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Beta-lactam resistance response triggered by inactivation of a nonessential penicillin-binding protein.

Moya B, Dötsch A, Juan C, Blázquez J, Zamorano L, Haussler S, Oliver A - PLoS Pathog. (2009)

Bottom Line: It has long been recognized that the modification of penicillin-binding proteins (PBPs) to reduce their affinity for beta-lactams is an important mechanism (target modification) by which Gram-positive cocci acquire antibiotic resistance.Using as a model Pseudomonas aeruginosa, high up on the list of pathogens causing life-threatening infections in hospitalized patients worldwide, we show that PBPs may also play a major role in beta-lactam resistance in GNR, but through a totally distinct mechanism.The inactivation of this PBP is shown to determine a highly efficient and complex beta-lactam resistance response, triggering overproduction of the chromosomal beta-lactamase AmpC and the specific activation of the CreBC (BlrAB) two-component regulator, which in turn plays a major role in resistance.

View Article: PubMed Central - PubMed

Affiliation: Servicio de Microbiología and Unidad de Investigación, Hospital Son Dureta, Instituto Universitario de Investigación en Ciencias de la Salud Palma de Mallorca, Spain.

ABSTRACT
It has long been recognized that the modification of penicillin-binding proteins (PBPs) to reduce their affinity for beta-lactams is an important mechanism (target modification) by which Gram-positive cocci acquire antibiotic resistance. Among Gram-negative rods (GNR), however, this mechanism has been considered unusual, and restricted to clinically irrelevant laboratory mutants for most species. Using as a model Pseudomonas aeruginosa, high up on the list of pathogens causing life-threatening infections in hospitalized patients worldwide, we show that PBPs may also play a major role in beta-lactam resistance in GNR, but through a totally distinct mechanism. Through a detailed genetic investigation, including whole-genome analysis approaches, we demonstrate that high-level (clinical) beta-lactam resistance in vitro, in vivo, and in the clinical setting is driven by the inactivation of the dacB-encoded nonessential PBP4, which behaves as a trap target for beta-lactams. The inactivation of this PBP is shown to determine a highly efficient and complex beta-lactam resistance response, triggering overproduction of the chromosomal beta-lactamase AmpC and the specific activation of the CreBC (BlrAB) two-component regulator, which in turn plays a major role in resistance. These findings are a major step forward in our understanding of beta-lactam resistance biology, and, more importantly, they open up new perspectives on potential antibiotic targets for the treatment of infectious diseases.

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Related in: MedlinePlus

Schematic representation of the regulation of the P. aeruginosa chromosomal β-lactamase AmpC and peptidoglycan recycling under different conditions.(A) Wild-type strain in the absence of β-lactams. During regular bacterial growth, the peptidoglycan degradation products, MurNac-peptides [N-acetylglucosaminyl-1,6-anhydro- N-acetylmuramyl-tri (or tetra) peptides], are generated in the periplasm through the activity of PBP4 and several other enzymes. These products are then internalized through the permease AmpG, and processed in the cytoplasm by the β-N-acetylglucosaminidase NagZ and the N-acetyl-anhydromuramyl-L-alanine amidase AmpD. P. aeruginosa has two additional AmpD proteins, AmpDh2 that it is apparently located in the outer membrane and AmpDh3 that is still of unknown location. The generated tripeptides are then incorporated to the murein biosynthesis pathway to yield the UDP-MurNac-pentapeptides that will be exported to the periplasm and incorporated to the peptidoglycan, to complete the recycling process. In the absence of β-lactam antibiotics, these UDP-MurNac-pentapeptides interact with AmpR, which functions as a negative regulator of ampC expression. (B) Growth of wild-type strain in the presence AmpC inducer β-lactams. During growth in the presence of certain β-lactams (AmpC inducers), such as cefoxitin or imipenem, AmpC is produced at high levels, conferring natural (intrinsic) resistance to the antibiotic, provided it is a good substrate for the enzyme (as occurs for cefoxitin but not for imipenem). The exact mechanism responsible for the induction of the expression of AmpC in the presence of these antibiotics is still not fully understood. One of the components of the induction process is apparently the saturation of AmpD, due to the enhanced generation of its substrate (MurNac-tripeptides). The accumulated MurNAc-tripeptides are thought to displace the UDP-MurNAc-pentapeptides from AmpR, converting it into an activator of ampC transcription. Our results, and other previous indirect evidences, suggest that the inhibition of PBP4 by these β-lactam antibiotics (known to be the most potent PBP4 inhibitors) plays a major role in the ampC induction process, and determines the activation of the CreBC (BlrAB) two-component regulator. The exact function of the signal transducer AmpE, located in the inner membrane, still needs to be elucidated, but apparently interacts with both AmpD and PBP4. (C) Growth of the AmpD and/or PBP4 mutants in the presence of AmpC non-inducer β-lactams (most antipseudomonal cephalosporins and penicillins, such as ceftazidime or piperacillin, respectively). The inactivation of PBP4 or AmpD produces a very similar constitutive ampC overexpression. Both mechanisms ultimately relay in the activation of AmpR, which changes its activity from negative to positive regulator of ampC expression. Independently of the mechanism, AmpC overexpression itself is shown to confer only moderate (low-level) acquired resistance to non-inducer β-lactams. Additionally, the inactivation of PBP4 specifically activates the CreBC (BlrAB) system, which drives, in conjunction with the AmpC overexpression, the high-level β-lactam resistance response.
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Related In: Results  -  Collection


