Composition and Diversity of CRISPR-Cas13a systems in the genus <i>Leptotrichia</i>

Watanabe, Shinya; Cui, Bintao; Kiga, Kotaro; Aiba, Yoshifumi; Tan, Xin-Ee; Sato’o, Yusuke; Kawauchi, Moriyuki; Boonsiri, Tanit; Thitiananpakorn, Kanate; Taki, Yusuke; Li, Fen-Yu; Azam, Aa Haeruman; Nakada, Yumi; Sasahara, Teppei; Cui, Longzhu

doi:10.1101/710533

Cited by 6 publications

(7 citation statements)

References 36 publications

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“…The generated genomic sequences were finally annotated using Microbial Genome Annotation Pipeline ( https://www.migap.org/ ). Meanwhile, the genomic sequences of in vitro-selected mutants with reduced oxacillin susceptibility were determined by sequencing paired-end reads as previously described 68 . The paired-end library was prepared using a Nextera XT library prep kit and sequenced using the Illumina MiSeq platform with the MiSeq reagent kit version 3.…”

Section: Methodsmentioning

confidence: 99%

Identification and characterization of mutations responsible for the β-lactam resistance in oxacillin-susceptible mecA-positive Staphylococcus aureus

Boonsiri

Watanabe

Tan

et al. 2020

Sci Rep

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Staphylococcus aureus strains that are susceptible to the β-lactam antibiotic oxacillin despite carrying mecA (OS-MRSA) cause serious clinical problems globally because of their ability to easily acquire β-lactam resistance. Understanding the genetic mechanism(s) of acquisition of the resistance is therefore crucial for infection control management. For this purpose, a whole-genome sequencing-based analysis was performed using 43 clinical OS-MRSA strains and 100 mutants with reduced susceptibility to oxacillin (MICs 1.0–256 µg/mL) generated from 26 representative OS-MRSA strains. Genome comparison between the mutants and their respective parent strains identified a total of 141 mutations in 46 genes and 8 intergenic regions. Among them, the mutations are frequently found in genes related to RNA polymerase (rpoBC), purine biosynthesis (guaA, prs, hprT), (p)ppGpp synthesis (relSau), glycolysis (pykA, fbaA, fruB), protein quality control (clpXP, ftsH), and tRNA synthase (lysS, gltX), whereas no mutations existed in mec and bla operons. Whole-genome transcriptional profile of the resistant mutants demonstrated that expression of genes associated with purine biosynthesis, protein quality control, and tRNA synthesis were significantly inhibited similar to the massive transcription downregulation seen in S. aureus during the stringent response, while the levels of mecA expression and PBP2a production were varied. We conclude that a combination effect of mecA upregulation and stringent-like response may play an important role in acquisition of β-lactam resistance in OS-MRSA.

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Section: Methodsmentioning

confidence: 99%

Identification and characterization of mutations responsible for the β-lactam resistance in oxacillin-susceptible mecA-positive Staphylococcus aureus

Boonsiri

Watanabe

Tan

et al. 2020

Sci Rep

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“…When phages infect bacteria, CRISPR-Cas13a recognizes transcript of phage genome, which leads to nonspecific degradation of bacterial transcripts and arrests bacterial cell growth to prevent the spread of infection. Recently, we identified four different subtypes of CRISPR-Cas13a systems from 11 strains of six Leptotrichia species 16 . Among them CRISPR-Cas13a form Leptotrichia shahii (LshCas13a) had a significant inhibition effect on bacterial growth, consistent with the observation by Abudayyeh et al 14 .…”

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confidence: 99%

Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria

et al. 2020

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The emergence of antimicrobial-resistant bacteria is an increasingly serious threat to global health, necessitating the development of innovative antimicrobials. Here we report the development of a series of CRISPR-Cas13a-based antibacterial nucleocapsids, termed Cap-sidCas13a(s), capable of sequence-specific killing of carbapenem-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus by recognizing corresponding antimicrobial resistance genes. CapsidCas13a constructs are generated by packaging programmed CRISPR-Cas13a into a bacteriophage capsid to target antimicrobial resistance genes. Contrary to Cas9-based antimicrobials that lack bacterial killing capacity when the target genes are located on a plasmid, the CapsidCas13a(s) exhibit strong bacterial killing activities upon recognizing target genes regardless of their location. Moreover, we also demonstrate that the CapsidCas13a(s) can be applied to detect bacterial genes through gene-specific depletion of bacteria without employing nucleic acid manipulation and optical visualization devices. Our data underscore the potential of CapsidCas13a(s) as both therapeutic agents against antimicrobial-resistant bacteria and nonchemical agents for detection of bacterial genes.

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“…Phenol-chloroform-based extraction of genomic DNA from S. aureus and purification of DNA using a QIAamp DNA mini kit (Qiagen, Hilden, Germany) was described previously (Watanabe et al, 2018). Genomic sequencing and assembly were performed as previously described with slight modifications (Watanabe et al, 2019), and default parameters were used except otherwise noted. Briefly, sequencing was performed using a combination SCCmec type sea seb sec seg sei sell selm seln selo selp selu tst…”

Section: Whole-genome Sequencingmentioning

confidence: 99%

The Association Between Onset of Staphylococcal Non-menstrual Toxic Shock Syndrome With Inducibility of Toxic Shock Syndrome Toxin-1 Production

et al. 2022

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Non-menstrual toxic shock syndrome (non-mTSS) is a life-threatening disease caused by Staphylococcus aureus strains producing superantigens, such as staphylococcal enterotoxins A, B, C, and toxic shock syndrome toxin-1 (TSST-1). However, little is known about why the TSS cases are rare, although S. aureus strains frequently carry a tst gene, which encodes TSST-1. To answer this question, the amount of TSST-1 produced by 541 clinical isolates was measured in both the presence and absence of serum supplementation to growth media. Then a set of S. aureus strains with similar genetic backgrounds isolated from patients presenting with non-mTSS and those with clinical manifestations other than non-mTSS was compared for their TSST-1 inducibility by human serum, and their whole-genome sequences were determined. Subsequently, the association of mutations identified in the tst promoter of non-mTSS strains with TSST-1 inducibility by human serum was evaluated by constructing promoter replacement mutants and green fluorescent protein (GFP) reporter recombinants. Results showed that 39 out of 541 clinical isolates (7.2%), including strains isolated from non-mTSS patients, had enhanced production of TSST-1 in the presence of serum. TSST-1 inducibility by human serum was more clearly seen in non-mTSS strains of clonal complex (CC)-5. Moreover, the whole-genome sequence analysis identified a set of sequence variations at a putative SarA-binding site of the tst promoter. This sequence variation was proven to be partially responsible for the induction of TSST-1 production by human serum. We conclude that the onset of staphylococcal toxic shock syndrome caused by TSST-1-producing CC-5 strains seem at least partially initiated by serum induction of TSST-1, which is regulated by the mutation of putative SarA-binding site at the tst promoter.

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Composition and Diversity of CRISPR-Cas13a systems in the genus Leptotrichia

Cited by 6 publications

References 36 publications

Identification and characterization of mutations responsible for the β-lactam resistance in oxacillin-susceptible mecA-positive Staphylococcus aureus

Identification and characterization of mutations responsible for the β-lactam resistance in oxacillin-susceptible mecA-positive Staphylococcus aureus

Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria

The Association Between Onset of Staphylococcal Non-menstrual Toxic Shock Syndrome With Inducibility of Toxic Shock Syndrome Toxin-1 Production

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