scholarly journals Expression of Pyridoxal 5′-Phosphate-Independent Racemases Can Reduce 2-Aminoacrylate Stress inSalmonella enterica

2018 ◽  
Vol 200 (9) ◽  
Author(s):  
Kelsey M. Hodge-Hanson ◽  
Allison Zoino ◽  
Diana M. Downs
Keyword(s):  
Mutant Strain ◽  
Metabolic Stress ◽  
Reactive Metabolites ◽  
Methanococcus Maripaludis ◽  
Content Type ◽  
Direct Quenching ◽  
Glutamate Racemase ◽  
Independent Mechanism ◽  
Domains Of Life

ABSTRACTThe RidA protein (PF01042) fromSalmonella entericais a deaminase that quenches 2-aminoacrylate (2AA) and other reactive metabolites. In the absence of RidA, 2AA accumulates, damages cellular enzymes, and compromises the metabolic network.In vitro, RidA homologs from all domains of life deaminate 2AA, and RidA proteins from plants, bacteria, yeast, and humans complement the mutant phenotype of aridAmutant strain ofS. enterica. In the present study, a methanogenic archaeon,Methanococcus maripaludisS2, was used to probe alternative mechanisms to restore metabolic balance.M. maripaludisMMP0739, which is annotated as an aspartate/glutamate racemase, complemented aridAmutant strain and reduced the intracellular 2AA burden. The aspartate/glutamate racemase YgeA fromEscherichia coliorS. enterica, when provided intrans, similarly restored wild-type growth to aridAmutant. These results uncovered a new mechanism to ameliorate metabolic stress, and they suggest that direct quenching by RidA is not the only strategy to quench 2AA.IMPORTANCE2-Aminoacrylate is an endogenously generated reactive metabolite that can damage cellular enzymes if not directly quenched by the conserved deaminase RidA. This study used an archaeon to identify a RidA-independent mechanism to prevent metabolic stress caused by 2AA. The data suggest that a gene product annotated as an aspartate/glutamate racemase (MMP0739) produces a metabolite that can quench 2AA, expanding our understanding of strategies available to quench reactive metabolites.

scholarly journals PA5339, a RidA Homolog, Is Required for Full Growth inPseudomonas aeruginosa

2018 ◽  
Vol 200 (22) ◽  
Author(s):  
Jessica Irons ◽  
Kelsey M. Hodge-Hanson ◽  
Diana M. Downs
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Salmonella Enterica ◽  
Metabolic Stress ◽  
Content Type ◽  
Growth Defects ◽  
Collective Data ◽  
Domains Of Life ◽  
Mutant Phenotypes

ABSTRACTThe Rid protein superfamily (YjgF/YER057c/UK114) is found in all domains of life. The archetypal protein, RidA fromSalmonella enterica, is a deaminase that quenches the reactive metabolite 2-aminoacrylate (2AA). 2AA deaminase activity is conserved in RidA proteins from humans, plants, yeast, archaea, and bacteria. Mutants ofSalmonella enterica,Escherichia coli, andSaccharomyces cerevisiaethat lack a functional RidA exhibit growth defects, suggesting that 2AA metabolic stress is similarly conserved. The PubSEED database showsPseudomonas aeruginosa(PAO1) encodes eight members of the Rid superfamily. Mutants ofP. aeruginosaPAO1 lacking each of five Rid proteins were screened, and the mutant phenotypes that arose in the absence of PA5339 were dissected. A PA5339::Tn mutant has growth, motility, and biofilm defects that can all be linked to the accumulation of 2AA. Further, the PA5339 protein was demonstrably a 2AA deaminasein vitroand restored metabolic balance to aS. enterica ridAmutantin vivo. The data presented here show that the RidA paradigm inPseudomonas aeruginosahad similarities to those described in other organisms but was distinct in that deleting only one of multiple homologs generated deficiencies. Based on the collective data presented here in, PA5339 was renamed RidA.IMPORTANCERidA is a widely conserved protein that prevents endogenous metabolic stress caused by 2-aminoacrylate (2AA) damage to pyridoxal 5′-phosphate (PLP)-dependent enzymes in prokaryotes and eukaryotes. The framework for understanding the accumulation of 2AA and its consequences have largely been defined inSalmonella enterica. We show here that inP. aeruginosa(PAO1), 2AA accumulation leads to reduced growth, compromised motility, and defective biofilm formation. This study expands our knowledge how the metabolic architecture of an organism contributes to the consequences of 2AA inactivation of PLP-dependent enzymes and identifies a key RidA protein inP. aeruginosa.

