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Effect of a β-lactamase inhibitor, tazobactam, on growth and penicillin-binding proteins of Borrelia burgdorferi

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Effect of a β-lactamase inhibitor, tazobactam, on growth and penicillin-binding proteins of Borrelia burgdorferi

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Abstract

The effects of tazobactam, a relatively new beta-lactamase inhibitor, were investigated on growth and penicillin-binding proteins (PBPs) of Borrellia burgdorferi. A previous communication from our group demonstrated several proteins capable of binding labelled penicillin in this organism. Of these proteins, 94-kDa and 57-kDa PBPs possessed the highest affinity for penicillin and were assumed to be essential proteins involved in cell-wall synthesis. In these experiments, tazobactam was used in competition binding experiments as well as on whole spirochetes. Only the 94-kDa and 57-kDa PBPs were affected by increasing amounts of tazobactam during competition-binding experiments and growth of B. burgdorferi was also inhibited. These results may explain the in vitro activity of beta-lactamase inhibitors in general and suggest a utility for these compounds when examining PBPs with hydrolysing activity and/or organisms with beta-lactamases.

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... Generally, the inhibitors do not inactivate PBPs, but notable exceptions include (i) the intrinsic activities of sulbactam against Bacteroides spp., Acinetobacter spp., and N. gonorrhoeae; (ii) clavulanate action against Haemophilus influenza and N. gonorrhoeae; and (iii) tazobactam inhibition of PBPs in Borrelia burgdorferi [Urban et al., 1991;Miller et al., 1978;Bush, 1988;Higgins et al., 2004;Levin, 2002;Neu and Fu, 1978;Urban et al., 1993;Williams, 1997]. As these "antibacterial effects" are relatively weak, the inhibitors are always combined with β-lactam antibiotics for clinical use. ...
Thesis
Antimicrobial resistance (AMR) has become a major threat to public health nowadays. The use and abuse of antibiotics is increasingly leading to selection and spread of resistance mechanisms worldwide, greatly compromising our capacity to treat infectious diseases. AMR might ultimately result in a future without effective antimicrobial therapy. Due to their safety and clinical efficacy, β-lactams are the most utilized antimicrobial therapy, and the most common resistance mechanism is the expression of β-lactamases. Therefore, the development of new antimicrobial drugs, for novel or already known targets, is of utmost importance. In particular, the development of novel inhibitors towards β-lactamases is also quite promising, as it would allow us to continue using the effective and safe antimicrobial drugs already available today. The biochemical and structural study of novel β-lactamases or synthetic mutants, through X-ray crystallography and various molecular modelling techniques (homology modelling, docking, molecular dynamics, water network analysis), can provide valuable information. In this context, we have characterized phenotypically, biochemically and structurally several β-lactamases.The CMY-136 β-lactamase possesses an unusual mutation, Y221H, as compared to CMY-2, in a position highly conserved among class C ß-lactamases. Crystallographic and molecular modelling experiments reveal a steric impediment around the mutated position 221 that may affect the conformation and dynamics of the Ω-loop, and therefore account for an increased turnover rate for bulky substrates and a decreased affinity for most substrates as compared to CMY-2.The crystal structure of the OXA-427, a novel class D carbapenemase, shows the Lys73 only partially carbamoylated, a very unusual characteristic for this class of β-lactamases, and an unexpected hydrophobic bridge in the vicinity of the active site. Moreover, molecular dynamics simulations revealed an extended and highly flexible β5-β6 loop. Altogether, these features may explain the unique hydrolytic profile determined experimentally for this enzyme.Modifications in the β5-β6 loop of the OXA-48 β-lactamase (alanine scanning, systematic deletions, replacement with the β5-β6 loop from OXA-18) result in profound changes in the hydrolytic profile, with gradual acquisition of cephalosporinase activity and decrease of carbapenemase activity in some cases. X-ray crystallography and molecular modelling studies suggest that the altered conformation and flexibility of this loop and of adjacent regions in these mutants may allow for the better accommodation of the bulkier cephalosporins, compared to OXA-48. Additionally, water dynamics analysis highlighted changes in the water network around and inside the active site cavity that may be responsible for the lower activity towards carbapenems. Together with studies on other naturally occurring mutants, results corroborate the relevance of the β5-β6 loop on the substrate profile of OXA-type enzymes. Crystal structure of the OXA-48 217ΔP mutant reveals an unexpected self-inhibited conformation induced by the presence of a nitrate ion, a previously unknown inhibitor of class D β-lactamases.Finally, the Beta-Lactamase DataBase (BLDB, http://bldb.eu) developed in our laboratory is a comprehensive, manually curated public resource providing up-to-date structural and functional information on β-lactamases. It contains all reported naturally-occurring β-lactamases and synthetic mutants, together with all available 3D structures from the PDB and the phenotypical characterization.Overall, these results constitute an essential foundation for a better understanding of the structure-function relationship of β-lactamases, which may prove crucial for the future rational development of β-lactamase inhibitors.
