scholarly journals A Type III-A CRISPR-Cas system employs degradosome nucleases to ensure robust immunity

2019 ◽  
Author(s):  
Lucy Chou-Zheng ◽  
Asma Hatoum-Aslan
Keyword(s):  
Immune Response ◽  
Staphylococcus Epidermidis ◽  
Mobile Genetic Elements ◽  
Genetic Elements ◽  
Type Iii ◽  
Robust Immune Response ◽  
Functional Assays ◽  
Dna And Rna ◽  
Biochemical Analyses ◽  
Phage Population

AbstractCRISPR-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.

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

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Lucy Chou-Zheng ◽  
Asma Hatoum-Aslan
Keyword(s):  
Immune Response ◽  
Staphylococcus Epidermidis ◽  
Mobile Genetic Elements ◽  
Genetic Elements ◽  
Type Iii ◽  
Robust Immune Response ◽  
Functional Assays ◽  
Dna And Rna ◽  
Biochemical Analyses ◽  
Phage Population

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.

scholarly journals Chromosomal Targeting by the Type III-A CRISPR-Cas System Can Reshape Genomes in Staphylococcus aureus

mSphere ◽  
2017 ◽  
Vol 2 (6) ◽  
Author(s):  
Jing Guan ◽  
Wanying Wang ◽  
Baolin Sun
Keyword(s):  
Staphylococcus Aureus ◽  
Molecular Mechanisms ◽  
Mobile Genetic Elements ◽  
Industrial Applications ◽  
Partial Deletion ◽  
Content Type ◽  
Genetic Elements ◽  
Type Iii ◽  
Wide Range ◽  
Novel Strategy

ABSTRACT Staphylococcus aureus is a pathogen that can cause a wide range of infections in humans. Studies have suggested that CRISPR-Cas systems can drive the loss of integrated mobile genetic elements (MGEs) by chromosomal targeting. Here we demonstrate that CRISPR-mediated cleavage contributes to the partial deletion of integrated SCCmec in methicillin-resistant S. aureus (MRSA), which provides a strategy for the treatment of MRSA infections. The spacer within artificial CRISPR arrays should contain more than 25 nucleotides for immunity, and consecutive trinucleotide pairings between a selected target and the 5′ tag of crRNA can block targeting. These findings add to our understanding of the molecular mechanisms of the type III-A CRISPR-Cas system and provide a novel strategy for the exploitation of engineered CRISPR immunity against integrated MGEs in bacteria for clinical and industrial applications. CRISPR-Cas (clustered regularly interspaced short palindromic repeat [CRISPR]-CRISPR-associated protein [Cas]) systems can provide protection against invading genetic elements by using CRISPR RNAs (crRNAs) as a guide to locate and degrade the target DNA. CRISPR-Cas systems have been classified into two classes and five types according to the content of cas genes. Previous studies have indicated that CRISPR-Cas systems can avoid viral infection and block plasmid transfer. Here we show that chromosomal targeting by the Staphylococcus aureus type III-A CRISPR-Cas system can drive large-scale genome deletion and alteration within integrated staphylococcal cassette chromosome mec (SCCmec). The targeting activity of the CRISPR-Cas system is associated with the complementarity between crRNAs and protospacers, and 10- to 13-nucleotide truncations of spacers partially block CRISPR attack and more than 13-nucleotide truncation can fully abolish targeting, suggesting that a minimal length is required to license cleavage. Avoiding base pairings in the upstream region of protospacers is also necessary for CRISPR targeting. Successive trinucleotide complementarity between the 5′ tag of crRNAs and protospacers can disrupt targeting. Our findings reveal that type III-A CRISPR-Cas systems can modulate bacterial genome stability and may serve as a high-efficiency tool for deleting resistance or virulence genes in bacteria. IMPORTANCE Staphylococcus aureus is a pathogen that can cause a wide range of infections in humans. Studies have suggested that CRISPR-Cas systems can drive the loss of integrated mobile genetic elements (MGEs) by chromosomal targeting. Here we demonstrate that CRISPR-mediated cleavage contributes to the partial deletion of integrated SCCmec in methicillin-resistant S. aureus (MRSA), which provides a strategy for the treatment of MRSA infections. The spacer within artificial CRISPR arrays should contain more than 25 nucleotides for immunity, and consecutive trinucleotide pairings between a selected target and the 5′ tag of crRNA can block targeting. These findings add to our understanding of the molecular mechanisms of the type III-A CRISPR-Cas system and provide a novel strategy for the exploitation of engineered CRISPR immunity against integrated MGEs in bacteria for clinical and industrial applications.

