Evaluation of the novel CRISPRi plasmid for <i>S</i>. <i>aureus</i>.

<p>2A. Transformation efficiencies of <i>S</i>. <i>aureus</i>. The transformation efficiencies of pBACi and pKFT are shown. Both plasmids were extracted from DH5α (<i>dcm</i>+) and BL21(DE3) (<i>dcm</i>-). Electroporation of all strains was performed at least three times. When no transformants were obtained until three trials, seven more electroporations (total n = 10) were conducted. *: Strains which could accept pKFT extracted from RN4220 (Transformation efficiency is 16.8~1118.2CFU/µg DNA). §: Strains which could not accept pKFT extracted from RN4220. 2B. Confirmation of correct transformation. The MW2 transformants were subjected to PCR. 1–3: Transformants using pBACi extracted from BL21(DE3). 4–6: Transformants using pBACi extracted from RN4220. 7: pBACi DNA. 8: No DNA (only purified water). M: 100bp marker. 2C. Growth curve of MW2/pBACi. Temporal measurement of OD₆₆₀ are shown. Three independent cultures were performed. After inoculation of 1/100 volume o/n pre-culture media into fresh media (OD₆₆₀: ~0.1), the temporal samplings (3 h: early-mid log phase, 6 h: late log phase, and 12 h: stationary phase) were performed. Vertical line: OD₆₆₀. Horizontal line: time (hours, h). Average and standard error (SE) are shown. 2D. Relative expression of <i>dcas9</i>. The temporal expression of <i>dcas9</i> in MW2/pBACi is shown. Three genes were used as reference genes. Gyrase subunit B (<i>gyrB</i>): white, glyceraldehyde 3-phosphate dehydrogenase (<i>gapDH</i>): gray and <i>femB</i>: black. Three independent samples of three cultures and three qPCR assays were conducted (n = 9/sample). The samplings were performed at three time points as described above. Average and SE are shown.</p

Repression of various virulence and antibiotic resistant factors in MW2 using CRISPRi.

<p>Knockdown strain names were as follows. C: MW2/pBACi (Vector control), 1. MW2/pYS69 (<i>icaA</i> silenced knockdown strain 1), 2: MW2/pYS70 (<i>icaA</i> silenced knockdown strain 2), 3: MW2/pYS106 (<i>sec</i> silenced knockdown strain 1), 4: MW2/pYS107 (<i>sec</i> silenced knockdown strain 2), 5: MW2/pYS108 (<i>sec</i> silenced knockdown strain 3), 6: MW2/pYS109 (<i>coa</i> silenced knockdown strain 1) 7: MW2/pYS110 (<i>coa</i> silenced knockdown strain 2), 8: MW2/pYS111 (<i>coa</i> silenced knockdown strain 3), 9: MW2/pYS112 (<i>blaZ</i> silenced knockdown strain 1), and 10: MW2/pYS113 (<i>blaZ</i> silenced knockdown strain 2). All phenotype assays were repeated at least three times (qPCR and ELISA: three independent cultures and three independent measurements. Other assays: three independent assays). Average and SE of each assay are shown in graphs. Statistics: Student’s <i>t</i>-test. 4A. crRNA binding regions. 100bp upstream sequences of four genes in MW2 and nucleotide sequences corresponding to spacer sequences designed in this study. Underline: spacer sequences, red boxes: PAM sequence (NGG), start codon: start codon of gene, capital letters: putative -35b, -10b and ribosome binding sites of genes. 4B. Biofilm formation on plastic surfaces. After 24 h culture, the amount of biofilm on surface was assayed. G-: without additional glucose, G+: with additional glucose. 4C. OD₅₉₀ value of extracts from G+ wells as shown in Fig 4B. 4D. Repression of <i>icaA</i> mRNA. 4E. SDS-PAGE of supernatants. Culture media were subjected to electrophoresis. Arrow head: Protein corresponding to SEC (SEC from MW2: 28kDa). M: molecular weight marker (kDa), 4F: Sandwich ELISA of supernatants. SEC production was quantified. 4G. Repression of <i>sec</i> mRNA. 4H. Coagulase test. White arrow indicates a fibrin clot. 4I. Repression of <i>coa</i> mRNA. 4J. Measurement of β-lactamase activity. OD₄₉₀ of nitrocephin degraded product was measured. The value of control was compared with silencing vectors (%, vertical line). 4K. Repression of <i>blaZ</i> mRNA.</p

Plasmid map of pBACi, CRISPRi plasmid for <i>S</i>. <i>aureus</i>.

