Genomic analysis of bifunctional Class C-Class D ?-lactamases in environmental bacteria.
ABSTRACT: ?-lactamases, which are found in several bacterial species and environments, are the main cause of resistance to ?-lactams in Gram-negative bacteria. In 2009, a protein (LRA-13) with two ?-lactamase domains (one class C domain and one class D domain) was experimentally characterised, and an extended action spectrum against ?-lactams consistent with two functional domains was found. Here, we present the results of searches in the non-redundant NCBI protein database that revealed the existence of a group of homologous bifunctional ?-lactamases in the genomes of environmental bacteria. These findings suggest that bifunctional ?-lactamases are widespread in nature; these findings also raise concern that bifunctional ?-lactamases may be transferred to bacteria of clinical importance through lateral gene transfer mechanisms.
Project description:Production of ?-lactamases of one of four molecular classes (A, B, C and D) is the major mechanism of bacterial resistance to ?-lactams, the largest class of antibiotics, which have saved countless lives since their inception 70 years ago. Although several hundred efficient class D enzymes have been identified in Gram-negative pathogens over the last four decades, none have been reported in Gram-positive bacteria. Here we demonstrate that efficient class D ?-lactamases capable of hydrolyzing a wide array of ?-lactam substrates are widely disseminated in various species of environmental Gram-positive organisms. Class D enzymes of Gram-positive bacteria have a distinct structural architecture and employ a unique substrate-binding mode that is quite different from that of all currently known class A, C and D ?-lactamases. These enzymes thus constitute a previously unknown reservoir of novel antibiotic-resistance enzymes.
Project description:Carbapenemases are beta-lactamases with versatile hydrolytic capacities. They have the ability to hydrolyze penicillins, cephalosporins, monobactams, and carbapenems. Bacteria producing these beta-lactamases may cause serious infections in which the carbapenemase activity renders many beta-lactams ineffective. Carbapenemases are members of the molecular class A, B, and D beta-lactamases. Class A and D enzymes have a serine-based hydrolytic mechanism, while class B enzymes are metallo-beta-lactamases that contain zinc in the active site. The class A carbapenemase group includes members of the SME, IMI, NMC, GES, and KPC families. Of these, the KPC carbapenemases are the most prevalent, found mostly on plasmids in Klebsiella pneumoniae. The class D carbapenemases consist of OXA-type beta-lactamases frequently detected in Acinetobacter baumannii. The metallo-beta-lactamases belong to the IMP, VIM, SPM, GIM, and SIM families and have been detected primarily in Pseudomonas aeruginosa; however, there are increasing numbers of reports worldwide of this group of beta-lactamases in the Enterobacteriaceae. This review updates the characteristics, epidemiology, and detection of the carbapenemases found in pathogenic bacteria.
Project description:Despite 70 years of clinical use, ?-lactam antibiotics still remain at the forefront of antimicrobial chemotherapy. The major challenge to these life-saving therapeutics is the presence of bacterial enzymes (i.e., ?-lactamases) that can hydrolyze the ?-lactam bond and inactivate the antibiotic. These enzymes can be grouped into four classes (A-D). Among the most genetically diverse are the class D ?-lactamases. In this class are ?-lactamases that can inactivate the entire spectrum of ?-lactam antibiotics (penicillins, cephalosporins, and carbapenems). Class D ?-lactamases are mostly found in Gram-negative bacteria such as Pseudomonas aeruginosa , Escherichia coli , Proteus mirabilis , and Acinetobacter baumannii . The active-sites of class D ?-lactamases contain an unusual N-carboxylated lysine post-translational modification. A strongly hydrophobic active-site helps create the conditions that allow the lysine to combine with CO2, and the resulting carbamate is stabilized by a number of hydrogen bonds. The carboxy-lysine plays a symmetric role in the reaction, serving as a general base to activate the serine nucleophile in the acylation reaction, and the deacylating water in the second step. There are more than 250 class D ?-lactamases described, and the full set of variants shows remarkable diversity with regard to substrate binding and turnover. Narrow-spectrum variants are most effective against the earliest generation penicillins and cephalosporins such as ampicillin and cephalothin. Extended-spectrum variants (also known as extended-spectrum ?-lactamases, ESBLs) pose a more dangerous clinical threat as they possess a small number of substitutions that allow them to bind and hydrolyze later generation cephalosporins that contain bulkier side-chain constituents (e.g., cefotaxime, ceftazidime, and cefepime). Mutations that permit this versatility seem to cluster in the area surrounding an active-site tryptophan resulting in a widened active-site to accommodate the oxyimino side-chains of these cephalosporins. More concerning are the class D ?-lactamases that hydrolyze clinically important carbapenem ?-lactam drugs (e.g., imipenem). Whereas carbapenems irreversibly acylate and inhibit narrow-spectrum ?-lactamases, class D carbapenemases are able to recruit and activate a deacylating water. The rotational orientation of the C6 hydroxyethyl group found on all carbapenem antibiotics likely plays a role in whether the deacylating water is effective or not. Inhibition of class D ?-lactamases is a current challenge. Commercially available inhibitors that are active against other classes of ?-lactamases are ineffective against class D enzymes. On the horizon are several compounds, consisting of both ?-lactam derivatives and non-?-lactams, that have the potential of providing novel leads to design new mechanism-based inactivators that are effective against the class D enzymes. Several act synergistically when given in combination with a ?-lactam antibiotic, and others show a unique mechanism of inhibition that is distinct from the traditional ?-lactamase inhibitors. These studies will bolster structure-based inhibitor design efforts to facilitate the optimization and development of these compounds as class D inactivators.