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ppat-1000353-g002: Schematic representation of the regulation of the P. aeruginosa chromosomal β-lactamase AmpC and peptidoglycan recycling under different conditions.(A) Wild-type strain in the absence of β-lactams. During regular bacterial growth, the peptidoglycan degradation products, MurNac-peptides [N-acetylglucosaminyl-1,6-anhydro- N-acetylmuramyl-tri (or tetra) peptides], are generated in the periplasm through the activity of PBP4 and several other enzymes. These products are then internalized through the permease AmpG, and processed in the cytoplasm by the β-N-acetylglucosaminidase NagZ and the N-acetyl-anhydromuramyl-L-alanine amidase AmpD. P. aeruginosa has two additional AmpD proteins, AmpDh2 that it is apparently located in the outer membrane and AmpDh3 that is still of unknown location. The generated tripeptides are then incorporated to the murein biosynthesis pathway to yield the UDP-MurNac-pentapeptides that will be exported to the periplasm and incorporated to the peptidoglycan, to complete the recycling process. In the absence of β-lactam antibiotics, these UDP-MurNac-pentapeptides interact with AmpR, which functions as a negative regulator of ampC expression. (B) Growth of wild-type strain in the presence AmpC inducer β-lactams. During growth in the presence of certain β-lactams (AmpC inducers), such as cefoxitin or imipenem, AmpC is produced at high levels, conferring natural (intrinsic) resistance to the antibiotic, provided it is a good substrate for the enzyme (as occurs for cefoxitin but not for imipenem). The exact mechanism responsible for the induction of the expression of AmpC in the presence of these antibiotics is still not fully understood. One of the components of the induction process is apparently the saturation of AmpD, due to the enhanced generation of its substrate (MurNac-tripeptides). The accumulated MurNAc-tripeptides are thought to displace the UDP-MurNAc-pentapeptides from AmpR, converting it into an activator of ampC transcription. Our results, and other previous indirect evidences, suggest that the inhibition of PBP4 by these β-lactam antibiotics (known to be the most potent PBP4 inhibitors) plays a major role in the ampC induction process, and determines the activation of the CreBC (BlrAB) two-component regulator. The exact function of the signal transducer AmpE, located in the inner membrane, still needs to be elucidated, but apparently interacts with both AmpD and PBP4. (C) Growth of the AmpD and/or PBP4 mutants in the presence of AmpC non-inducer β-lactams (most antipseudomonal cephalosporins and penicillins, such as ceftazidime or piperacillin, respectively). The inactivation of PBP4 or AmpD produces a very similar constitutive ampC overexpression. Both mechanisms ultimately relay in the activation of AmpR, which changes its activity from negative to positive regulator of ampC expression. Independently of the mechanism, AmpC overexpression itself is shown to confer only moderate (low-level) acquired resistance to non-inducer β-lactams. Additionally, the inactivation of PBP4 specifically activates the CreBC (BlrAB) system, which drives, in conjunction with the AmpC overexpression, the high-level β-lactam resistance response.

Mentions: Figure 2 summarizes the current knowledge on P. aeruginosa ampC regulation, peptidoglycan recycling, and the described similarities and differences of the β-lactam resistance response driven by AmpD inactivation or PBP4 mutation.


