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The Involvement of Mycobacterium Type III-A CRISPR-Cas System in Oxidative Stress.

ABSTRACT: 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.

SUBMITTER: Yang F

PROVIDER: S-EPMC8696179 | biostudies-literature |

REPOSITORIES: biostudies-literature

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The involvement of Mycobacterium type III-A CRISPR-Cas system in oxidative stress and intracellular fitness

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.

2021-02-05 | GSE166137 | GEO

The involvement of Mycobacterium type III-A CRISPR-Cas system in oxidative stress and intracellular fitness

Project description:The involvement of Mycobacterium type III-A CRISPR-Cas system in oxidative stress and intracellular fitness

| PRJNA699439 | ENA

Lactococcus lactis type III-A CRISPR-Cas system cleaves bacteriophage RNA.

Project description:CRISPR-Cas defends microbial cells against invading nucleic acids including viral genomes. Recent studies have shown that type III-A CRISPR-Cas systems target both RNA and DNA in a transcription-dependent manner. We previously found a type III-A system on a conjugative plasmid in Lactococcus lactis which provided resistance against virulent phages of the Siphoviridae family. Its naturally occurring spacers are oriented to generate crRNAs complementary to target phage mRNA, suggesting transcription-dependent targeting. Here, we show that only constructs whose spacers produce crRNAs complementary to the phage mRNA confer phage resistance in L. lactis. In vivo nucleic acid cleavage assays showed that cleavage of phage dsDNA genome was not detected within phage-infected L. lactis cells. On the other hand, Northern blots indicated that the lactococcal CRISPR-Cas cleaves phage mRNA in vivo. These results cannot exclude that single-stranded phage DNA is not being targeted, but phage DNA replication has been shown to be impaired.

| S-EPMC6546414 | biostudies-literature

Type III CRISPR-Cas Systems: Deciphering the Most Complex Prokaryotic Immune System.

Project description:The emergence and persistence of selfish genetic elements is an intrinsic feature of all living systems. Cellular organisms have evolved a plethora of elaborate defense systems that limit the spread of such genetic parasites. CRISPR-Cas are RNA-guided defense systems used by prokaryotes to recognize and destroy foreign nucleic acids. These systems acquire and store fragments of foreign nucleic acids and utilize the stored sequences as guides to recognize and destroy genetic invaders. CRISPR-Cas systems have been extensively studied, as some of them are used in various genome editing technologies. Although Type III CRISPR-Cas systems are among the most common CRISPR-Cas systems, they are also some of the least investigated ones, mostly due to the complexity of their action compared to other CRISPR-Cas system types. Type III effector complexes specifically recognize and cleave RNA molecules. The recognition of the target RNA activates the effector large subunit - the so-called CRISPR polymerase - which cleaves DNA and produces small cyclic oligonucleotides that act as signaling molecules to activate auxiliary effectors, notably non-specific RNases. In this review, we provide a historical overview of the sometimes meandering pathway of the Type III CRISPR research. We also review the current data on the structures and activities of Type III CRISPR-Cas systems components, their biological roles, and evolutionary history. Finally, using structural modeling with AlphaFold2, we show that the archaeal HRAMP signature protein, which heretofore has had no assigned function, is a degenerate relative of Type III CRISPR-Cas signature protein Cas10, suggesting that HRAMP systems have descended from Type III CRISPR-Cas systems or their ancestors.

| S-EPMC8527444 | biostudies-literature

Target sequence requirements of a type III-B CRISPR-Cas immune system.

Project description:CRISPR-Cas systems are RNA-based immune systems that protect many prokaryotes from invasion by viruses and plasmids. Type III CRISPR systems are unique, as their targeting mechanism requires target transcription. Upon transcript binding, DNA cleavage by type III effector complexes is activated. Type III systems must differentiate between invader and native transcripts to prevent autoimmunity. Transcript origin is dictated by the sequence that flanks the 3' end of the RNA target site (called the PFS). However, how the PFS is recognized may vary among different type III systems. Here, using purified proteins and in vitro assays, we define how the type III-B effector from the hyperthermophilic bacterium Thermotoga maritima discriminates between native and invader transcripts. We show that native transcripts are recognized by base pairing at positions -2 to -5 of the PFS and by a guanine at position -1, which is not recognized by base pairing. We also show that mismatches with the RNA target are highly tolerated in this system, except for those nucleotides adjacent to the PFS. These findings define the target requirement for the type III-B system from T. maritima and provide a framework for understanding the target requirements of type III systems as a whole.

| S-EPMC6664175 | biostudies-literature

Genetic characterization of antiplasmid immunity through a type III-A CRISPR-Cas system.

Project description:Many prokaryotes possess an adaptive immune system encoded by clustered regularly interspaced short palindromic repeats (CRISPRs). CRISPR loci produce small guide RNAs (crRNAs) that, in conjunction with flanking CRISPR-associated (cas) genes, combat viruses and block plasmid transfer by an antisense targeting mechanism. CRISPR-Cas systems have been classified into three types (I to III) that employ distinct mechanisms of crRNA biogenesis and targeting. The type III-A system in Staphylococcus epidermidis RP62a blocks the transfer of staphylococcal conjugative plasmids and harbors nine cas-csm genes. Previous biochemical analysis indicated that Cas10, Csm2, Csm3, Csm4, and Csm5 form a crRNA-containing ribonucleoprotein complex; however, the roles of these genes toward antiplasmid targeting remain unknown. Here, we determined the cas-csm genes that are required for antiplasmid immunity and used genetic and biochemical analyses to investigate the functions of predicted motifs and domains within these genes. We found that many mutations affected immunity by impacting the formation of the Cas10-Csm complex or crRNA biogenesis. Surprisingly, mutations in the predicted nuclease domains of the members of the Cas10-Csm complex had no detectable effect on antiplasmid immunity or crRNA biogenesis. In contrast, the deletion of csm6 and mutations in the cas10 Palm polymerase domain prevented CRISPR immunity without affecting either complex formation or crRNA production, suggesting their involvement in target destruction. By delineating the genetic requirements of this system, our findings further contribute to the mechanistic understanding of type III CRISPR-Cas systems.