scholarly journals Competing Substrates for the Bifunctional Diaminopimelic Acid Epimerase/Glutamate Racemase Modulate Peptidoglycan Synthesis in Chlamydia trachomatis

2020 ◽  
Vol 89 (1) ◽  
pp. e00401-20
Author(s):  
Raghuveer Singh ◽  
Jessica A. Slade ◽  
Mary Brockett ◽  
Daniel Mendez ◽  
George W. Liechti ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Diaminopimelic Acid ◽  
Competition Strategy ◽  
Content Type ◽  
E Coli ◽  
Glutamate Racemase ◽  
Heterologous System ◽  
Substrate Competition

ABSTRACTThe Chlamydia trachomatis genome encodes multiple bifunctional enzymes, such as DapF, which is capable of both diaminopimelic acid (DAP) epimerase and glutamate racemase activity. Our previous work demonstrated the bifunctional activity of chlamydial DapF in vitro and in a heterologous system (Escherichia coli). In the present study, we employed a substrate competition strategy to demonstrate DapFCt function in vivo in C. trachomatis. We reasoned that, because DapFCt utilizes a shared substrate-binding site for both racemase and epimerase activities, only one activity can occur at a time. Therefore, an excess of one substrate relative to another must determine which activity is favored. We show that the addition of excess l-glutamate or meso-DAP (mDAP) to C. trachomatis resulted in 90% reduction in bacterial titers, compared to untreated controls. Excess l-glutamate reduced in vivo synthesis of mDAP by C. trachomatis to undetectable levels, thus confirming that excess racemase substrate led to inhibition of DapFCt DAP epimerase activity. We previously showed that expression of dapFCt in a murI (racemase) ΔdapF (epimerase) double mutant of E. coli rescues the d-glutamate auxotrophic defect. Addition of excess mDAP inhibited growth of this strain, but overexpression of dapFCt allowed the mutant to overcome growth inhibition. These results confirm that DapFCt is the primary target of these mDAP and l-glutamate treatments. Our findings demonstrate that suppression of either the glutamate racemase or epimerase activity of DapF compromises the growth of C. trachomatis. Thus, a substrate competition strategy can be a useful tool for in vivo validation of an essential bifunctional enzyme.

scholarly journals Cofactor Selectivity in Methylmalonyl Coenzyme A Mutase, a Model Cobamide-Dependent Enzyme

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Olga M. Sokolovskaya ◽  
Kenny C. Mok ◽  
Jong Duk Park ◽  
Jennifer L. A. Tran ◽  
Kathryn A. Quanstrom ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Bacterial Growth ◽  
Microbial Interactions ◽  
Vitamin B ◽  
Content Type ◽  
Domains Of Life ◽  
Enzyme Cofactors ◽  
Lower Ligand ◽  
Enzyme Selectivity

ABSTRACT Cobamides, a uniquely diverse family of enzyme cofactors related to vitamin B12, are produced exclusively by bacteria and archaea but used in all domains of life. While it is widely accepted that cobamide-dependent organisms require specific cobamides for their metabolism, the biochemical mechanisms that make cobamides functionally distinct are largely unknown. Here, we examine the effects of cobamide structural variation on a model cobamide-dependent enzyme, methylmalonyl coenzyme A (CoA) mutase (MCM). The in vitro binding affinity of MCM for cobamides can be dramatically influenced by small changes in the structure of the lower ligand of the cobamide, and binding selectivity differs between bacterial orthologs of MCM. In contrast, variations in the lower ligand have minor effects on MCM catalysis. Bacterial growth assays demonstrate that cobamide requirements of MCM in vitro largely correlate with in vivo cobamide dependence. This result underscores the importance of enzyme selectivity in the cobamide-dependent physiology of bacteria. IMPORTANCE Cobamides, including vitamin B12, are enzyme cofactors used by organisms in all domains of life. Cobamides are structurally diverse, and microbial growth and metabolism vary based on cobamide structure. Understanding cobamide preference in microorganisms is important given that cobamides are widely used and appear to mediate microbial interactions in host-associated and aquatic environments. Until now, the biochemical basis for cobamide preferences was largely unknown. In this study, we analyzed the effects of the structural diversity of cobamides on a model cobamide-dependent enzyme, methylmalonyl-CoA mutase (MCM). We found that very small changes in cobamide structure could dramatically affect the binding affinity of cobamides to MCM. Strikingly, cobamide-dependent growth of a model bacterium, Sinorhizobium meliloti, largely correlated with the cofactor binding selectivity of S. meliloti MCM, emphasizing the importance of cobamide-dependent enzyme selectivity in bacterial growth and cobamide-mediated microbial interactions.

