DNA microarray-based identification of genes associated with glycopeptide resistance in Staphylococcus aureus

Longzhu Cui, Jian Qi Lian, Hui Min Neoh, Ethel Reyes, Keiichi Hiramatsu

Research output: Contribution to journalArticle

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Abstract

Six pairs of transcription profiles between glycopeptide-intermediate S. aureus (GISA [or vancomycin-intermediate S. aureus; VISA]) and glycopeptide-susceptible S. aureus (vancomycin-susceptible S. aureus [VSSA], including glycopeptide-susceptible isogenic mutants from VISA) strains were compared using a microarray. Ninety-two open reading frames which were or tended to be increased in transcription in VISA in at least five out of six array combination pairs were evaluated for their effects on glycopeptide susceptibility by introducing these genes one by one into VSSA strain N315 to construct an overexpression library. By screening the library, 17 genes including 8 novel genes were identified as associated with glycopeptide resistance since their experimental overexpression reduced vancomycin and/or teicoplanin susceptibility of N315. The raised MICs of vancomycin and teicoplanin were 1.25 to 3.0 and 1.5 to 6.0 mg/liter, respectively, as compared to 1.0 mg/liter of N315. Three of these genes, namely graF, msrA2, and mgrA, also raised the oxacillin MIC from 8.0 mg/liter for N315 to 64 to ∼128 mg/liter when they were overexpressed in N315. Their contribution to vancomycin and beta-lactam resistance was further supported by gene knockout and trans-complementation assay. By using a plasmid-based promoter-green fluorescent protein gene (gfp) transcriptional fusion system, graF promoter-activated cells were purified, and subsequent susceptibility tests and Northern blot analysis demonstrated that the cells with up-regulated activity of graF promoter showed reduced susceptibility to vancomycin, teicoplanin, and oxacillin. In addition, cell morphology studies showed that graF and msrA2 overexpression increased cell wall thickness of N315 by factors of 23.91 and 22.27%, respectively, accompanied by glycopeptide MIC increments of 3- to 6-fold, when they were overexpressed in N315. Moreover, extended experiments and analyses indicate that many of the genes identified above are related to the cell wall biosynthetic pathway, including active nutrient transport systems. We propose that the genes which raise glycopeptide resistance in S. aureus function toward altering the cell wall metabolic pathway.

Original languageEnglish
Pages (from-to)3404-3413
Number of pages10
JournalAntimicrobial Agents and Chemotherapy
Volume49
Issue number8
DOIs
Publication statusPublished - Aug 2005
Externally publishedYes

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Glycopeptides
Vancomycin
Oligonucleotide Array Sequence Analysis
Staphylococcus aureus
Teicoplanin
Genes
Cell Wall
Oxacillin
beta-Lactam Resistance
Gene Knockout Techniques
Active Biological Transport
Gene Fusion
Biosynthetic Pathways
Metabolic Networks and Pathways
Green Fluorescent Proteins
Gene Library
Northern Blotting
Open Reading Frames
Libraries
Plasmids

ASJC Scopus subject areas

  • Pharmacology (medical)

Cite this

DNA microarray-based identification of genes associated with glycopeptide resistance in Staphylococcus aureus. / Cui, Longzhu; Lian, Jian Qi; Neoh, Hui Min; Reyes, Ethel; Hiramatsu, Keiichi.

In: Antimicrobial Agents and Chemotherapy, Vol. 49, No. 8, 08.2005, p. 3404-3413.

Research output: Contribution to journalArticle

Cui, Longzhu ; Lian, Jian Qi ; Neoh, Hui Min ; Reyes, Ethel ; Hiramatsu, Keiichi. / DNA microarray-based identification of genes associated with glycopeptide resistance in Staphylococcus aureus. In: Antimicrobial Agents and Chemotherapy. 2005 ; Vol. 49, No. 8. pp. 3404-3413.
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