Beta-lactam resistance response triggered by inactivation of a nonessential penicillin-binding protein.

Moya B, Dötsch A, Juan C, Blázquez J, Zamorano L, Haussler S, Oliver A - PLoS Pathog. (2009)

Schematic representation of the regulation of the P. aeruginosa chromosomal β-lactamase AmpC and peptidoglycan recycling under different conditions.(A) Wild-type strain in the absence of β-lactams. During regular bacterial growth, the peptidoglycan degradation products, MurNac-peptides [N-acetylglucosaminyl-1,6-anhydro- N-acetylmuramyl-tri (or tetra) peptides], are generated in the periplasm through the activity of PBP4 and several other enzymes. These products are then internalized through the permease AmpG, and processed in the cytoplasm by the β-N-acetylglucosaminidase NagZ and the N-acetyl-anhydromuramyl-L-alanine amidase AmpD. P. aeruginosa has two additional AmpD proteins, AmpDh2 that it is apparently located in the outer membrane and AmpDh3 that is still of unknown location. The generated tripeptides are then incorporated to the murein biosynthesis pathway to yield the UDP-MurNac-pentapeptides that will be exported to the periplasm and incorporated to the peptidoglycan, to complete the recycling process. In the absence of β-lactam antibiotics, these UDP-MurNac-pentapeptides interact with AmpR, which functions as a negative regulator of ampC expression. (B) Growth of wild-type strain in the presence AmpC inducer β-lactams. During growth in the presence of certain β-lactams (AmpC inducers), such as cefoxitin or imipenem, AmpC is produced at high levels, conferring natural (intrinsic) resistance to the antibiotic, provided it is a good substrate for the enzyme (as occurs for cefoxitin but not for imipenem). The exact mechanism responsible for the induction of the expression of AmpC in the presence of these antibiotics is still not fully understood. One of the components of the induction process is apparently the saturation of AmpD, due to the enhanced generation of its substrate (MurNac-tripeptides). The accumulated MurNAc-tripeptides are thought to displace the UDP-MurNAc-pentapeptides from AmpR, converting it into an activator of ampC transcription. Our results, and other previous indirect evidences, suggest that the inhibition of PBP4 by these β-lactam antibiotics (known to be the most potent PBP4 inhibitors) plays a major role in the ampC induction process, and determines the activation of the CreBC (BlrAB) two-component regulator. The exact function of the signal transducer AmpE, located in the inner membrane, still needs to be elucidated, but apparently interacts with both AmpD and PBP4. (C) Growth of the AmpD and/or PBP4 mutants in the presence of AmpC non-inducer β-lactams (most antipseudomonal cephalosporins and penicillins, such as ceftazidime or piperacillin, respectively). The inactivation of PBP4 or AmpD produces a very similar constitutive ampC overexpression. Both mechanisms ultimately relay in the activation of AmpR, which changes its activity from negative to positive regulator of ampC expression. Independently of the mechanism, AmpC overexpression itself is shown to confer only moderate (low-level) acquired resistance to non-inducer β-lactams. Additionally, the inactivation of PBP4 specifically activates the CreBC (BlrAB) system, which drives, in conjunction with the AmpC overexpression, the high-level β-lactam resistance response.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654508&req=5