scholarly journals Glutamate Racemase Is the Primary Target of β-Chloro-d-Alanine in Mycobacterium tuberculosis

2016 ◽  
Vol 60 (10) ◽  
pp. 6091-6099 ◽  
Author(s):  
Gareth A. Prosser ◽  
Anne Rodenburg ◽  
Hania Khoury ◽  
Cesira de Chiara ◽  
Steve Howell ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Early Stage ◽  
Human Pathogens ◽  
Intact Cells ◽  
Content Type ◽  
Peptidoglycan Biosynthesis ◽  
Glutamate Racemase ◽  
Starting Point ◽  
Effective Drugs

ABSTRACTThe increasing global prevalence of drug resistance among many leading human pathogens necessitates both the development of antibiotics with novel mechanisms of action and a better understanding of the physiological activities of preexisting clinically effective drugs. Inhibition of peptidoglycan (PG) biosynthesis and cross-linking has traditionally enjoyed immense success as an antibiotic target in multiple bacterial pathogens, except inMycobacterium tuberculosis, where it has so far been underexploited.d-Cycloserine, a clinically approved antituberculosis therapeutic, inhibits enzymes within thed-alanine subbranch of the PG-biosynthetic pathway and has been a focus in our laboratory for understanding peptidoglycan biosynthesis inhibition and for drug development in studies ofM. tuberculosis. During our studies on alternative inhibitors of thed-alanine pathway, we discovered that the canonical alanine racemase (Alr) inhibitor β-chloro–d-alanine (BCDA) is a very poor inhibitor of recombinantM. tuberculosisAlr, despite having potent antituberculosis activity. Through a combination of enzymology, microbiology, metabolomics, and proteomics, we show here that BCDA does not inhibit thed-alanine pathway in intact cells, consistent with its poorin vitroactivity, and that it is instead a mechanism-based inactivator of glutamate racemase (MurI), an upstream enzyme in the same early stage of PG biosynthesis. This is the first report to our knowledge of inhibition of MurI inM. tuberculosisand thus provides a valuable tool for studying this essential and enigmatic enzyme and a starting point for future MurI-targeted antibacterial development.

scholarly journals Altered Murine Tissue Colonization by Borrelia burgdorferi following Targeted Deletion of Linear Plasmid 17-Carried Genes

2012 ◽  
Vol 80 (5) ◽  
pp. 1773-1782 ◽  
Author(s):  
Timothy Casselli ◽  
Yvonne Tourand ◽  
Troy Bankhead
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Mutant Strain ◽  
Genetic Manipulation ◽  
Infectious Clone ◽  
Linear Plasmid ◽  
Peripheral Tissues ◽  
Content Type ◽  
Immunodeficient Mice

ABSTRACTThe causative agent of Lyme disease,Borrelia burgdorferi, possesses a segmented genome comprised of a single linear chromosome and upwards of 23 linear and circular plasmids. Much of what is known about plasmid-borne genes comes from studying laboratory clones that have spontaneously lost one or more plasmids duringin vitropassage. Some plasmids, including the linear plasmid lp17, are never or rarely reported to be lost during routine culture; therefore, little is known about the requirement of these conserved plasmids for infectivity. In this study, the effects of deleting regions of lp17 were examined bothin vitroandin vivo. A mutant strain lacking the genesbbd16tobbd25showed no deficiency in the ability to establish infection or disseminate to the bloodstream of mice; however, colonization of peripheral tissues was delayed. Despite the ability to colonize ear, heart, and joint tissues, this mutant exhibited a defect in bladder tissue colonization for up to 56 days postinfection. This phenotype was not observed in immunodeficient mice, suggesting that bladder colonization by the mutant strain was inhibited by an adaptive immune-based mechanism. Moreover, the mutant displayed increased expression of outer surface protein Cin vitro, which was correlated with the absence of the genebbd18. To our knowledge, this is the first report involving genetic manipulation of lp17 in an infectious clone ofB. burgdorferiand reveals for the first time the effects of lp17 gene deletion during murine infection by the Lyme disease spirochete.

