell envelope integrity.
2022-08-25 | GSE204976 | GEO
Project description:Plant cell walls maintain their functional integrity during growth, defense and turgor generation through a dedicated cell wall integrity (CWI) maintenance mechanism. Upon detection of integrity impairment due to cell wall damage (CWD) compensatory responses are activated to maintain CWI. Responses include production of salicylic acid (SA), jasmonic acid (JA) and lignin as well as growth inhibition. Here we investigated the processes responsible for inducing growth inhibition upon CWD in Arabidopsis thaliana seedlings. Genetic studies implicated NITRATE REDUCTASE1 2 (NIA1 2) in the response to CWD. RNAseq-based transcriptomics showed that nearly all the CWD-induced transcript changes detectable in Col-0 were absent in nia1 2 seedlings.
2018-01-25 | GSE109613 | GEO
Project description:The cell wall is essential for viability of fungi and is an effective drug target in pathogens such as Candida albicans. The contribution of posttranscriptional gene regulators to cell wall integrity in C. albicans is unknown. We show that the C. albicans Ccr4-Pop2 mRNA deadenylase, a regulator of mRNA stability and translation, is required for cell wall integrity. The ccr4/pop2 mutants display reduced wall β-glucans and sensitivity to the echinocandin caspofungin. Moreover, the deadenylase mutants are compromised for filamentation and virulence. We demonstrate that defective cell walls in the ccr4/pop2 mutants are linked to dysfunctional mitochondria and phospholipid imbalance. To further understand mitochondrial function in cell wall integrity, we screened a Saccharomyces cerevisiae collection of mitochondrial mutants. We identify several mitochondrial proteins required for caspofungin tolerance and find a connection between mitochondrial phospholipid homeostasis and caspofungin sensitivity. We focus on the mitochondrial outer membrane SAM complex subunit Sam37, demonstrating it is required for both trafficking of phospholipids between the ER and mitochondria and cell wall integrity. Moreover, in C. albicans also Sam37 is essential for caspofungin tolerance. Our study provides the basis for an integrative view of mitochondrial function in fungal cell wall biogenesis and resistance to echinocandin antifungal drugs. Two-color experimental design comparing cells with a double-knockout of the CCR4 genes to cells with a reintegrated CCR4 gene.
2010-12-10 | E-GEOD-25891 | biostudies-arrayexpress
Project description:Intraflagellar transport (IFT) is a microtubule-based system that supports the assembly and maintenance of cilia, an essential organelle for development and maintenance of cellular integrity. Dysfunction of IFT was shown to lead to a wide spectrum of disease phenotypes, so called ciliopathies of variable severity. In previous studies two distinct complexes (IFT-A; IFT-B) were identified, containing multiple proteins that are part of the IFT machinery. Two of the IFT-B components are TTC30A and TTC30B and in contrast to all other IFT components, only these two are paralogues. To investigate whether these two proteins have a similar and essential ciliary or constitute redundant functions, CRIPSR/Cas9 was used to specifically knock-out TTC30A or B and to generate a double knock-out. Localization studies showed a redundancy of both proteins in ciliogenesis while polyglutamylation of cilia was affected by both single knockouts. Further, the localization of other IFT components was not affected by the depletion of a single paralogue. A loss of both proteins led to a severe ciliogenesis defect, resulting in no cilia formation which can be rescued by expression of TTC30A or B. The redundancy can be explained by highly similar interaction patterns of both paralogues, both equally interact with the IFT-B machinery. In summary our study demonstrates that a loss of one TTC30 paralogue can mostly compensate severe ciliary defects, due to redundancy. However, cells assemble shorter cilia which are potentially limited in their functions, especially because of impaired polyglutamylation. A complete loss of both proteins leads to a deficit in IFT complex B integrity followed by disrupted IFT and subsequently no cilia.
2023-12-28 | PXD033614 | Pride
Project description:Intraflagellar transport (IFT) is a microtubule-based system that supports the assembly and maintenance of cilia, an essential organelle for development and maintenance of cellular integrity. Dysfunction of IFT was shown to lead to a wide spectrum of disease phenotypes, so called ciliopathies of variable severity. In previous studies two distinct complexes (IFT-A; IFT-B) were identified, containing multiple proteins that are part of the IFT machinery. Two of the IFT-B components are TTC30A and TTC30B and in contrast to all other IFT components, only these two are paralogues. To investigate whether these two proteins have a similar and essential ciliary or constitute redundant functions, CRIPSR/Cas9 was used to specifically knock-out TTC30A or B and to generate a double knock-out. Localization studies showed a redundancy of both proteins in ciliogenesis while polyglutamylation of cilia was affected by both single knockouts. Further, the localization of other IFT components was not affected by the depletion of a single paralogue. A loss of both proteins led to a severe ciliogenesis defect, resulting in no cilia formation which can be rescued by expression of TTC30A or B. The redundancy can be explained by highly similar interaction patterns of both paralogues, both equally interact with the IFT-B machinery. In summary our study demonstrates that a loss of one TTC30 paralogue can mostly compensate severe ciliary defects, due to redundancy. However, cells assemble shorter cilia which are potentially limited in their functions, especially because of impaired polyglutamylation. A complete loss of both proteins leads to a deficit in IFT complex B integrity followed by disrupted IFT and subsequently no cilia formation
2022-08-12 | PXD034412 | Pride
Project description:The human pathogen Staphylococcus aureus encodes a specialised type VII secretion system (T7SS), which plays an important role in bacterial virulence during infection. However, the functions the T7SS during infection and in bacterial physiology remain unclear. Here we demonstrate that S. aureus strains lacking the transporter EssC as well as the T7SS effectors, EsxC and EsxA were highly sensitive to the important last resort drug, daptomycin as well as other membrane-targeting antibiotics, including gramicidin and bithionol. To understand how T7SS mediates increased antibiotic sensitivity, we investigated the impact of T7SS on the staphylococcal cell envelope. Interestingly, T7SS mutants displayed decreased membrane fluidity with altered localisation of cell membrane proteins such as flotillin under normal growth conditions. LC/MS analysis showed different protein profiles in the mutant membrane preparations compared to the WT, suggesting that T7SS impacts membrane homeostasis. Scanning electron microscopy analysis demonstrated distinct cell surface morphologies for the T7SS mutants, with altered cell wall synthesis as well cell surface charge. In line with the increased negative surface charge, daptomycin binding was enhanced in the mutants, which demonstrated increased membrane permeability when treated with the drug. T7SS mutants were more sensitive to daptomycin during intracellular infection, and furthermore, in a murine skin infection model, esxC mutants survived less than the WT when treated with daptomycin. Thus, our data show that the T7SS impacts sensitivity of S. aureus to membrane-acting drugs such as daptomycin through modulation of cell membrane integrity, indicating its potential as a drug target
2024-04-19 | PXD047096 | Pride
Project description:A phosphoproteomic evaluation of the cellular response to micafungin (cell wall perturbant) in the filamentous fungi, A. nidulans, reveals putative, currently unknown cell wall integrity signaling (CWIS) associated proteins. This study used thirteen timepoints were taken from 0 to 10 minutes after cell wall exposure with two biological and two technical replicates. All data was processed using label free quantification with MaxQuant software.
2020-07-16 | PXD015038 | Pride