Impact of normalization method on experimental outcome using RT-qPCR in Staphylococcus aureus

Valihrach, Lukás; Demnerova, Kateřina

doi:10.1016/j.mimet.2012.05.008

Cited by 61 publications

(43 citation statements)

References 7 publications

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“…The quantity of cDNA measured by real-time PCR was normalized to the abundance of hu cDNA (36). All qRT-PCR assays were repeated at least three times.…”

Section: Total Rna Isolation and Real-time Quantitative Reverse Transmentioning

confidence: 99%

SpoVG Regulates Cell Wall Metabolism and Oxacillin Resistance in Methicillin-Resistant Staphylococcus aureus Strain N315

Liu

Zhang

Sun

2016

Antimicrob Agents Chemother

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bIncreasing cases of infections caused by methicillin-resistant Staphylococcus aureus (MRSA) strains in healthy individuals have raised concerns worldwide. MRSA strains are resistant to almost the entire family of ␤-lactam antibiotics due to the acquisition of an extra penicillin-binding protein, PBP2a. Studies have shown that spoVG is involved in oxacillin resistance, while the regulatory mechanism remains elusive. In this study, we have found that SpoVG plays a positive role in oxacillin resistance through promoting cell wall synthesis and inhibiting cell wall degradation in MRSA strain N315. Deletion of spoVG in strain N315 led to a significant decrease in oxacillin resistance and a dramatic increase in Triton X-100-induced autolytic activity simultaneously. Real-time quantitative reverse transcription-PCR revealed that the expression of 8 genes related to cell wall metabolism or oxacillin resistance was altered in the spoVG mutant. Electrophoretic mobility shift assay indicated that SpoVG can directly bind to the putative promoter regions of lytN (murein hydrolase), femA, and lytSR (the two-component system). These findings suggest a molecular mechanism in which SpoVG modulates oxacillin resistance by regulating cell wall metabolism in MRSA. Staphylococcus aureus is a versatile and dangerous pathogen in humans, especially while the frequency of staphylococcal infections caused by methicillin-resistant Staphylococcus aureus (MRSA) increases steadily (1). S. aureus has four penicillin-binding proteins (PBPs) which are involved in the last stages of peptidoglycan synthesis (2). MRSA strains have acquired an extra PBP (PBP2a), encoded by the mecA gene on the staphylococcal cassette chromosome mec (SCCmec), which has a remarkably lower affinity for ␤-lactams than does PBP2 and is responsible for resisting these drugs (3). Besides the expression of PBP2a, which is responsible for a higher level of ␤-lactam resistance, S. aureus has another primary ␤-lactam resistance mechanism: the expression of ␤-lactamase enzymes, encoded by the blaZ gene, which hydrolyze ␤-lactams such as penicillin. In addition, several additional native genes, such as vraSR, pbp4, and fem (factor essential for methicillin resistance), have been identified as being essential to the full expression of oxacillin resistance (4-6). Inactivation of femA and pbp4 genes in the presence of an intact mecA gene usually results in strains with a low resistance to oxacillin. The VraS/VraR twocomponent regulatory system regulates the genes which are associated with cell wall biosynthesis, such as pbp2, sgtB, and murZ (7).Most MRSA and glycopeptide-intermediately resistant Staphylococcus aureus (GISA) isolates are resistant to Triton X-100-induced autolysis, while instances of reduced resistance have been observed with concomitant increases of the autolysis rate (8-11). The expression of autolytic enzymes, including Atl, LytM, LytN, and Sle1 (12-15), is controlled by pleiotropic regulators, such as MgrA, SarV, SarA, LytSR, and ArlSR (autolysis-related locus...