scholarly journals Phosphinothricin Acetyltransferases Identified UsingIn Vivo,In Vitro, and Bioinformatic Analyses

2016 ◽  
Vol 82 (24) ◽  
pp. 7041-7051 ◽  
Author(s):  
Chelsey M. VanDrisse ◽  
Kristy L. Hentchel ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Deinococcus Radiodurans ◽  
Phylogenetic Analyses ◽  
Critical Role ◽  
Metabolic Stress ◽  
Physiological Role ◽  
Methionine Sulfoximine ◽  
Acinetobacter Baylyi ◽  
Content Type

ABSTRACTAcetylation of small molecules is widespread in nature, and in some cases, cells use this process to detoxify harmful chemicals.Streptomycesspecies utilize aGcn5N-acetyltransferase (GNAT), known as Bar, to acetylate and detoxify a self-produced toxin,phosphinothricin (PPT), a glutamate analogue. Bar homologues, such as MddA fromSalmonella enterica, acetylate methionine analogues such as methionine sulfoximine (MSX) and methionine sulfone (MSO), but not PPT, even though Bar homologues are annotated as PPT acetyltransferases.S. entericawas used as a heterologous host to determine whether or not putative PPT acetyltransferases from various sources could acetylate PPT, MSX, and MSO.In vitroandin vivoanalyses identified substrates acetylated by putative PPT acetyltransferases fromDeinococcus radiodurans(DR_1057 and DR_1182) andGeobacillus kaustophilus(GK0593 and GK2920).In vivo, synthesis of DR_1182, GK0593, and GK2920 blocked the inhibitory effects of PPT, MSX, and MSO. In contrast, DR_1057 did not detoxify any of the above substrates. Results ofin vitrostudies were consistent with thein vivoresults. In addition, phylogenetic analyses were used to predict the functionality of annotated PPT acetyltransferases inBurkholderia xenovorans,Bacillus subtilis,Staphylococcus aureus,Acinetobacter baylyi, andEscherichia coli.IMPORTANCEThe work reported here provides an example of the use of a heterologous system for the identification of enzyme function. Many members of this superfamily of proteins do not have a known function, or it has been annotated solely on the basis of sequence homology to previously characterized enzymes. The critical role ofGcn5N-acetyltransferases (GNATs) in the modulation of central metabolic processes, and in controlling metabolic stress, necessitates approaches that can reveal their physiological role. The combination ofin vivo,in vitro, and bioinformatics approaches reported here identified GNATs that can acetylate and detoxify phosphinothricin.

scholarly journals Abbreviated Pathway for Biosynthesis of 2-Thiouridine in Bacillus subtilis

2015 ◽  
Vol 197 (11) ◽  
pp. 1952-1962 ◽  
Author(s):  
Katherine A. Black ◽  
Patricia C. Dos Santos
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Cysteine Desulfurase ◽  
Content Type ◽  
E Coli ◽  
Wobble Position ◽  
Lysine Trna ◽  
Domains Of Life

ABSTRACTThe 2-thiouridine (s2U) modification of the wobble position in glutamate, glutamine, and lysine tRNA molecules serves to stabilize the anticodon structure, improving ribosomal binding and overall efficiency of the translational process. Biosynthesis of s2U inEscherichia colirequires a cysteine desulfurase (IscS), a thiouridylase (MnmA), and five intermediate sulfur-relay enzymes (TusABCDE). TheE. coliMnmA adenylates and subsequently thiolates tRNA to form the s2U modification.Bacillus subtilislacks IscS and the intermediate sulfur relay proteins, yet its genome contains a cysteine desulfurase gene,yrvO, directly adjacent tomnmA. The genomic synteny ofyrvOandmnmAcombined with the absence of the Tus proteins indicated a potential functionality of these proteins in s2U formation. Here, we provide evidence that theB. subtilisYrvO and MnmA are sufficient for s2U biosynthesis. A conditionalB. subtilisknockout strain showed that s2U abundance correlates with MnmA expression, andin vivocomplementation studies inE. coliIscS- or MnmA-deficient strains revealed the competency of these proteins in s2U biosynthesis.In vitroexperiments demonstrated s2U formation by YrvO and MnmA, and kinetic analysis established a partnership between theB. subtilisproteins that is contingent upon the presence of ATP. Furthermore, we observed that the slow-growth phenotype ofE. coliΔiscSand ΔmnmAstrains associated with s2U depletion is recovered byB. subtilis yrvOandmnmA. These results support the proposal that the involvement of a devoted cysteine desulfurase, YrvO, in s2U synthesis bypasses the need for a complex biosynthetic pathway by direct sulfur transfer to MnmA.IMPORTANCEThe 2-thiouridine (s2U) modification of the wobble position in glutamate, glutamine, and lysine tRNA is conserved in all three domains of life and stabilizes the anticodon structure, thus guaranteeing fidelity in translation. The biosynthesis of s2U inEscherichia colirequires seven proteins: the cysteine desulfurase IscS, the thiouridylase MnmA, and five intermediate sulfur-relay enzymes (TusABCDE).Bacillus subtilisand most Gram-positive bacteria lack a complete set of biosynthetic components. Interestingly, themnmAcoding sequence is located adjacent toyrvO, encoding a cysteine desulfurase. In this work, we provide evidence that theB. subtilisYrvO is able to transfer sulfur directly to MnmA. Both proteins are sufficient for s2U biosynthesis in a pathway independent of the one used inE. coli.

