β-Lactam Resistance Response Triggered by Inactivation of a Nonessential Penicillin-Binding Protein

Article (PDF Available)inPLoS Pathogens 5(3):e1000353 · April 2009with51 Reads
DOI: 10.1371/journal.ppat.1000353 · Source: PubMed
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.


    • "Indeed, our experiments using an ampG mutant of strain PAO1 and a plasmid-mediated ampC demonstrated that fitness-virulence impairment is produced by the blocking of peptidoglycan recycling and simultaneous ampC overexpression. Finally, we also ruled out the alternative hypothesis that the preservation of fitness and virulence of the dacB mutants was driven by the previously documented activation of the creBC system (shown to play a major role in fitness and response to -lactam challenge [22, 26] ), since knocking out this twocomponent regulatory system did not produce a significant effect on fitness and virulence. Regardless of the underlying mechanisms, our results are helpful to understand the natural occurrence of AmpC-hyperproducing mutants among clinical strains, since while ampD dacB double mutants have been well documented (22, 34), ampD triple mutants have not been reported so far. "
    [Show abstract] [Hide abstract] ABSTRACT: Understanding the interplay between antibiotic resistance and bacterial fitness and virulence is essential to guide individual treatments and improve global antibiotic policies. A paradigmatic example of a resistance mechanism is the intrinsic inducible chromosomal β-lactamase AmpC from multiple Gram-negative bacteria, including Pseudomonas aeruginosa, a major nosocomial pathogen. The regulation of ampC expression is intimately linked to peptidoglycan recycling, and AmpC-mediated β-lactam resistance is frequently mediated by inactivating mutations in ampD, encoding an N-acetyl-anhydromuramyl-l-alanine amidase, affecting the levels of ampC-activating muropeptides. Here we dissect the impact of the multiple pathways causing AmpC hyperproduction on P. aeruginosa fitness and virulence. Through a detailed analysis, we demonstrate that the lack of all three P. aeruginosa AmpD amidases causes a dramatic effect in fitness and pathogenicity, severely compromising growth rates, motility, and cytotoxicity; the latter effect is likely achieved by repressing key virulence factors, such as protease LasA, phospholipase C, or type III secretion system components. We also show that ampC overexpression is required but not sufficient to confer the growth-motility-cytotoxicity impaired phenotype and that alternative pathways leading to similar levels of ampC hyperexpression and resistance, such as those involving PBP4, had no fitness-virulence cost. Further analysis indicated that fitness-virulence impairment is caused by overexpressing ampC in the absence of cell wall recycling, as reproduced by expressing ampC from a plasmid in an AmpG (muropeptide permease)-deficient background. Thus, our findings represent a major step in the understanding of β-lactam resistance biology and its interplay with fitness and pathogenesis.
    Full-text · Article · Nov 2016
    • "Recent studies stressed the complexity of this relationship, in which the role of the amidase AmpD is definitely known, the role of the transcriptional regulator AmpR is partially recognised with some degree of certainty, while researchers have speculated on the function of LMM-PBP4 based on information available for coding gene of this protein in the PAO1 and UCBPP- PA14 strains. The putative nature of this information has been partly resolved in recent research showing, for example, that LMM-PBP4 of Pseudomonas aeruginosa exercises control over the function of AmpR, presumably as a result of its ability to create or destroy a particular muropeptide chain subunit [21, 23, 38]. A study aimed to characterize the role of LMM-PBPs in peptidoglycan composition, ?-lactam resistance, and AmpC regulation, indicated that PBP4 play a significant role as D,D-carboxipeptidase only when PBP5 is absent; on the other hand, the peptidoglycan structure of PBP4 and PBP7 single and double mutants showed that these proteins have D,D-endopeptidase activity [39]. "
    [Show abstract] [Hide abstract] ABSTRACT: Background Community and nosocomial infections by Pseudomonas aeruginosa still create a major therapeutic challenge. The resistance of this opportunist pathogen to β-lactam antibiotics is determined mainly by production of the inactivating enzyme AmpC, a class C cephalosporinase with a regulation system more complex than those found in members of the Enterobacteriaceae family. This regulatory system also participates directly in peptidoglycan turnover and recycling. One of the regulatory mechanisms for AmpC expression, recently identified in clinical isolates, is the inactivation of LMM-PBP4 (Low-Molecular-Mass Penicillin-Binding Protein 4), a protein whose catalytic activity on natural substrates has remained uncharacterized until now. Results We carried out in vivo activity trials for LMM-PBP4 of Pseudomonas aeruginosa on macromolecular peptidoglycan of Escherichia coli and Pseudomonas aeruginosa. The results showed a decrease in the relative quantity of dimeric, trimeric and anhydrous units, and a smaller reduction in monomer disaccharide pentapeptide (M5) levels, validating the occurrence of D,D-carboxypeptidase and D,D-endopeptidase activities. Under conditions of induction for this protein and cefoxitin treatment, the reduction in M5 is not fully efficient, implying that LMM-PBP4 of Pseudomonas aeruginosa presents better behaviour as a D,D-endopeptidase. Kinetic evaluation of the direct D,D-peptidase activity of this protein on natural muropeptides M5 and D45 confirmed this bifunctionality and the greater affinity of LMM-PBP4 for its dimeric substrate. A three-dimensional model for the monomeric unit of LMM-PBP4 provided structural information which supports its catalytic performance. Conclusions LMM-PBP4 of Pseudomonas aeruginosa is a bifunctional enzyme presenting both D,D-carboxypeptidase and D,D-endopeptidase activities; the D,D-endopeptidase function is predominant. Our study provides unprecedented functional and structural information which supports the proposal of this protein as a potential hydrolase-autolysin associated with peptidoglycan maturation and recycling. The fact that mutant PBP4 induces AmpC, may indicate that a putative muropeptide-subunit product of the DD-EPase activity of PBP4 could be a negative regulator of the pathway. This data contributes to understanding of the regulatory aspects of resistance to β-lactam antibiotics in this bacterial model. Electronic supplementary material The online version of this article (doi:10.1186/s12866-016-0853-x) contains supplementary material, which is available to authorized users.
    Full-text · Article · Oct 2016
    • "Park and Uehara also suggested that the free pentapeptide may be an effector of AmpR (Park and Uehara, 2008). The enhancement of -lactam resistance in P. aeruginosa caused by the inactivation of PBP4, a noncritical carboxypeptidase encoded by dacB, seems to indicate that the pentapeptide is a more likely candidate, but the identity of the true signal molecule(s) has not yet been determined unequivocally (Moya et al., 2009; Zamorano et al., 2010). The tetrapeptide is unlikely to be the signal molecule because it is known that tetrapeptides do not accumulate in the cytoplasm. "
    [Show abstract] [Hide abstract] ABSTRACT: Antimicrobial resistance is one of the most serious health threats. Cell-wall remodeling processes are tightly regulated to warrant bacterial survival and in some cases are directly linked to antibiotic resistance. Remodeling produces cell-wall fragments that are recycled but can also act as messengers for bacterial communication, as effector molecules in immune response and as signaling molecules triggering antibiotic resistance. This review is intended to provide state-of-the-art information about the molecular mechanisms governing this process and gather structural information of the different macromolecular machineries involved in peptidoglycan recycling in Gram-negative bacteria. The growing body of literature on the 3D structures of the corresponding macromolecules reveals an extraordinary complexity. Considering the increasing incidence and widespread emergence of Gram-negative multidrug-resistant pathogens in clinics, structural information on the main actors of the recycling process paves the way for designing novel antibiotics disrupting cellular communication in the recycling-resistance pathway.
    Full-text · Article · Jul 2016
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