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Exposure of clinical MRSA heterogeneous strains to β-lactams redirects metabolism to optimize energy production through the TCA cycle.

Keaton MA, Rosato RR, Plata KB, Singh CR, Rosato AE - PLoS ONE (2013)

Bottom Line: The mechanism associated with heterogeneous expression requires both increase expression of mecA and a mutational event that involved the triggering of a β-lactam-mediated SOS response and related lexA and recA genes.We found unique features present in the oxacillin-selected SA13011-HoR derivative when compared to the corresponding SA13011-HeR parental strain that included significant increases in tricarboxyl citric acid (TCA) cycle intermediates and a concomitant decrease in fermentative pathways.These results provide evidence of both the metabolic cost and the adaptation that HeR-MRSA clinical strains undergo when exposed to β-lactam pressure, indicating that the energy production is redirected to supply the cell wall synthesis/metabolism, which in turn contributes to the survival response in the presence of β-lactam antibiotics.

View Article: PubMed Central - PubMed

Affiliation: Metabolon, Inc, Durham, North Carolina, United States of America.

ABSTRACT
Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as one of the most important pathogens both in health care and community-onset infections. The prerequisite for methicillin resistance is mecA, which encodes a β-lactam-insensitive penicillin binding protein PBP2a. A characteristic of MRSA strains from hospital and community associated infections is their heterogeneous expression of resistance to β-lactam (HeR) in which only a small portion (≤ 0.1%) of the population expresses resistance to oxacillin (OXA) ≥ 10 µg/ml, while in other isolates, most of the population expresses resistance to a high level (homotypic resistance, HoR). The mechanism associated with heterogeneous expression requires both increase expression of mecA and a mutational event that involved the triggering of a β-lactam-mediated SOS response and related lexA and recA genes. In the present study we investigated the cellular physiology of HeR-MRSA strains during the process of β-lactam-mediated HeR/HoR selection at sub-inhibitory concentrations by using a combinatorial approach of microarray analyses and global biochemical profiling employing gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) to investigate changes in metabolic pathways and the metabolome associated with β-lactam-mediated HeR/HoR selection in clinically relevant heterogeneous MRSA. We found unique features present in the oxacillin-selected SA13011-HoR derivative when compared to the corresponding SA13011-HeR parental strain that included significant increases in tricarboxyl citric acid (TCA) cycle intermediates and a concomitant decrease in fermentative pathways. Inactivation of the TCA cycle enzyme cis-aconitase gene in the SA13011-HeR strain abolished β-lactam-mediated HeR/HoR selection demonstrating the significance of altered TCA cycle activity during the HeR/HoR selection. These results provide evidence of both the metabolic cost and the adaptation that HeR-MRSA clinical strains undergo when exposed to β-lactam pressure, indicating that the energy production is redirected to supply the cell wall synthesis/metabolism, which in turn contributes to the survival response in the presence of β-lactam antibiotics.

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Analysis of biochemicals associated with the TCA cycle determined by global biochemical profiling across SA13011-HeR and SA13011-HoR (SA13011+ OXA 0.5 µg/ml) derivative.
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pone-0071025-g003: Analysis of biochemicals associated with the TCA cycle determined by global biochemical profiling across SA13011-HeR and SA13011-HoR (SA13011+ OXA 0.5 µg/ml) derivative.

Mentions: Consistent with the upregulation of several TCA cycle genes during HoR selection, many of the TCA intermediates were elevated in the SA13011-HoR strain as compared to SA13011-HeR, with significant increases in the levels of cis-aconitate and malate (Figure 3; Table S1). These changes suggest that the SA13011-HoR strain is potentially capable of higher energy production and increased biosynthetic capabilities than the parental SA13011-HeR strain that may allow the HoR-derivative strain to respond to inhibition of wall synthesis by β-lactams. SA13011-HoR also displayed significantly higher levels of acetyl-CoA, which can be generated from glycolysis, β-oxidation of fatty acids, or acetate. Glycolysis did not appear to increase during the selection process, as 3-phosphoglycerate and phophoenolpyruvate were unchanged (Table S1). Instead, the majority of medium chain and long chain free fatty acids were found to be significantly decreased in SA13011-HoR, indicating increased utilization of this energy source (Figure 4). Consistent with an increase in fatty acid oxidation and TCA activity, acetyl-CoA (Figure 3) and NADH (Figure 5) levels rose after OXA-mediated HeR/HoR selection (SA13011-HoR). This was accompanied by a decrease in NAD levels (Figure 5) suggesting that the increased NADH was not being utilized. Together with diminished phosphate levels (Figure 5) and the decreased expression of a number of genes involved in oxidative phosphorylation in SA13011-HoR cells, including a subunit of FoF1-ATP synthase (atpC) and a protein required for cytochrome oxidase assembly (ctaA; Table 1), these changes indicate a decrease in oxidative phosphorylation during the selection process.