scholarly journals RidA Proteins Prevent Metabolic Damage Inflicted by PLP-Dependent Dehydratases in All Domains of Life

mBio ◽  
2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Jennifer A. Lambrecht ◽  
George E. Schmitz ◽  
Diana M. Downs
Keyword(s):  
Cellular Damage ◽  
Cellular Functions ◽  
Metabolic Intermediate ◽  
Content Type ◽  
Cellular Environment ◽  
Serovar Typhimurium ◽  
Domains Of Life ◽  
Branched Chain

ABSTRACTPyridoxal 5′-phosphate (PLP) is a coenzyme synthesized by all forms of life. Relevant to the work reported here is the mechanism of the PLP-dependent threonine/serine dehydratases, which generate reactive enamine/imine intermediates that are converted to keto acids by members of the RidA family of enzymes. The RidA protein ofSalmonella entericaserovar Typhimurium LT2 is the founding member of this broadly conserved family of proteins (formerly known as YjgF/YER057c/UK114). RidA proteins were recently shown to be enamine deaminases. Here we demonstrate the damaging potential of enamines in the absence of RidA proteins. Notably,S. entericastrains lacking RidA have decreased activity of the PLP-dependent transaminase B enzyme IlvE, an enzyme involved in branched-chain amino acid biosynthesis. We reconstituted the threonine/serine dehydratase (IlvA)-dependent inhibition of IlvEin vitro, show that thein vitrosystem reflects the mechanism of RidA functionin vivo, and show that IlvE inhibition is prevented by RidA proteins from all domains of life. We conclude that 2-aminoacrylate (2AA) inhibition represents a new type of metabolic damage, and this finding provides an important physiological context for the role of the ubiquitous RidA family of enamine deaminases in preventing damage by 2AA.IMPORTANCEExternal stresses that disrupt metabolic components can perturb cellular functions and affect growth. A similar consequence is expected if endogenously generated metabolites are reactive and persist in the cellular environment. Here we show that the metabolic intermediate 2-aminoacrylate (2AA) causes significant cellular damage if allowed to accumulate aberrantly. Furthermore, we show that the widely conserved protein RidA prevents this accumulation by facilitating conversion of 2AA to a stable metabolite. This work demonstrates that the reactive metabolite 2AA, previously considered innocuous in the cell due to a short half-life in aqueous solution, can survive in the cellular environment long enough to cause damage. This work provides insights into the roles and persistence of reactive metabolitesin vivoand shows that the RidA family of proteins is able to prevent damage caused by a reactive intermediate that is created as a consequence of PLP-dependent chemistry.

scholarly journals In Bacillus subtilis, the SatA (Formerly YyaR) Acetyltransferase Detoxifies Streptothricin via Lysine Acetylation

2017 ◽  
Vol 83 (21) ◽  
Author(s):  
Rachel M. Burckhardt ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Bacillus Subtilis ◽  
Bacillus Anthracis ◽  
Antimicrobial Agents ◽  
Ectopic Expression ◽  
Lysine Acetylation ◽  
Acetyl Coa ◽  
Content Type ◽  
Domains Of Life ◽  
Necessary And Sufficient