Project description:The Class D β-lactamases have emerged as a prominent resistance mechanism against β-lactam antibiotics that previously had efficacy against infections caused by pathogenic bacteria, especially by Acinetobacter baumannii and the Enterobacteriaceae. The phenotypic and structural characteristics of these enzymes correlate to activities that are classified either as a narrow spectrum, an extended spectrum, or a carbapenemase spectrum. We focus on Class D β-lactamases that are carried on plasmids and, thus, present particular clinical concern. Following a historical perspective, the susceptibility and kinetics patterns of the important plasmid-encoded Class D β-lactamases and the mechanisms for mobilization of the chromosomal Class D β-lactamases are discussed.
Project description:The ?-lactams retain a central place in the antibacterial armamentarium. In Gram-negative bacteria, ?-lactamase enzymes that hydrolyze the amide bond of the four-membered ?-lactam ring are the primary resistance mechanism, with multiple enzymes disseminating on mobile genetic elements across opportunistic pathogens such as Enterobacteriaceae (e.g., Escherichia coli) and non-fermenting organisms (e.g., Pseudomonas aeruginosa). ?-Lactamases divide into four classes; the active-site serine ?-lactamases (classes A, C and D) and the zinc-dependent or metallo-?-lactamases (MBLs; class B). Here we review recent advances in mechanistic understanding of each class, focusing upon how growing numbers of crystal structures, in particular for ?-lactam complexes, and methods such as neutron diffraction and molecular simulations, have improved understanding of the biochemistry of ?-lactam breakdown. A second focus is ?-lactamase interactions with carbapenems, as carbapenem-resistant bacteria are of grave clinical concern and carbapenem-hydrolyzing enzymes such as KPC (class A) NDM (class B) and OXA-48 (class D) are proliferating worldwide. An overview is provided of the changing landscape of ?-lactamase inhibitors, exemplified by the introduction to the clinic of combinations of ?-lactams with diazabicyclooctanone and cyclic boronate serine ?-lactamase inhibitors, and of progress and strategies toward clinically useful MBL inhibitors. Despite the long history of ?-lactamase research, we contend that issues including continuing unresolved questions around mechanism; opportunities afforded by new technologies such as serial femtosecond crystallography; the need for new inhibitors, particularly for MBLs; the likely impact of new ?-lactam:inhibitor combinations and the continuing clinical importance of ?-lactams mean that this remains a rewarding research area.
Project description:Worldwide dissemination of pathogens resistant to almost all available antibiotics represent a real problem preventing efficient treatment of infectious diseases. Among antimicrobial used in therapy, ?-lactam antibiotics represent 40% thus playing a crucial role in the management of infections treatment. We report a small series of phenylboronic acids derivatives (BAs) active against class A carbapenemases KPC-2 and GES-5, and class C cephalosporinases AmpC. The inhibitory profile of our BAs against class A and C was investigated by means of molecular docking, enzyme kinetics and X-ray crystallography. We were interested in the mechanism of recognition among class A and class C to direct the design of broad serine ?-Lactamases (SBLs) inhibitors. Molecular modeling calculations vs GES-5 and crystallographic studies vs AmpC reasoned, respectively, the ortho derivative 2 and the meta derivative 3 binding affinity. The ability of our BAs to protect ?-lactams from BLs hydrolysis was determined in biological assays conducted against clinical strains: Fractional inhibitory concentration index (FICI) tests confirmed their ability to be synergic with ?-lactams thus restoring susceptibility to meropenem. Considering the obtained results and the lack of cytotoxicity, our derivatives represent validated probe for the design of SBLs inhibitors.
