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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 acnA mRNA (A) and TCA cycle-associated genes (B) by Real-Time RT-PCR.RNAs were prepared from SA13011-HeR/HoR, acnA- mutant LMR15 and LMR15 complemented with either the empty-vector (LMR15-EV) or wild-type acnA (LMR17), grown in the absence or presence of OXA (0.5 µg/ml). Cells were collected at exponential phase of growth as described in Materials and Methods. Relative fold change values versus SA13011-HeR ( = 1) of specific mRNAs are shown in the vertical axis; 16rRNA was used as an internal control. *, significantly different than SA13011-HeR (P<0.001).h.
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pone-0071025-g007: Quantitation of acnA mRNA (A) and TCA cycle-associated genes (B) by Real-Time RT-PCR.RNAs were prepared from SA13011-HeR/HoR, acnA- mutant LMR15 and LMR15 complemented with either the empty-vector (LMR15-EV) or wild-type acnA (LMR17), grown in the absence or presence of OXA (0.5 µg/ml). Cells were collected at exponential phase of growth as described in Materials and Methods. Relative fold change values versus SA13011-HeR ( = 1) of specific mRNAs are shown in the vertical axis; 16rRNA was used as an internal control. *, significantly different than SA13011-HeR (P<0.001).h.

Mentions: The results from gene expression and metabolomics analyses described above demonstrated both a marked increase in the expression of genes corresponding to the TCA cycle and a redirection of metabolic activity toward the cycle (see summary Fig. 6). To further investigate its functional role and the contribution to the β-lactam mediated HeR/HoR selection, the aconitase gene acnA-citB, which encodes the second enzyme of TCA cycle, was inactivated in SA13011-HeR strain (SA13011 ΔacnA::tetM, LMR15; Table 4). Expression of acnA in mutant strains was monitored by Real-Time RT-PCR (Fig. 7A). Phenotypic analysis of acnA- mutant LMR15 showed no changes in the susceptibility to OXA after exposure to sub-inhibitory concentrations of the antibiotic, i.e. MIC: 1 µg/ml before selection vs. 0.75 µg/ml after OXA exposure (Table 3, Fig. 8A). These results indicated that inactivation of the acnA gene impaired β-lactam-mediated HeR/HoR selection. Complementation of acnA- mutant LMR15 with a cloned full-length acnA (LMR17) resulted in transcription levels similar to those corresponding to SA13011-HoR (Fig. 7A). As a control, LMR15 was complemented with the corresponding empty-vector (LMR15-EV) and, as expected, this did not rescue acnA expression (Fig. 7A). Importantly, complementation with full-length acnA restored the selection of the OXA resistant derivative, although not to the same degree observed in SA13011-HoR strain [MICs: 32 µg/ml for LMR18 (LMR17+ OXA) vs. 256 µg/ml, for SA13011-HoR, Table 3, Fig. 8B). However, higher MICs values were achieved when full-length acnA-complemented LMR17 strain undergoing selection with OXA was simultaneously supplemented with glucose (10mM) to maximize optimal glycolysis coverage, which resulted in LMR18 (LMR17+ OXA) MIC of 256 µg/ml (Fig. 8B). Similar results were obtained when pyruvate (20mM) or ribose (12mM) were used instead of glucose (data not shown). Supplementation with carbon sources had no effect per se in terms of resistance acquisition either in SA13011-HeR or LMR17 (Table 3). Expression analysis by Real-Time RT-PCR demonstrated altered regulation of TCA cycle genes in the absence of acnA during β-lactam-mediated HeR/HoR selection (Fig. 7B). After acnA inactivation, primary TCA cycle-related genes including citZ, citB, acyP and acsA were down-regulated in LMR16 (LMR15+ OXA), while complementation of the acnA mutant with the cloned full-length gene determined a regain in their corresponding expression in LMR18 (LMR17+ OXA). Together, these results demonstrate the requirement of an active TCA cycle and its key functional role during during β-lactam-mediated HeR/HoR 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)