... The structure of BLIs confers some measure of intrinsic antimicrobial action, a fact often overlooked because of their use as enzymatic inhibitors. For example, SLB has useful in vitro and clinical activity against strains of Acinetobacter baumannii [7,8] and Burkholderia cepacia [9], while TAZ has direct activity against the Lyme disease spirochete Borrelia burgdorferi [10]. Best studied for SLB, these antimicrobial activities are mediated by direct binding to penicillin-binding proteins (PBPs), essential transpeptidases involved in cell wall biosynthesis, leading to morphologic effects such as filamentation, spherocyte formation, or bacterial lysis [11,12]. ...
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Infections caused by New Delhi metallo-β-lactamases (NDM)-producing strains of multidrug-resistant (MDR) Klebsiella pneumoniae (KP) are a global public health threat lacking reliable therapies. NDM is impervious to all existing β-lactamase inhibitor (BLI) drugs, including the non-β-lactam structure BLI, avibactam (AVI). Though lacking direct activity against NDM enzymes, AVI can interact with penicillin-binding protein 2 in a manner that may influence cell wall dynamics. We found that exposure of NDM KP to AVI led to striking bactericidal interactions with human cathelicidin antimicrobial peptide LL-37, a frontline component of host innate immunity. Moreover, AVI markedly sensitized NDM KP to killing by freshly isolated human neutrophils, platelets, and serum when complement was active. Finally, AVI monotherapy reduced lung NDM KP counts in a murine pulmonary challenge model. AVI has immune sensitizing activities against NDM KP not appreciated by standard antibiotic testing and meriting further study.
... All three β-lactamase inhibitor compounds share structural similarity with penicillin; are effective against many susceptible organisms expressing class A β-lactamases (including CTX-M and the ESBL derivatives of TEM-1,TEM-2, and SHV-1), and are generally less effective against class B, C, and D β-lactamases [8,29]. Sulbactam :PenamSulfone Class AInactivator [30] Tazobactam:Penamsulfone Class AInactivator [30] Clavulanate :3-Alkylideneoxapenam Class AInactivator [30] and (iii) tazobactam inhibition of PBPs in Borrelia burgdorferi3132333435363738. As these " antibacterial effects " are relatively weak, the inhibitors are always combined with βlactam antibiotics for clinical use. ...