<p>Plasmid map of pBACi, CRISPRi plasmid for <i>S</i>. <i>aureus</i>.</p

Staphylococcal protein A (Spa) silenced using CRISPRi.

<p>No significant growth inhibition was observed between the control and the silenced knockdown strains. pBACi (C): Vector control, pYS103 [103]: <i>spa</i> silenced knockdown vector 1, pYS104 [104]: <i>spa</i> silenced knockdown vector 2, pYS105 [105]: <i>spa</i> silenced knockdown vector 3. 3A. The upstream sequence of <i>spa</i> in MW2 and nucleotide sequences corresponding to spacer sequences. The partial sequence between <i>spa</i> (MW0084) and <i>sarS</i> (MW0085) in MW2 (Accession number: BA000033) is shown (101,008 bp-101,207 bp in MW2). Underline: spacer sequence [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185987#pone.0185987.ref001" target="_blank">1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185987#pone.0185987.ref003" target="_blank">3</a>], red boxes: PAM sequence (NGG), start codon: start codon of <i>spa</i> (ttg), capital letters: putative -35b, -10b and SD sequence (ribosome binding site) of <i>spa</i> in MW2. 3B. Western blotting to detect Spa in vector controls and silenced knockdown strains. Seven strains were analyzed. Three independent cultures from a single colony were performed and representative data are shown. Brackets indicate Protein A. Due to posttranslational processing of the cell surface-exposed ProteinA, some strains show double bands. “Plasmid No.” indicates serial pYS number plasmid used in this study (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185987#pone.0185987.s001" target="_blank">S1 File</a>). M: molecular marker. 75, 50 and 37 mean 75kDa, 50kDa and 37kDa bands, respectively. C: pBACi control vector (No crRNA coding region for <i>spa</i> silencing), pYS103-105: silencing CRISPRi vector containing the nucleotide sequence 1–3 corresponding to Fig 3A. Arrow heads: Spa. Two bands were found. This might result from processed/non-processed bands [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185987#pone.0185987.ref052" target="_blank">52</a>]. 3C. Repression of <i>spa</i> mRNA in MW2 using CRISPRi. Relative <i>spa</i> gene expression (/<i>gyrB</i>) is shown. MW2/pBACi, MW2/pYS103, MW2/pYS104 and MW2/pYS105 were independently cultured three times. Three independent qPCR were performed (n = 9/strain). The average and SE are shown. Statistics: Student’s <i>t</i>-test.</p

Tailor-made gene silencing of <i>Staphylococcus aureus</i> clinical isolates by CRISPR interference

<div><p>Preparing the genetically modified organisms have required much time and labor, making it the rate-limiting step but CRISPR/Cas9 technology appearance has changed this difficulty. Although reports on CRISPR/Cas9 technology such as genome editing and CRISPR interference (CRISPRi) in eukaryotes increased, those in prokaryotes especially in Staphylococci were limited. Thus, its potential in the bacteriology remains unexplored. This is attributed to ecological difference between eukaryotes and prokaryotes. Here, we constructed a novel CRISPRi plasmid vector, pBACi for <i>Staphylococcus aureus</i>. The transformation efficiency of <i>S</i>. <i>aureus</i> was ~10⁴ CFU/μg DNA using a vector extracted from <i>dcm</i> negative, which encoded one of DNA modification genes, <i>E</i>. <i>coli</i>. Further, pBACi was introduced into various clinical isolates including that not accepting the conventional temperature-sensitive vector. <i>dcas9</i> in the vector was expressed throughout the growth phases of <i>S</i>. <i>aureus</i> and this vector decreased various gene mRNA expressions based on the crRNA targeting sequences and altered the knockdown strains’ phenotypes. The targeted genes included various virulence and antibiotic resistant genes. Bioinformatics suggest this vector can be introduced into wide range of low-GC Gram-positive bacteria. Because this new CRISPR/Cas9-based vector can easily prepare knockdown strains, we believe the novel vector will facilitate the characterization of the function of genes from <i>S</i>. <i>aureus</i> and other Gram-positive bacteria.</p></div

