The involvement of Mycobacterium type III-A CRISPR-Cas system in oxidative stress and intracellular fitness
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA699439 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR13627324_1.fastq.gz | Fastqsanger.gz | |||
| SRR13627324_2.fastq.gz | Fastqsanger.gz | |||
| SRR13627325_1.fastq.gz | Fastqsanger.gz | |||
| SRR13627325_2.fastq.gz | Fastqsanger.gz | |||
| SRR13627326_1.fastq.gz | Fastqsanger.gz |
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Project description:Previous studies suggest that type I and type II CRISPR-Cas systems were employed to evade host immunity by targeting interference of bacteria’s own genes. Although Mycobacterium bovis (M. bovis) and Mycobacterium tuberculosis (M. tuberculosis) possess integrated type III-A CRISPR-Cas system, its role in mycobacteria remains obscure. Here, we observed that seven cas genes (csm2~6, cas10, cas6) were significantly upregulated under oxidative stress treatment. To explore the function of type III-A CRSIRP-Cas system, Total CRISPR system (TCR) mutant strain was generated in M. bovis Bacille Calmette-Guérin (BCG). Deletion of TCR results in increased sensitivity in response to H2O2 and reduced cell envelope integrity. By using RNA-Seq, 590 differential expression genes were found in mutant for TCR, of which 220 genes were upregulated and 370 genes were downregulated, indicating an important role of TCR in controlling gene expression in mycobacteria. Consistently, disrupting TCR results in poor intracellular survival in vivo and in vitro. Moreover, we showed for the first time that TCR contributed to the regulation of regulatory T cell population, supporting a role of TCR in modulating host immunity. These observations demonstrate that type III-A CRISPR-Cas system as an important factor for intracellular survival and host immunoregulation in mycobacteria, may be a potential target for therapy.
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Project description:Type I and type II CRISPR-Cas systems are employed to evade host immunity by targeting interference of bacteria's own genes. Although Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis, possesses integrated type III-A CRISPR-Cas system, its role in mycobacteria remains obscure. Here, we observed that seven cas genes (csm2∼5, cas10, cas6) were upregulated in Mycobacterium bovis BCG under oxidative stress treatment, indicating the role of type III-A CRISPR-Cas system in oxidative stress. To explore the functional role of type III-A CRISPR-Cas system, TCC (Type III-A CRISPR-Cas system, including cas6, cas10, and csm2-6) mutant was generated. Deletion of TCC results in increased sensitivity in response to hydrogen peroxide and reduced cell envelope integrity. Analysis of RNA-seq dataset revealed that TCC impacted on the oxidation-reduction process and the composition of cell wall which is essential for mycobacterial envelop integrity. Moreover, disrupting TCC led to poor intracellular survival in vivo and in vitro. Finally, we showed for the first time that TCC contributed to the regulation of regulatory T cell population, supporting a role of TCC in modulating host immunity. Our finding reveals the important role of TCC in cell envelop homeostasis. Our work also highlights type III-A CRISPR-Cas system as an important factor for intracellular survival and host immunoregulation in mycobacteria, thus may be a potential target for therapy.
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Project description:Bacteria protect themselves from infection by bacteriophages (phages) using different defence systems, such as CRISPR-Cas. Although CRISPR-Cas provides phage resistance, fitness costs are incurred, such as through autoimmunity. CRISPR-Cas regulation can optimise defence and minimise these costs. We recently developed a genome-wide functional genomics approach (SorTn-seq) for high-throughput discovery of regulators of bacterial gene expression. Here, we applied SorTn-seq to identify loci influencing expression of the two type III-A Serratia CRISPR arrays. Multiple genes affected CRISPR expression, including those involved in outer membrane and lipopolysaccharide synthesis. By comparing loci affecting type III CRISPR arrays and cas operon expression, we identified PigU (LrhA) as a repressor that co-ordinately controls both arrays and cas genes. By repressing type III-A CRISPR-Cas expression, PigU shuts off CRISPR-Cas interference against plasmids and phages. PigU also represses interference and CRISPR adaptation by the type I-F system, which is also present in Serratia. RNA sequencing demonstrated that PigU is a global regulator that controls secondary metabolite production and motility, in addition to CRISPR-Cas immunity. Increased PigU also resulted in elevated expression of three Serratia prophages, indicating their likely induction upon sensing PigU-induced cellular changes. In summary, PigU is a major regulator of CRISPR-Cas immunity in Serratia.
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Project description:CRISPR-Cas technology has transformed functional genomics, yet understanding of how individual exons differentially shape cellular phenotypes remains limited. Here, we optimized and conducted massively parallel exon deletion and splice-site mutation screens in human cell lines to identify exons that regulate cellular fitness. Fitness-promoting exons are prevalent in essential and highly expressed genes and commonly overlap with protein domains and interaction interfaces. Conversely, fitness-suppressing exons are enriched in nonessential genes, exhibiting lower inclusion levels, and overlap with intrinsically disordered regions and disease-associated mutations. In-depth mechanistic investigation of the screen-hit TAF5 alternative exon-8 revealed that its inclusion is required for assembly of the TFIID general transcription initiation complex, thereby regulating global gene expression output. Collectively, our orthogonal exon perturbation screens established a comprehensive repository of phenotypically important exons and uncovered regulatory mechanisms governing cellular fitness and gene expression.
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