ppat-1000353-g002: Schematic representation of the regulation of the P. aeruginosa chromosomal β-lactamase AmpC and peptidoglycan recycling under different conditions.(A) Wild-type strain in the absence of β-lactams. During regular bacterial growth, the peptidoglycan degradation products, MurNac-peptides [N-acetylglucosaminyl-1,6-anhydro- N-acetylmuramyl-tri (or tetra) peptides], are generated in the periplasm through the activity of PBP4 and several other enzymes. These products are then internalized through the permease AmpG, and processed in the cytoplasm by the β-N-acetylglucosaminidase NagZ and the N-acetyl-anhydromuramyl-L-alanine amidase AmpD. P. aeruginosa has two additional AmpD proteins, AmpDh2 that it is apparently located in the outer membrane and AmpDh3 that is still of unknown location. The generated tripeptides are then incorporated to the murein biosynthesis pathway to yield the UDP-MurNac-pentapeptides that will be exported to the periplasm and incorporated to the peptidoglycan, to complete the recycling process. In the absence of β-lactam antibiotics, these UDP-MurNac-pentapeptides interact with AmpR, which functions as a negative regulator of ampC expression. (B) Growth of wild-type strain in the presence AmpC inducer β-lactams. During growth in the presence of certain β-lactams (AmpC inducers), such as cefoxitin or imipenem, AmpC is produced at high levels, conferring natural (intrinsic) resistance to the antibiotic, provided it is a good substrate for the enzyme (as occurs for cefoxitin but not for imipenem). The exact mechanism responsible for the induction of the expression of AmpC in the presence of these antibiotics is still not fully understood. One of the components of the induction process is apparently the saturation of AmpD, due to the enhanced generation of its substrate (MurNac-tripeptides). The accumulated MurNAc-tripeptides are thought to displace the UDP-MurNAc-pentapeptides from AmpR, converting it into an activator of ampC transcription. Our results, and other previous indirect evidences, suggest that the inhibition of PBP4 by these β-lactam antibiotics (known to be the most potent PBP4 inhibitors) plays a major role in the ampC induction process, and determines the activation of the CreBC (BlrAB) two-component regulator. The exact function of the signal transducer AmpE, located in the inner membrane, still needs to be elucidated, but apparently interacts with both AmpD and PBP4. (C) Growth of the AmpD and/or PBP4 mutants in the presence of AmpC non-inducer β-lactams (most antipseudomonal cephalosporins and penicillins, such as ceftazidime or piperacillin, respectively). The inactivation of PBP4 or AmpD produces a very similar constitutive ampC overexpression. Both mechanisms ultimately relay in the activation of AmpR, which changes its activity from negative to positive regulator of ampC expression. Independently of the mechanism, AmpC overexpression itself is shown to confer only moderate (low-level) acquired resistance to non-inducer β-lactams. Additionally, the inactivation of PBP4 specifically activates the CreBC (BlrAB) system, which drives, in conjunction with the AmpC overexpression, the high-level β-lactam resistance response.
Mentions: Figure 2 summarizes the current knowledge on P. aeruginosa ampC regulation, peptidoglycan recycling, and the described similarities and differences of the β-lactam resistance response driven by AmpD inactivation or PBP4 mutation.

Bottom Line: It has long been recognized that the modification of penicillin-binding proteins (PBPs) to reduce their affinity for beta-lactams is an important mechanism (target modification) by which Gram-positive cocci acquire antibiotic resistance.Using as a model Pseudomonas aeruginosa, high up on the list of pathogens causing life-threatening infections in hospitalized patients worldwide, we show that PBPs may also play a major role in beta-lactam resistance in GNR, but through a totally distinct mechanism.The inactivation of this PBP is shown to determine a highly efficient and complex beta-lactam resistance response, triggering overproduction of the chromosomal beta-lactamase AmpC and the specific activation of the CreBC (BlrAB) two-component regulator, which in turn plays a major role in resistance.

View Article: PubMed Central - PubMed

Affiliation: Servicio de Microbiología and Unidad de Investigación, Hospital Son Dureta, Instituto Universitario de Investigación en Ciencias de la Salud Palma de Mallorca, Spain.

ABSTRACT
It has long been recognized that the modification of penicillin-binding proteins (PBPs) to reduce their affinity for beta-lactams is an important mechanism (target modification) by which Gram-positive cocci acquire antibiotic resistance. Among Gram-negative rods (GNR), however, this mechanism has been considered unusual, and restricted to clinically irrelevant laboratory mutants for most species. Using as a model Pseudomonas aeruginosa, high up on the list of pathogens causing life-threatening infections in hospitalized patients worldwide, we show that PBPs may also play a major role in beta-lactam resistance in GNR, but through a totally distinct mechanism. Through a detailed genetic investigation, including whole-genome analysis approaches, we demonstrate that high-level (clinical) beta-lactam resistance in vitro, in vivo, and in the clinical setting is driven by the inactivation of the dacB-encoded nonessential PBP4, which behaves as a trap target for beta-lactams. The inactivation of this PBP is shown to determine a highly efficient and complex beta-lactam resistance response, triggering overproduction of the chromosomal beta-lactamase AmpC and the specific activation of the CreBC (BlrAB) two-component regulator, which in turn plays a major role in resistance. These findings are a major step forward in our understanding of beta-lactam resistance biology, and, more importantly, they open up new perspectives on potential antibiotic targets for the treatment of infectious diseases.

Show MeSH
Related in: MedlinePlus