Shuttle Vectors Derived from pN315 for Study of Essential Genes in Staphylococcus aureus

Matsuo, Miki; Kurokawa, Kenji; Lee, Bok-Luel; Sekimizu, Kazuhisa

doi:10.1248/bpb.33.198

Cited by 9 publications

(9 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…pMNS contains the transcription terminators, Pspac promoter, lacZ , and lacI from pMutinT3 and pE194 ori , pUC ori , and the spectinomycin resistance gene from pTetON. pMNS is compatible with pKE516 [38]. Plasmids used in this study are shown in Table 4.…”

Section: Methodsmentioning

confidence: 99%

Mobile Genetic Element SCCmec-encoded psm-mec RNA Suppresses Translation of agrA and Attenuates MRSA Virulence

et al. 2013

Self Cite

View full text Add to dashboard Cite

Community acquired-methicillin resistant Staphylococcus aureus (CA-MRSA) is a socially problematic pathogen that infects healthy individuals, causing severe disease. CA-MRSA is more virulent than hospital associated-MRSA (HA-MRSA). The underlying mechanism for the high virulence of CA-MRSA is not known. The transcription product of the psm-mec gene, located in the mobile genetic element SCCmec of HA-MRSA, but not CA-MRSA, suppresses the expression of phenol-soluble modulin α (PSMα), a cytolytic toxin of S. aureus. Here we report that psm-mec RNA inhibits translation of the agrA gene encoding a positive transcription factor for the PSMα gene via specific binding to agrA mRNA. Furthermore, 25% of 325 clinical MRSA isolates had a mutation in the psm-mec promoter that attenuated transcription, and 9% of the strains had no psm-mec. In most of these psm-mec-mutated or psm-mec-deleted HA-MRSAs, PSMα expression was increased compared with strains carrying intact psm-mec, and some mutated strains produced high amounts of PSMα comparable with that of CA-MRSA. Deletion of psm-mec from HA-MRSA strains carrying intact psm-mec increased the expression of AgrA protein and PSMα, and virulence in mice. Thus, psm-mec RNA suppresses MRSA virulence via inhibition of agrA translation and the absence of psm-mec function in CA-MRSA causes its high virulence property.

show abstract

Section: Methodsmentioning

confidence: 99%

Mobile Genetic Element SCCmec-encoded psm-mec RNA Suppresses Translation of agrA and Attenuates MRSA Virulence

et al. 2013

Self Cite

View full text Add to dashboard Cite

show abstract

“…To construct plasmid pNcmk, cmk (SAHV_1466) of Mu3 chromosomal DNA was amplified using the cmk-CP1 and cmk-CP2 primers (see Table S1 in the supplemental material) and was inserted into the BamHI site in the pND50 plasmid vector (12). The integrity of the cloned cmk was ascertained by sequencing the recombinant plasmid using the pND-P1 and pND-P2 primers.…”

Section: Methodsmentioning

confidence: 99%

Comprehensive Identification of Mutations Responsible for Heterogeneous Vancomycin-Intermediate Staphylococcus aureus (hVISA)-to-VISA Conversion in Laboratory-Generated VISA Strains Derived from hVISA Clinical Strain Mu3