Quantitation of acnA mRNA (A) and TCA cycle-associated genes (B) by Real-Time RT-PCR.RNAs were prepared from SA13011-HeR/HoR, acnA- mutant LMR15 and LMR15 complemented with either the empty-vector (LMR15-EV) or wild-type acnA (LMR17), grown in the absence or presence of OXA (0.5 µg/ml). Cells were collected at exponential phase of growth as described in Materials and Methods. Relative fold change values versus SA13011-HeR ( = 1) of specific mRNAs are shown in the vertical axis; 16rRNA was used as an internal control. *, significantly different than SA13011-HeR (P<0.001).h.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3733780&req=5

pone-0071025-g007: Quantitation of acnA mRNA (A) and TCA cycle-associated genes (B) by Real-Time RT-PCR.RNAs were prepared from SA13011-HeR/HoR, acnA- mutant LMR15 and LMR15 complemented with either the empty-vector (LMR15-EV) or wild-type acnA (LMR17), grown in the absence or presence of OXA (0.5 µg/ml). Cells were collected at exponential phase of growth as described in Materials and Methods. Relative fold change values versus SA13011-HeR ( = 1) of specific mRNAs are shown in the vertical axis; 16rRNA was used as an internal control. *, significantly different than SA13011-HeR (P<0.001).h.
Mentions: The results from gene expression and metabolomics analyses described above demonstrated both a marked increase in the expression of genes corresponding to the TCA cycle and a redirection of metabolic activity toward the cycle (see summary Fig. 6). To further investigate its functional role and the contribution to the β-lactam mediated HeR/HoR selection, the aconitase gene acnA-citB, which encodes the second enzyme of TCA cycle, was inactivated in SA13011-HeR strain (SA13011 ΔacnA::tetM, LMR15; Table 4). Expression of acnA in mutant strains was monitored by Real-Time RT-PCR (Fig. 7A). Phenotypic analysis of acnA- mutant LMR15 showed no changes in the susceptibility to OXA after exposure to sub-inhibitory concentrations of the antibiotic, i.e. MIC: 1 µg/ml before selection vs. 0.75 µg/ml after OXA exposure (Table 3, Fig. 8A). These results indicated that inactivation of the acnA gene impaired β-lactam-mediated HeR/HoR selection. Complementation of acnA- mutant LMR15 with a cloned full-length acnA (LMR17) resulted in transcription levels similar to those corresponding to SA13011-HoR (Fig. 7A). As a control, LMR15 was complemented with the corresponding empty-vector (LMR15-EV) and, as expected, this did not rescue acnA expression (Fig. 7A). Importantly, complementation with full-length acnA restored the selection of the OXA resistant derivative, although not to the same degree observed in SA13011-HoR strain [MICs: 32 µg/ml for LMR18 (LMR17+ OXA) vs. 256 µg/ml, for SA13011-HoR, Table 3, Fig. 8B). However, higher MICs values were achieved when full-length acnA-complemented LMR17 strain undergoing selection with OXA was simultaneously supplemented with glucose (10mM) to maximize optimal glycolysis coverage, which resulted in LMR18 (LMR17+ OXA) MIC of 256 µg/ml (Fig. 8B). Similar results were obtained when pyruvate (20mM) or ribose (12mM) were used instead of glucose (data not shown). Supplementation with carbon sources had no effect per se in terms of resistance acquisition either in SA13011-HeR or LMR17 (Table 3). Expression analysis by Real-Time RT-PCR demonstrated altered regulation of TCA cycle genes in the absence of acnA during β-lactam-mediated HeR/HoR selection (Fig. 7B). After acnA inactivation, primary TCA cycle-related genes including citZ, citB, acyP and acsA were down-regulated in LMR16 (LMR15+ OXA), while complementation of the acnA mutant with the cloned full-length gene determined a regain in their corresponding expression in LMR18 (LMR17+ OXA). Together, these results demonstrate the requirement of an active TCA cycle and its key functional role during during β-lactam-mediated HeR/HoR 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