ISSR-PCR markers and mobile genetic elements in horse (Equus caballus) genome

2014 ◽  
pp. 42-57 ◽  
Author(s):  
N.V. Bardukov ◽  
◽  
A.V. Feofilov ◽  
T.T. Glazko ◽  
V.I. Glazko ◽  
...  
Keyword(s):  
Equus Caballus ◽  
Mobile Genetic Elements ◽  
Pcr Markers ◽  
Genetic Elements ◽  
Issr Pcr

scholarly journals SCOPE enables type III CRISPR-Cas diagnostics using flexible targeting and stringent CARF ribonuclease activation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jurre A. Steens ◽  
Yifan Zhu ◽  
David W. Taylor ◽  
Jack P. K. Bravo ◽  
Stijn H. P. Prinsen ◽  
...  
Keyword(s):  
Immune Response ◽  
Diagnostic Tool ◽  
Nasal Swab ◽  
Rna Binding ◽  
Seed Region ◽  
Base Pairing ◽  
Specific Detection ◽  
Type Iii ◽  
Single Host ◽  
Target Rna

AbstractCharacteristic properties of type III CRISPR-Cas systems include recognition of target RNA and the subsequent induction of a multifaceted immune response. This involves sequence-specific cleavage of the target RNA and production of cyclic oligoadenylate (cOA) molecules. Here we report that an exposed seed region at the 3′ end of the crRNA is essential for target RNA binding and cleavage, whereas cOA production requires base pairing at the 5′ end of the crRNA. Moreover, we uncover that the variation in the size and composition of type III complexes within a single host results in variable seed regions. This may prevent escape by invading genetic elements, while controlling cOA production tightly to prevent unnecessary damage to the host. Lastly, we use these findings to develop a new diagnostic tool, SCOPE, for the specific detection of SARS-CoV-2 from human nasal swab samples, revealing sensitivities in the atto-molar range.

scholarly journals Genomic evolution of antimicrobial resistance in Escherichia coli

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Pimlapas Leekitcharoenphon ◽  
Markus Hans Kristofer Johansson ◽  
Patrick Munk ◽  
Burkhard Malorny ◽  
Magdalena Skarżyńska ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Resistance ◽  
Virulence Genes ◽  
Mobile Genetic Elements ◽  
Whole Genome ◽  
Sequencing Analysis ◽  
Metagenomic Sequencing ◽  
Fecal Samples ◽  
E Coli ◽  
Genetic Elements

AbstractThe emergence of antimicrobial resistance (AMR) is one of the biggest health threats globally. In addition, the use of antimicrobial drugs in humans and livestock is considered an important driver of antimicrobial resistance. The commensal microbiota, and especially the intestinal microbiota, has been shown to have an important role in the emergence of AMR. Mobile genetic elements (MGEs) also play a central role in facilitating the acquisition and spread of AMR genes. We isolated Escherichia coli (n = 627) from fecal samples in respectively 25 poultry, 28 swine, and 15 veal calf herds from 6 European countries to investigate the phylogeny of E. coli at country, animal host and farm levels. Furthermore, we examine the evolution of AMR in E. coli genomes including an association with virulence genes, plasmids and MGEs. We compared the abundance metrics retrieved from metagenomic sequencing and whole genome sequenced of E. coli isolates from the same fecal samples and farms. The E. coli isolates in this study indicated no clonality or clustering based on country of origin and genetic markers; AMR, and MGEs. Nonetheless, mobile genetic elements play a role in the acquisition of AMR and virulence genes. Additionally, an abundance of AMR was agreeable between metagenomic and whole genome sequencing analysis for several AMR classes in poultry fecal samples suggesting that metagenomics could be used as an indicator for surveillance of AMR in E. coli isolates and vice versa.