Matsuo

Cui

Kim

et al. 2013

Antimicrob Agents Chemother

Self Cite

View full text Add to dashboard Cite

bHeterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) spontaneously produces VISA cells within its cell population at a frequency of 10 ؊6 or greater. We established a total of 45 VISA mutant strains independently obtained from hVISA Mu3 and its related strains by one-step vancomycin selection. We then performed high-throughput whole-genome sequencing of the 45 strains and their parent strains to identify the genes involved in the hVISA-to-VISA phenotypic conversion. A comparative genome study showed that all the VISA strains tested carried a unique set of mutations. All of the 45 VISA strains carried 1 to 4 mutations possibly affecting the expression of a total of 48 genes. Among them, 32 VISA strains carried only one gene affected by a single mutation. As many as 20 genes in more than eight functional categories were affected in the 32 VISA strains, which explained the extremely high rates of the hVISA-to-VISA phenotypic conversion. Five genes, rpoB, rpoC, walK, pbp4, and pp2c, were previously reported as being involved in vancomycin resistance. Fifteen remaining genes were newly identified as associated with vancomycin resistance in this study. The gene most frequently affected (6 out of 32 strains) was cmk, which encodes cytidylate kinase, followed closely by rpoB (5 out of 32), encoding the ␤ subunit of RNA polymerase. A mutation prevalence study also revealed a sizable number of cmk mutants among clinical VISA strains (7 out of 38 [18%]). Reduced cytidylate kinase activity in cmk mutant strains is proposed to contribute to the hVISA-to-VISA phenotype conversion by thickening the cell wall and reducing the cell growth rate.

show abstract

“…From these cells, targeted integration of pMltaS at the chromosomal ltaS gene was confirmed by PCR, resulting in T772 (DltaS::pMltaS, Dspa::phleo/pM102-ltaS). Finally, T772 was transformed with pM101 (13), which is incompatible with pM102-ltaS because they possess the same replicon, but has a different marker from pM102 (namely, kanamycin resistance). A kanamycin-resistant transformant, T775 (DltaS::pMltaS, Dspa::phleo/pM101), was obtained at 30˚C.…”

Section: Protein Sera and Bacteriamentioning

confidence: 99%

Specific Serum Ig Recognizing Staphylococcal Wall Teichoic Acid Induces Complement-Mediated Opsonophagocytosis against Staphylococcus aureus

Jung

Kurokawa

et al. 2012

The Journal of Immunology

Self Cite

View full text Add to dashboard Cite

Wall teichoic acid (WTA) of Staphylococcus aureus is a major cell envelope-associated glycopolymer that is a key molecule in promoting colonization during S. aureus infection. The complement system plays a key role in the opsonization and clearance of pathogens. We recently reported that S. aureus WTA functions as a ligand of human serum mannose-binding lectin (MBL), a recognition molecule of the lectin complement pathway. Intriguingly, serum MBL in adults does not bind to WTA because of an inhibitory effect of serum anti–WTA-IgG. In this study, serum anti–WTA-IgG was purified to homogeneity using a purified S. aureus WTA-coupled affinity column to examine the biological function of human anti–WTA-IgG. The purified anti–WTA-IgG contained the IgG2 subclass as a major component and specifically induced C4 and C3 deposition on the S. aureus surface in the anti–WTA-IgG–depleted serum, but not in C1q-deficient serum. Furthermore, the anti–WTA-IgG–dependent C3 deposition induced phagocytosis of S. aureus cells by human polymorphonuclear leukocytes. These results demonstrate that serum anti–WTA-IgG is a real trigger for the induction of classical complement-dependent opsonophagocytosis against S. aureus. Our results also support the fact that a lack of the lectin complement pathway in MBL-deficient adults is compensated by Ag-specific, Ab-mediated adaptive immunity.

show abstract

Shuttle Vectors Derived from pN315 for Study of Essential Genes in Staphylococcus aureus

Cited by 9 publications

References 27 publications

Mobile Genetic Element SCCmec-encoded psm-mec RNA Suppresses Translation of agrA and Attenuates MRSA Virulence

Mobile Genetic Element SCCmec-encoded psm-mec RNA Suppresses Translation of agrA and Attenuates MRSA Virulence

Comprehensive Identification of Mutations Responsible for Heterogeneous Vancomycin-Intermediate Staphylococcus aureus (hVISA)-to-VISA Conversion in Laboratory-Generated VISA Strains Derived from hVISA Clinical Strain Mu3

Specific Serum Ig Recognizing Staphylococcal Wall Teichoic Acid Induces Complement-Mediated Opsonophagocytosis against Staphylococcus aureus

Contact Info

Product

Resources

About