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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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Quantitation of mRNA levels of TCA cycle-, amino-acid catabolism-, carbohydrate catabolism- and cell wall-associated genes by real-time RT-PCR.RNA was prepared from SA13011-HeR and its highly resistant derivative SA13011-HoR (SA13011+ OXA 0.5 µg/ml) cells, collected at exponential phase of growth, as described in Materials and Methods. Relative fold change values of specific mRNAs in SA13011-HoR vs. SA13011-HeR (reference value = 1) are shown on the vertical axis. Relative fold change values representing the means of at least three biological replicates of specific mRNAs ± standard error of the mean (SEM), sampled in triplicate to minimize error by inter- and intra-samples, are shown on the vertical axis; 16S rRNA was used as an internal control. Differences between the mean values were analyzed using a one-way analysis of variance (ANOVA). A P value of <0.01 was considered statistically significant. Oligonucleotide primers are shown in Table S2.
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pone-0071025-g001: Quantitation of mRNA levels of TCA cycle-, amino-acid catabolism-, carbohydrate catabolism- and cell wall-associated genes by real-time RT-PCR.RNA was prepared from SA13011-HeR and its highly resistant derivative SA13011-HoR (SA13011+ OXA 0.5 µg/ml) cells, collected at exponential phase of growth, as described in Materials and Methods. Relative fold change values of specific mRNAs in SA13011-HoR vs. SA13011-HeR (reference value = 1) are shown on the vertical axis. Relative fold change values representing the means of at least three biological replicates of specific mRNAs ± standard error of the mean (SEM), sampled in triplicate to minimize error by inter- and intra-samples, are shown on the vertical axis; 16S rRNA was used as an internal control. Differences between the mean values were analyzed using a one-way analysis of variance (ANOVA). A P value of <0.01 was considered statistically significant. Oligonucleotide primers are shown in Table S2.

Mentions: Validation of the metabolic changes in gene expression regulated during HeR/HoR selection identified by microarray analysis was performed by Real-Time RT-PCR by using RNAs collected from SA13011-HeR and -HoR cells. Consistent with the microarray analysis, we observed a 10-fold increase in citZ and a 6-fold increase in citB expression (Figure 1A). We also measured expression of mqo2 which encodes malate dehydrogenase and found its expression was also elevated in SA13011-HoR. We confirmed changes reporting on acetate metabolism (acyP, acsA, and ackA;Figure 1A), proline and ornithine metabolism [rocA, (1-pyroline-5-carboxylate dehydrogenase); rocD (ornithine-oxo-acid transaminase; Figure 1B], and ribose metabolism (rbsD;Figure 1C) that were observed in the microarray analysis (Table 1). Additionally, we determined increased expression of dltA [D-alanine-poly (phosphoribitol) ligase subunit 1] and dltC [D-alanine-poly (phosphoribitol) ligase subunit 2] (Figure 1C), both genes encoding for enzymes involved in ribitol metabolism [25]. Expression analysis of cell wall associated genes included genes related to the expression of methicillin resistance including PBP2a (mecA, 6-fold increase) and PBP2 (pbp2, 4-fold increase), as well as genes associated with peptidoglycan cross-linking (femA; 5.8-fold increase) (Figure 1D). Consistent with their role, expression of glucosamine-6-phosphate synthase (glmS), important for production of a major building block of peptidoglycan, was also found up-regulated in SA13011-HoR strain (Figure 1D). Similar results were obtained during HeR/HoR selection of the heterogeneous MRSA strain SA43002 (phenotypically similar to SA13011) (Fig. S3).


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)

Quantitation of mRNA levels of TCA cycle-, amino-acid catabolism-, carbohydrate catabolism- and cell wall-associated genes by real-time RT-PCR.RNA was prepared from SA13011-HeR and its highly resistant derivative SA13011-HoR (SA13011+ OXA 0.5 µg/ml) cells, collected at exponential phase of growth, as described in Materials and Methods. Relative fold change values of specific mRNAs in SA13011-HoR vs. SA13011-HeR (reference value = 1) are shown on the vertical axis. Relative fold change values representing the means of at least three biological replicates of specific mRNAs ± standard error of the mean (SEM), sampled in triplicate to minimize error by inter- and intra-samples, are shown on the vertical axis; 16S rRNA was used as an internal control. Differences between the mean values were analyzed using a one-way analysis of variance (ANOVA). A P value of <0.01 was considered statistically significant. Oligonucleotide primers are shown in Table S2.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3733780&req=5

pone-0071025-g001: Quantitation of mRNA levels of TCA cycle-, amino-acid catabolism-, carbohydrate catabolism- and cell wall-associated genes by real-time RT-PCR.RNA was prepared from SA13011-HeR and its highly resistant derivative SA13011-HoR (SA13011+ OXA 0.5 µg/ml) cells, collected at exponential phase of growth, as described in Materials and Methods. Relative fold change values of specific mRNAs in SA13011-HoR vs. SA13011-HeR (reference value = 1) are shown on the vertical axis. Relative fold change values representing the means of at least three biological replicates of specific mRNAs ± standard error of the mean (SEM), sampled in triplicate to minimize error by inter- and intra-samples, are shown on the vertical axis; 16S rRNA was used as an internal control. Differences between the mean values were analyzed using a one-way analysis of variance (ANOVA). A P value of <0.01 was considered statistically significant. Oligonucleotide primers are shown in Table S2.
Mentions: Validation of the metabolic changes in gene expression regulated during HeR/HoR selection identified by microarray analysis was performed by Real-Time RT-PCR by using RNAs collected from SA13011-HeR and -HoR cells. Consistent with the microarray analysis, we observed a 10-fold increase in citZ and a 6-fold increase in citB expression (Figure 1A). We also measured expression of mqo2 which encodes malate dehydrogenase and found its expression was also elevated in SA13011-HoR. We confirmed changes reporting on acetate metabolism (acyP, acsA, and ackA;Figure 1A), proline and ornithine metabolism [rocA, (1-pyroline-5-carboxylate dehydrogenase); rocD (ornithine-oxo-acid transaminase; Figure 1B], and ribose metabolism (rbsD;Figure 1C) that were observed in the microarray analysis (Table 1). Additionally, we determined increased expression of dltA [D-alanine-poly (phosphoribitol) ligase subunit 1] and dltC [D-alanine-poly (phosphoribitol) ligase subunit 2] (Figure 1C), both genes encoding for enzymes involved in ribitol metabolism [25]. Expression analysis of cell wall associated genes included genes related to the expression of methicillin resistance including PBP2a (mecA, 6-fold increase) and PBP2 (pbp2, 4-fold increase), as well as genes associated with peptidoglycan cross-linking (femA; 5.8-fold increase) (Figure 1D). Consistent with their role, expression of glucosamine-6-phosphate synthase (glmS), important for production of a major building block of peptidoglycan, was also found up-regulated in SA13011-HoR strain (Figure 1D). Similar results were obtained during HeR/HoR selection of the heterogeneous MRSA strain SA43002 (phenotypically similar to SA13011) (Fig. S3).

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