A Novel Repressor of the ica Locus Discovered in Clinically Isolated Super-Biofilm-Elaborating Staphylococcus aureus

ABSTRACT Staphylococcus aureus TF2758 is a clinical isolate from an atheroma and a super-biofilm-elaborating/polysaccharide intercellular adhesin (PIA)/poly-N-acetylglucosamine (PNAG)-overproducing strain (L. Shrestha et al., Microbiol Immunol 60:148–159, 2016, https://doi.org/10.1111/1348-0421.12359). A microarray analysis and DNA genome sequencing were performed to identify the mechanism underlying biofilm overproduction by TF2758. We found high transcriptional expression levels of a 7-gene cluster (satf2580 to satf2586) and the ica operon in TF2758. Within the 7-gene cluster, a putative transcriptional regulator gene designated rob had a nonsense mutation that caused the truncation of the protein. The complementation of TF2758 with rob from FK300, an rsbU-repaired derivative of S. aureus strain NCTC8325-4, significantly decreased biofilm elaboration, suggesting a role for rob in this process. The deletion of rob in non-biofilm-producing FK300 significantly increased biofilm elaboration and PIA/PNAG production. In the search for a gene(s) in the 7-gene cluster for biofilm elaboration controlled by rob, we identified open reading frame (ORF) SAOUHSC_2898 (satf2584). Our results suggest that ORF SAOUHSC_2898 (satf2584) and icaADBC are required for enhanced biofilm elaboration and PIA/PNAG production in the rob deletion mutant. Rob bound to a palindromic sequence within its own promoter region. Furthermore, Rob recognized the TATTT motif within the icaR-icaA intergenic region and bound to a 25-bp DNA stretch containing this motif, which is a critically important short sequence regulating biofilm elaboration in S. aureus. Our results strongly suggest that Rob is a long-sought repressor that recognizes and binds to the TATTT motif and is an important regulator of biofilm elaboration through its control of SAOUHSC_2898 (SATF2584) and Ica protein expression in S. aureus

Complete genome sequencing of three human clinical isolates of Staphylococcus caprae reveals virulence factors similar to those of S. epidermidis and S. capitis

Abstract Background Staphylococcus caprae is an animal-associated bacterium regarded as part of goats’ microflora. Recently, S. caprae has been reported to cause human nosocomial infections such as bacteremia and bone and joint infections. However, the mechanisms responsible for the development of nosocomial infections remain largely unknown. Moreover, the complete genome sequence of S. caprae has not been determined. Results We determined the complete genome sequences of three methicillin-resistant S. caprae strains isolated from humans and compared these sequences with the genomes of S. epidermidis and S. capitis, both of which are closely related to S. caprae and are inhabitants of human skin capable of causing opportunistic infections. The genomes showed that S. caprae JMUB145, JMUB590, and JMUB898 strains contained circular chromosomes of 2,618,380, 2,629,173, and 2,598,513 bp, respectively. JMUB145 carried type V SCCmec, while JMUB590 and JMUB898 had type IVa SCCmec. A genome-wide phylogenetic SNP tree constructed using 83 complete genome sequences of 24 Staphylococcus species and 2 S. caprae draft genome sequences confirmed that S. caprae is most closely related to S. epidermidis and S. capitis. Comparative complete genome analysis of eight S. epidermidis, three S. capitis and three S. caprae strains revealed that they shared similar virulence factors represented by biofilm formation genes. These factors include wall teichoic acid synthesis genes, poly-gamma-DL-glutamic acid capsule synthesis genes, and other genes encoding nonproteinaceous adhesins. The 17 proteinases/adhesins and extracellular proteins known to be associated with biofilm formation in S. epidermidis were also conserved in these three species, and their biofilm formation could be detected in vitro. Moreover, two virulence-associated gene clusters, the type VII secretion system and capsular polysaccharide biosynthesis gene clusters, identified in S. aureus were present in S. caprae but not in S. epidermidis and S. capitis genomes. Conclusion The complete genome sequences of three methicillin-resistant S. caprae isolates from humans were determined for the first time. Comparative genome analysis revealed that S. caprae is closely related to S. epidermidis and S. capitis at the species level, especially in the ability to form biofilms, which may lead to increased virulence during the development of S. caprae infections