scholarly journals Development of SARS-CoV-2 Specific IgG and Virus-Neutralizing Antibodies after Infection with Variants of Concern or Vaccination

Vaccines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 700
Author(s):  
Franziska Neumann ◽  
Ruben Rose ◽  
Janine Römpke ◽  
Olaf Grobe ◽  
Thomas Lorentz ◽  
...  
Keyword(s):  
Immune Response ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Neutralization Test ◽  
Vaccine Dose ◽  
High Avidity ◽  
Receptor Binding Domain ◽  
Robust Immune Response ◽  
Specific Igg ◽  
Nucleoprotein N

The humoral immunity after SARS-CoV-2 infection or vaccination was examined. Convalescent sera after infection with variants of concern (VOCs: B.1.1.7, n = 10; B.1.351, n = 1) and sera from 100 vaccinees (Pfizer/BioNTech, BNT162b2, n = 33; Moderna, mRNA-1273, n = 11; AstraZeneca, ChAdOx1 nCoV-19/AZD1222, n = 56) were tested for the presence of immunoglobulin G (IgG) directed against the viral spike (S)-protein, its receptor-binding domain (RBD), the nucleoprotein (N) and for virus-neutralizing antibodies (VNA). For the latter, surrogate assays (sVNT) and a Vero-cell based neutralization test (cVNT) were used. Maturity of IgG was determined by measuring the avidity in an immunoblot (IB). Past VOC infection resulted in a broad reactivity of anti-S IgG (100%), anti-RBD IgG (100%), and anti-N IgG (91%), while latter were absent in 99% of vaccinees. Starting approximately two weeks after the first vaccine dose, anti-S IgG (75–100%) and particularly anti-RBD IgG (98–100%) were detectable. After the second dose, their titers increased and were higher than in the convalescents. The sVNT showed evidence of VNA in 91% of convalescents and in 80–100%/100% after first/second vaccine dose, respectively. After the second dose, an increase in VNA titer and IgGs of high avidity were demonstrated by cVNT and IB, respectively. Re-vaccination contributes to a more robust immune response.

scholarly journals Microevolution within ST11 group Clostridioides difficile isolates through mobile genetic elements based on complete genome sequencing

BMC Genomics ◽  
2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Yuan Wu ◽  
Lin Yang ◽  
Wen-Ge Li ◽  
Wen Zhu Zhang ◽  
Zheng Jie Liu ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Resistance Genes ◽  
Point Mutations ◽  
Evolutionary Process ◽  
Mobile Genetic Elements ◽  
Genetic Elements ◽  
Clostridioides Difficile ◽  
Hospitalized Elderly ◽  
Great Heterogeneity ◽  
High Level

Abstract Background Clade 5 Clostridioides difficile diverges significantly from the other clades and is therefore, attracting increasing attention due its great heterogeneity. In this study, we used third-generation sequencing techniques to sequence the complete whole genomes of three ST11 C. difficile isolates, RT078 and another two new ribotypes (RTs), obtained from three independent hospitalized elderly patients undergoing antibiotics treatment. Mobile genetic elements (MGEs), antibiotic-resistance, drug resistance genes, and virulent-related genes were analyzed and compared within these three isolates. Results Isolates 10,010 and 12,038 carried a distinct deletion in tcdA compared with isolate 21,062. Furthermore, all three isolates had identical deletions and point-mutations in tcdC, which was once thought to be a unique characteristic of RT078. Isolate 21,062 (RT078) had a unique plasmid, different numbers of transposons and genetic organization, and harboring special CRISPR spacers. All three isolates retained high-level sensitivity to 11 drugs and isolate 21,062 (RT078) carried distinct drug-resistance genes and loss of numerous flagellum-related genes. Conclusions We concluded that capillary electrophoresis based PCR-ribotyping is important for confirming RT078. Furthermore, RT078 isolates displayed specific MGEs, indicating an independent evolutionary process. In the further study, we could testify these findings with more RT078 isolates of divergent origins.

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