| S-EPMC3911255 | biostudies-literature

Reprogramming the endogenous type III-A CRISPR-Cas system for genome editing and RNA interference in Mycobacterium tuberculosis

Project description:Purpose: Understanding the functional genomics of M.tb (Mycobacterium tuberculosis) and development of novel anti-M.tb drugs and vaccines needs an efficient gene edit tool. The aim of this study was to describe an easy and efficient gene edit tool for functional genomics study of M.tb. Method: A plasmid was designed containing mini CRISPR array and 400bp up and downside homologues DNA sequences from the target gene interposed by EGFP or BFP. This plasmid was transformed into M.tb that guided the endogenous CRISPR system of M.tb to cut the target gene and insert EGFP or BFP through homologues ends joining. The EGFP/BFP insertion was confirmed and the total genomic was extracted from mutated and wild type strains and subjected to High-throughput DNA sequencing. Results: The raw data was filtered by Trimmomatic and the clean reads were mapped to M.tb H37Ra reference genome with bwa 99.94%, and 99.97% genome of wag31 and esxQ deletion strains were covered respectively.

2020-08-30 | GSE103276 | GEO

Spacer acquisition by Type III CRISPR-Cas system during bacteriophage infection of Thermus thermophilus.

Project description:Type III CRISPR-Cas systems provide immunity to foreign DNA by targeting its transcripts. Target recognition activates RNases and DNases that may either destroy foreign DNA directly or elicit collateral damage inducing death of infected cells. While some Type III systems encode a reverse transcriptase to acquire spacers from foreign transcripts, most contain conventional spacer acquisition machinery found in DNA-targeting systems. We studied Type III spacer acquisition in phage-infected Thermus thermophilus, a bacterium that lacks either a standalone reverse transcriptase or its fusion to spacer integrase Cas1. Cells with spacers targeting a subset of phage transcripts survived the infection, indicating that Type III immunity does not operate through altruistic suicide. In the absence of selection spacers were acquired from both strands of phage DNA, indicating that no mechanism ensuring acquisition of RNA-targeting spacers exists. Spacers that protect the host from the phage demonstrate a very strong strand bias due to positive selection during infection. Phages that escaped Type III interference accumulated deletions of integral number of codons in an essential gene and much longer deletions in a non-essential gene. This and the fact that Type III immunity can be provided by plasmid-borne mini-arrays open ways for genomic manipulation of Thermus phages.

| S-EPMC7515739 | biostudies-literature

A type III-A CRISPR-Cas system employs degradosome nucleases to ensure robust immunity.

Project description:CRISPR-Cas systems provide sequence-specific immunity against phages and mobile genetic elements using CRISPR-associated nucleases guided by short CRISPR RNAs (crRNAs). Type III systems exhibit a robust immune response that can lead to the extinction of a phage population, a feat coordinated by a multi-subunit effector complex that destroys invading DNA and RNA. Here, we demonstrate that a model type III system in Staphylococcus epidermidis relies upon the activities of two degradosome-associated nucleases, PNPase and RNase J2, to mount a successful defense. Genetic, molecular, and biochemical analyses reveal that PNPase promotes crRNA maturation, and both nucleases are required for efficient clearance of phage-derived nucleic acids. Furthermore, functional assays show that RNase J2 is essential for immunity against diverse mobile genetic elements originating from plasmid and phage. Altogether, our observations reveal the evolution of a critical collaboration between two nucleic acid degrading machines which ensures cell survival when faced with phage attack.

| S-EPMC6447361 | biostudies-literature

Programmable type III-A CRISPR-Cas DNA targeting modules.

Project description:The CRISPR-Cas systems provide invader defense in a wide variety of prokaryotes, as well as technologies for many powerful applications. The Type III-A or Csm CRISPR-Cas system is one of the most widely distributed across prokaryotic phyla, and cleaves targeted DNA and RNA molecules. In this work, we have constructed modules of Csm systems from 3 bacterial species and heterologously expressed the functional modules in E. coli. The modules include a Cas6 protein and a CRISPR locus for crRNA production, and Csm effector complex proteins. The expressed modules from L. lactis, S. epidermidis and S. thermophilus specifically eliminate invading plasmids recognized by the crRNAs of the systems. Characteristically, activation of plasmid targeting activity depends on transcription of the plasmid sequence recognized by the crRNA. Activity was not observed when transcription of the crRNA target sequence was blocked, or when the opposite strand or a non-target sequence was transcribed. Moreover, the Csm module can be programmed to recognize plasmids with novel target sequences by addition of appropriate crRNA coding sequences to the module. These systems provide a platform for investigation of Type III-A CRISPR-Cas systems in E. coli, and for introduction of programmable transcription-activated DNA targeting into novel organisms.

| S-EPMC5404769 | biostudies-literature

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