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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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Comparative genome hybridization of four spontaneous ceftazidime-resistant mutants revealing mutations in gene PA3047.Genomic DNA from mutants 1A1, 1A5, 1D4, and 1A7 was analyzed on a whole genome DNA tiling microarray and compared to the parental wildtype PAO1. Data points (stems) represent the log2 ratio of signal intensity of each mutant against the wildtype signal. Mutants 1A5 and 1A7 showed strong decreases in signal at three consecutive positions (*), indicating deletions. In mutant 1A1 and 1D4, a slight decrease in signal (+) pointed towards a small genetic change, e.g., a single point mutation.
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ppat-1000353-g001: Comparative genome hybridization of four spontaneous ceftazidime-resistant mutants revealing mutations in gene PA3047.Genomic DNA from mutants 1A1, 1A5, 1D4, and 1A7 was analyzed on a whole genome DNA tiling microarray and compared to the parental wildtype PAO1. Data points (stems) represent the log2 ratio of signal intensity of each mutant against the wildtype signal. Mutants 1A5 and 1A7 showed strong decreases in signal at three consecutive positions (*), indicating deletions. In mutant 1A1 and 1D4, a slight decrease in signal (+) pointed towards a small genetic change, e.g., a single point mutation.

Mentions: In an attempt to detect the mutations in the gene(s) yet unknown to be involved in β-lactam resistance, we followed a whole-genome analysis approach. Four of the PAO1 ceftazidime resistant in vitro mutants were analyzed by comparative hybridization on a recently described microarray for the discovery of single nucleotide polymorphisms (SNPs) in P. aeruginosa [20] using the parental PAO1 strain as reference. As shown in Figure 1, major decreases in hybridization ratios (indicating deletions of 50–100 base pairs) were detected for two of the mutants in the gene PA3047, the E. coli dacB ortholog, encoding the nonessential low molecular mass PBP4 [16],[17]. PCR and sequencing confirmed the presence of the deletions in this gene [(nts 1149–1231 for one of the mutants (1A5) and nts 1069–1138 for the other (1D7)] (Figure 1, Table S1). Furthermore, the two remaining mutants (1A1 and 1D4) also revealed a less pronounced decrease of hybridization ratio at a single position in gene PA3047 (Figure 1); PCR and sequencing identified as well the mutations, a G to A change in nt 819 leading to a premature stop codon (W273X) for 1A1 and a A to C change in nt 235 leading to a missense mutation (T79P) for 1D4 (Table S1).


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)

Comparative genome hybridization of four spontaneous ceftazidime-resistant mutants revealing mutations in gene PA3047.Genomic DNA from mutants 1A1, 1A5, 1D4, and 1A7 was analyzed on a whole genome DNA tiling microarray and compared to the parental wildtype PAO1. Data points (stems) represent the log2 ratio of signal intensity of each mutant against the wildtype signal. Mutants 1A5 and 1A7 showed strong decreases in signal at three consecutive positions (*), indicating deletions. In mutant 1A1 and 1D4, a slight decrease in signal (+) pointed towards a small genetic change, e.g., a single point mutation.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000353-g001: Comparative genome hybridization of four spontaneous ceftazidime-resistant mutants revealing mutations in gene PA3047.Genomic DNA from mutants 1A1, 1A5, 1D4, and 1A7 was analyzed on a whole genome DNA tiling microarray and compared to the parental wildtype PAO1. Data points (stems) represent the log2 ratio of signal intensity of each mutant against the wildtype signal. Mutants 1A5 and 1A7 showed strong decreases in signal at three consecutive positions (*), indicating deletions. In mutant 1A1 and 1D4, a slight decrease in signal (+) pointed towards a small genetic change, e.g., a single point mutation.
Mentions: In an attempt to detect the mutations in the gene(s) yet unknown to be involved in β-lactam resistance, we followed a whole-genome analysis approach. Four of the PAO1 ceftazidime resistant in vitro mutants were analyzed by comparative hybridization on a recently described microarray for the discovery of single nucleotide polymorphisms (SNPs) in P. aeruginosa [20] using the parental PAO1 strain as reference. As shown in Figure 1, major decreases in hybridization ratios (indicating deletions of 50–100 base pairs) were detected for two of the mutants in the gene PA3047, the E. coli dacB ortholog, encoding the nonessential low molecular mass PBP4 [16],[17]. PCR and sequencing confirmed the presence of the deletions in this gene [(nts 1149–1231 for one of the mutants (1A5) and nts 1069–1138 for the other (1D7)] (Figure 1, Table S1). Furthermore, the two remaining mutants (1A1 and 1D4) also revealed a less pronounced decrease of hybridization ratio at a single position in gene PA3047 (Figure 1); PCR and sequencing identified as well the mutations, a G to A change in nt 819 leading to a premature stop codon (W273X) for 1A1 and a A to C change in nt 235 leading to a missense mutation (T79P) for 1D4 (Table S1).

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