ABSTRACT Soil is a complex niche, where survival of microorganisms is at risk due to the presence of antimicrobial agents. Many microbes chemically modify cytotoxic compounds to block their deleterious effects. Streptothricin is a broad-spectrum antibiotic produced by streptomycetes that affects Gram-positive and Gram-negative bacteria alike. Here we identify the SatA (for streptothricin acetyltransferase A, formerly YyaR) enzyme of Bacillus subtilis as the mechanism used by this soil bacterium to detoxify streptothricin. B. subtilis strains lacking satA were susceptible to streptothricin. Ectopic expression of satA + restored streptothricin resistance to B. subtilis satA (BsSatA) strains. Purified BsSatA acetylated streptothricin in vitro at the expense of acetyl-coenzyme A (acetyl-CoA). A single acetyl moiety transferred onto streptothricin by SatA blocked the toxic effects of the antibiotic. SatA bound streptothricin with high affinity (Kd [dissociation constant] = 1 μM), and did not bind acetyl-CoA in the absence of streptothricin. Expression of B. subtilis satA + in Salmonella enterica conferred streptothricin resistance, indicating that SatA was necessary and sufficient to detoxify streptothricin. Using this heterologous system, we showed that the SatA homologue from Bacillus anthracis also had streptothricin acetyltransferase activity. Our data highlight the physiological relevance of lysine acetylation for the survival of B. subtilis in the soil. IMPORTANCE Experimental support is provided for the functional assignment of gene products of the soil-dwelling bacilli Bacillus subtilis and Bacillus anthracis. This study focuses on one enzyme that is necessary and sufficient to block the cytotoxic effects of a common soil antibiotic. The enzyme alluded to is a member of a family of proteins that are broadly distributed in all domains of life but poorly studied in B. subtilis and B. anthracis. The initial characterization of the enzyme provides insights into its mechanism of catalysis.

scholarly journals Penicillin Binding Protein 1 Is Important in the Compensatory Response of Staphylococcus aureus to Daptomycin-Induced Membrane Damage and Is a Potential Target for β-Lactam–Daptomycin Synergy

2015 ◽  
Vol 60 (1) ◽  
pp. 451-458 ◽  
Author(s):  
Andrew D. Berti ◽  
Erin Theisen ◽  
John-Demian Sauer ◽  
Poochit Nonejuie ◽  
Joshua Olson ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Binding Protein ◽  
Membrane Damage ◽  
Metabolic Stress ◽  
Inducible Promoter ◽  
Penicillin Binding Protein ◽  
Compensatory Response ◽  
Content Type ◽  
Antimicrobial Efficiency

ABSTRACTThe activity of daptomycin (DAP) against methicillin-resistantStaphylococcus aureus(MRSA) is enhanced in the presence of β-lactam antibiotics. This effect is more pronounced with β-lactam antibiotics that exhibit avid binding to penicillin binding protein 1 (PBP1). Here, we present evidence that PBP1 has a significant role in responding to DAP-induced stress on the cell. Expression of thepbpAtranscript, encoding PBP1, was specifically induced by DAP exposure whereas expression ofpbpB,pbpC, andpbpD, encoding PBP2, PBP3, and PBP4, respectively, remained unchanged. Using a MRSA COL strain withpbpAunder an inducible promoter, increasedpbpAtranscription was accompanied by reduced susceptibility to, and killing by, DAPin vitro. Exposure to β-lactams that preferentially inactivate PBP1 was not associated with increased DAP binding, suggesting that synergy in the setting of anti-PBP1 pharmacotherapy results from increased DAP potency on a per-molecule basis. Combination exposure in anin vitropharmacokinetic/pharmacodynamic model system with β-lactams that preferentially inactivate PBP1 (DAP-meropenem [MEM] or DAP-imipenem [IPM]) resulted in more-rapid killing than did combination exposure with DAP-nafcillin (NAF) (nonselective), DAP-ceftriaxone (CRO) or DAP-cefotaxime (CTX) (PBP2 selective), DAP-cefaclor (CEC) (PBP3 selective), or DAP-cefoxitin (FOX) (PBP4 selective). Compared to β-lactams with poor PBP1 binding specificity, exposure ofS. aureusto DAP plus PBP1-selective β-lactams resulted in an increased frequency of septation and cell wall abnormalities. These data suggest that PBP1 activity may contribute to survival during DAP-induced metabolic stress. Therefore, targeted inactivation of PBP1 may enhance the antimicrobial efficiency of DAP, supporting the use of DAP–β-lactam combination therapy for serious MRSA infections, particularly when the β-lactam undermines the PBP1-mediated compensatory response.

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