Exposure of clinical MRSA heterogeneous strains to β-lactams redirects metabolism to optimize energy production through the TCA cycle.

Keaton MA, Rosato RR, Plata KB, Singh CR, Rosato AE - PLoS ONE (2013)

Analysis of biochemicals associated with the TCA cycle determined by global biochemical profiling across SA13011-HeR and SA13011-HoR (SA13011+ OXA 0.5 µg/ml) derivative.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3733780&req=5

pone-0071025-g003: Analysis of biochemicals associated with the TCA cycle determined by global biochemical profiling across SA13011-HeR and SA13011-HoR (SA13011+ OXA 0.5 µg/ml) derivative.
Mentions: Consistent with the upregulation of several TCA cycle genes during HoR selection, many of the TCA intermediates were elevated in the SA13011-HoR strain as compared to SA13011-HeR, with significant increases in the levels of cis-aconitate and malate (Figure 3; Table S1). These changes suggest that the SA13011-HoR strain is potentially capable of higher energy production and increased biosynthetic capabilities than the parental SA13011-HeR strain that may allow the HoR-derivative strain to respond to inhibition of wall synthesis by β-lactams. SA13011-HoR also displayed significantly higher levels of acetyl-CoA, which can be generated from glycolysis, β-oxidation of fatty acids, or acetate. Glycolysis did not appear to increase during the selection process, as 3-phosphoglycerate and phophoenolpyruvate were unchanged (Table S1). Instead, the majority of medium chain and long chain free fatty acids were found to be significantly decreased in SA13011-HoR, indicating increased utilization of this energy source (Figure 4). Consistent with an increase in fatty acid oxidation and TCA activity, acetyl-CoA (Figure 3) and NADH (Figure 5) levels rose after OXA-mediated HeR/HoR selection (SA13011-HoR). This was accompanied by a decrease in NAD levels (Figure 5) suggesting that the increased NADH was not being utilized. Together with diminished phosphate levels (Figure 5) and the decreased expression of a number of genes involved in oxidative phosphorylation in SA13011-HoR cells, including a subunit of FoF1-ATP synthase (atpC) and a protein required for cytochrome oxidase assembly (ctaA; Table 1), these changes indicate a decrease in oxidative phosphorylation during the selection process.

Bottom Line: The mechanism associated with heterogeneous expression requires both increase expression of mecA and a mutational event that involved the triggering of a β-lactam-mediated SOS response and related lexA and recA genes.We found unique features present in the oxacillin-selected SA13011-HoR derivative when compared to the corresponding SA13011-HeR parental strain that included significant increases in tricarboxyl citric acid (TCA) cycle intermediates and a concomitant decrease in fermentative pathways.These results provide evidence of both the metabolic cost and the adaptation that HeR-MRSA clinical strains undergo when exposed to β-lactam pressure, indicating that the energy production is redirected to supply the cell wall synthesis/metabolism, which in turn contributes to the survival response in the presence of β-lactam antibiotics.

View Article: PubMed Central - PubMed

Affiliation: Metabolon, Inc, Durham, North Carolina, United States of America.

ABSTRACT
Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as one of the most important pathogens both in health care and community-onset infections. The prerequisite for methicillin resistance is mecA, which encodes a β-lactam-insensitive penicillin binding protein PBP2a. A characteristic of MRSA strains from hospital and community associated infections is their heterogeneous expression of resistance to β-lactam (HeR) in which only a small portion (≤ 0.1%) of the population expresses resistance to oxacillin (OXA) ≥ 10 µg/ml, while in other isolates, most of the population expresses resistance to a high level (homotypic resistance, HoR). The mechanism associated with heterogeneous expression requires both increase expression of mecA and a mutational event that involved the triggering of a β-lactam-mediated SOS response and related lexA and recA genes. In the present study we investigated the cellular physiology of HeR-MRSA strains during the process of β-lactam-mediated HeR/HoR selection at sub-inhibitory concentrations by using a combinatorial approach of microarray analyses and global biochemical profiling employing gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) to investigate changes in metabolic pathways and the metabolome associated with β-lactam-mediated HeR/HoR selection in clinically relevant heterogeneous MRSA. We found unique features present in the oxacillin-selected SA13011-HoR derivative when compared to the corresponding SA13011-HeR parental strain that included significant increases in tricarboxyl citric acid (TCA) cycle intermediates and a concomitant decrease in fermentative pathways. Inactivation of the TCA cycle enzyme cis-aconitase gene in the SA13011-HeR strain abolished β-lactam-mediated HeR/HoR selection demonstrating the significance of altered TCA cycle activity during the HeR/HoR selection. These results provide evidence of both the metabolic cost and the adaptation that HeR-MRSA clinical strains undergo when exposed to β-lactam pressure, indicating that the energy production is redirected to supply the cell wall synthesis/metabolism, which in turn contributes to the survival response in the presence of β-lactam antibiotics.

Show MeSH
Related in: MedlinePlus