Thesis
The worldwide spread of extended-spectrum β-lactamases (ESBLs), class A, B and D carbapenemases, as well as the presence of chromosomal/plasmid mediated AmpC β- lactamases in some Gram-negative bacilli has reduced the utility of the β-lactam antibiotics and contributed to the increase in difficult- to-treat multidrug-resistant (MDR) organisms. Paring a β-lactam antibiotic with a β-lactamase inhibitor could be an effective way to combat β-lactam resistance. The span of coverage of the inhibitor however determines the extent of antibacterial activity of the β-lactam-β-lactamase inhibitor combination. The spectrum of inhibition of the currently clinical available inhibitors is limited largely to class A enzymes (mostly ESBL genes) and as a result their β-lactam combinations are liable to resistance from expression of class B, C and D enzymes, thus, prompting a need for novel inhibitors with broader spectrum of activity. Avibactam is one of these novel inhibitors, which demonstrates good activity against class A, C and D lactamases. The scope of this work was to construct an isogenetic background for cloning and producing different types of β-lactamases in order to test the inhibitory activity of avibactam against these enzymes. Overall, TEM-134, TEM-184, CTX-32-M, CTX-56-M, and ACT-2 β-lactamases of different classes were cloned using pLBII plasmid vector and Escherichia coli SNO3 as host. The inhibitory activity of Avibactam in combination with ceftazidime and other comparator antibiotics was tested against SNO3 strain of E. coli expressing a single β-lactamase enzyme of either molecular class A, C, or D. With the exception of E. coli expressing OXA-40, ceftazidime and comparator antibiotics in combination with Avibactam showed several fold reduction in MICs compared with when they were used alone (4-4266 fold reduction in MIC). Meropenem on the other hand retained its activity against all the enzymes (MIC <0.015μg/ml) used in this work. Avibactam extended the effect of ceftazidime and other comparator antibiotics to bacteria expressing β-lactamase enzyme. In future, it could be worth further investigating the combination of other β-lactam antibiotics with avibactam, since it appears as a promising drug in our antibacterial armamentarium.
... ␤-Lactam-␤-lactamase inhibitor combinations: clinical use. Generally, the inhibitors do not inactivate PBPs, but notable exceptions include (i) the intrinsic activities of sulbactam against Bacteroides spp., Acinetobacter spp., and N. gonorrhoeae; (ii) clavulanate action against Haemophilus influenzae and N. gonorrhoeae; and (iii) tazobactam inhibition of PBPs in Borrelia burgdorferi (53,167,222,264,285,423,424,447). As these "antibacterial effects" are relatively weak, the inhibitors are always combined with ␤-lactam antibiotics for clinical use. ...
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Since the introduction of penicillin, beta-lactam antibiotics have been the antimicrobial agents of choice. Unfortunately, the efficacy of these life-saving antibiotics is significantly threatened by bacterial beta-lactamases. beta-Lactamases are now responsible for resistance to penicillins, extended-spectrum cephalosporins, monobactams, and carbapenems. In order to overcome beta-lactamase-mediated resistance, beta-lactamase inhibitors (clavulanate, sulbactam, and tazobactam) were introduced into clinical practice. These inhibitors greatly enhance the efficacy of their partner beta-lactams (amoxicillin, ampicillin, piperacillin, and ticarcillin) in the treatment of serious Enterobacteriaceae and penicillin-resistant staphylococcal infections. However, selective pressure from excess antibiotic use accelerated the emergence of resistance to beta-lactam-beta-lactamase inhibitor combinations. Furthermore, the prevalence of clinically relevant beta-lactamases from other classes that are resistant to inhibition is rapidly increasing. There is an urgent need for effective inhibitors that can restore the activity of beta-lactams. Here, we review the catalytic mechanisms of each beta-lactamase class. We then discuss approaches for circumventing beta-lactamase-mediated resistance, including properties and characteristics of mechanism-based inactivators. We next highlight the mechanisms of action and salient clinical and microbiological features of beta-lactamase inhibitors. We also emphasize their therapeutic applications. We close by focusing on novel compounds and the chemical features of these agents that may contribute to a "second generation" of inhibitors. The goal for the next 3 decades will be to design inhibitors that will be effective for more than a single class of beta-lactamases.
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In this study we further delineate human T and B cell responses in Lyme borreliosis and present a model that addresses interactions of T cells and B cells in developing anti-B. burgdorferi reactivity.
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This review centers on studies of the penicillin-binding proteins (PBPs) as the targets of β-lactam antibiotics and their roles in bacterial physiology as elucidated by morphological observations, genetic studies, structural analysis, and biochemical investigations of their interactions both with cell wall substrates and with β-lactam antibiotics.
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