Project description:Since the discovery and use of penicillin, the increase of antibiotic resistance among bacterial pathogens has become a major health concern. The most prevalent resistance mechanism in Gram-negative bacteria is due to ?-lactamase expression. Class D ?-lactamases are of particular importance due to their presence in multidrug-resistant Acinetobacter baumannii and Pseudomonas aeruginosa. The class D enzymes were initially characterized by their ability to efficiently hydrolyze isoxazolyl-type ?-lactams like oxacillin. Due to this substrate preference, these enzymes are traditionally referred to as oxacillinases or OXAs. However, this class is comprised of subfamilies characterized by diverse activities that include oxacillinase, carbapenemase, or cephalosporinase substrate specificity. OXA-1 represents one subtype of class D enzyme that efficiently hydrolyzes oxacillin, and OXA-24/40 represents another with weak oxacillinase, but increased carbapenemase, activity. To examine the structural basis for the substrate selectivity differences between OXA-1 and OXA-24/40, the X-ray crystal structures of deacylation-deficient mutants of these enzymes (Lys70Asp for OXA-1; Lys84Asp for OXA-24) in complexes with oxacillin were determined to 1.4 Å and 2.4 Å, respectively. In the OXA-24/40/oxacillin structure, the hydrophobic R1 side chain of oxacillin disrupts the bridge between Tyr112 and Met223 present in the apo OXA-24/40 structure, causing the main chain of the Met223-containing loop to adopt a completely different conformation. In contrast, in the OXA-1/oxacillin structure, a hydrophobic pocket consisting of Trp102, Met99, Phe217, Leu161, and Leu255 nicely complements oxacillin's nonpolar R1 side chain. Comparison of the OXA-1/oxacillin and OXA-24/40/oxacillin complexes provides novel insight on how substrate selectivity is achieved among subtypes of class D ?-lactamases. By elucidating important active site interactions, these findings can also inform the design of novel antibiotics and inhibitors.
Project description:Class D beta-lactamase-mediated resistance to beta-lactams has been increasingly reported during the last decade. Those enzymes also known as oxacillinases or OXAs are widely distributed among Gram negatives. Genes encoding class D beta-lactamases are known to be intrinsic in many Gram-negative rods, including Acinetobacter baumannii and Pseudomonas aeruginosa, but play a minor role in natural resistance phenotypes. The OXAs (ca. 150 variants reported so far) are characterized by an important genetic diversity and a great heterogeneity in terms of beta-lactam hydrolysis spectrum. The acquired OXAs possess either a narrow spectrum or an expanded spectrum of hydrolysis, including carbapenems in several instances. Acquired class D beta-lactamase genes are mostly associated to class 1 integron or to insertion sequences.
Project description:?-Lactams are the most widely used and effective antibiotics for the treatment of infectious diseases. Unfortunately, bacteria have developed several mechanisms to combat these therapeutic agents. One of the major resistance mechanisms involves the production of ?-lactamase that hydrolyzes the ?-lactam ring thereby inactivating the drug. To overcome this threat, the small molecule ?-lactamase inhibitors (e.g., clavulanic acid, sulbactam and tazobactam) have been used in combination with ?-lactams for treatment. However, the bacterial resistance to this kind of combination therapy has evolved recently. Therefore, multiple attempts have been made to discover and develop novel broad-spectrum ?-lactamase inhibitors that sufficiently work against ?-lactamase producing bacteria. ?-lactamase inhibitory proteins (BLIPs) (e.g., BLIP, BLIP-I and BLIP-II) are potential inhibitors that have been found from soil bacterium Streptomyces spp. BLIPs bind and inhibit a wide range of class A ?-lactamases from a diverse set of Gram-positive and Gram-negative bacteria, including TEM-1, PC1, SME-1, SHV-1 and KPC-2. To the best of our knowledge, this article represents the first systematic review on ?-lactamase inhibitors with a particular focus on BLIPs and their inherent properties that favorably position them as a source of biologically-inspired drugs to combat antimicrobial resistance. Furthermore, an extensive compilation of binding data from ?-lactamase?BLIP interaction studies is presented herein. Such information help to provide key insights into the origin of interaction that may be useful for rationally guiding future drug design efforts.
Project description:?-Lactamases, the major resistance determinant for ?-lactam antibiotics in Gram-negative bacteria, are ancient enzymes whose origins can be traced back millions of years ago. These well-studied enzymes, currently numbering almost 2,800 unique proteins, initially emerged from environmental sources, most likely to protect a producing bacterium from attack by naturally occurring ?-lactams. Their ancestors were presumably penicillin-binding proteins that share sequence homology with ?-lactamases possessing an active-site serine. Metallo-?-lactamases also exist, with one or two catalytically functional zinc ions. Although penicillinases in Gram-positive bacteria were reported shortly after penicillin was introduced clinically, transmissible ?-lactamases that could hydrolyze recently approved cephalosporins, monobactams, and carbapenems later became important in Gram-negative pathogens. Nomenclature is based on one of two major systems. Originally, functional classifications were used, based on substrate and inhibitor profiles. A later scheme classifies ?-lactamases according to amino acid sequences, resulting in class A, B, C, and D enzymes. A more recent nomenclature combines the molecular and biochemical classifications into 17 functional groups that describe most ?-lactamases. Some of the most problematic enzymes in the clinical community include extended-spectrum ?-lactamases (ESBLs) and the serine and metallo-carbapenemases, all of which are at least partially addressed with new ?-lactamase inhibitor combinations. New enzyme variants continue to be described, partly because of the ease of obtaining sequence data from whole-genome sequencing studies. Often, these new enzymes are devoid of any phenotypic descriptions, making it more difficult for clinicians and antibiotic researchers to address new challenges that may be posed by unusual ?-lactamases.