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“…The quantity of cDNA measured by real-time PCR was normalized to the abundance of hu cDNA (36). All qRT-PCR assays were repeated at least three times.…”

Section: Total Rna Isolation and Real-time Quantitative Reverse Transmentioning

confidence: 99%

SpoVG Regulates Cell Wall Metabolism and Oxacillin Resistance in Methicillin-Resistant Staphylococcus aureus Strain N315

Liu

Zhang

Sun

2016

Antimicrob Agents Chemother

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“…The obtained cDNA was used for real-time amplification with specific primers (Table 2) and 20 ng of cDNA/reaction. mRNA levels were normalized against the mRNA level of gyrB, which is constitutively expressed under the conditions analyzed (21). The amounts of different transcripts were expressed as the n-fold difference relative to the control gene (2 Ϫ⌬CT , where ⌬C T represents the difference in threshold cycle between the target and control genes).…”

Section: Construction Of the B Subtilis Ccpc Promoter-b Subtilis CCmentioning

confidence: 99%

Catabolite Control Protein E (CcpE) Is a LysR-type Transcriptional Regulator of Tricarboxylic Acid Cycle Activity in Staphylococcus aureus

Hartmann

Zhang

Baronian

et al. 2013

Journal of Biological Chemistry

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Background:The TCA cycle is a central metabolic pathway that facilitates the adaption of bacteria to a nutrient-limited environment. Results: Inactivation of CcpE in Staphylococcus aureus resulted in a decreased transcription of the aconitase encoding gene citB, and reduced TCA cycle activity. Conclusion: CcpE affects the TCA cycle via direct transcriptional control of citB. Significance: This is the first positive regulator of TCA cycle activity identified in this pathogen.

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“…Primer pairs for genes tested are listed in Table S3. Data analysis was performed according to Pfaffl (52), with hu used for normalization (53).…”

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confidence: 99%

Genome-wide screen for genes involved in eDNA release during biofilm formation by Staphylococcus aureus

DeFrancesco

Masloboeva

Syed

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Staphylococcus aureus is a leading cause of both nosocomial and community-acquired infection. Biofilm formation at the site of infection reduces antimicrobial susceptibility and can lead to chronic infection. During biofilm formation, a subset of cells liberate cytoplasmic proteins and DNA, which are repurposed to form the extracellular matrix that binds the remaining cells together in large clusters. Using a strain that forms robust biofilms in vitro during growth under glucose supplementation, we carried out a genome-wide screen for genes involved in the release of extracellular DNA (eDNA). A high-density transposon insertion library was grown under biofilm-inducing conditions, and the relative frequency of insertions was compared between genomic DNA (gDNA) collected from cells in the biofilm and eDNA from the matrix. Transposon insertions into genes encoding functions necessary for eDNA release were identified by reduced representation in the eDNA. On direct testing, mutants of some of these genes exhibited markedly reduced levels of eDNA and a concomitant reduction in cell clustering. Among the genes with robust mutant phenotypes were gdpP, which encodes a phosphodiesterase that degrades the second messenger cyclic-di-AMP, and xdrA, the gene for a transcription factor that, as revealed by RNA-sequencing analysis, influences the expression of multiple genes, including many involved in cell wall homeostasis. Finally, we report that growth in biofilm-inducing medium lowers cyclicdi-AMP levels and does so in a manner that depends on the gdpP phosphodiesterase gene.Staphylococcus aureus | biofilm | eDNA | cyclic-di-AMP

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Impact of normalization method on experimental outcome using RT-qPCR in Staphylococcus aureus

Cited by 61 publications

References 7 publications

SpoVG Regulates Cell Wall Metabolism and Oxacillin Resistance in Methicillin-Resistant Staphylococcus aureus Strain N315

SpoVG Regulates Cell Wall Metabolism and Oxacillin Resistance in Methicillin-Resistant Staphylococcus aureus Strain N315

Catabolite Control Protein E (CcpE) Is a LysR-type Transcriptional Regulator of Tricarboxylic Acid Cycle Activity in Staphylococcus aureus

Genome-wide screen for genes involved in eDNA release during biofilm formation by Staphylococcus aureus

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