β-Lactamases: A Focus on Current Challenges.

b-Lactamases: A Focus on Current Challenges Robert A. Bonomo1,2 1 Department of Medicine, Case Western Reserve University School of Medicine, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, Ohio 44120 2 Departments of Pharmacology, Molecular Biology and Microbiology, Biochemistry, and Proteomics and Bioinformatics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44120 www.perspectivesinmedicine.org Correspondence: b-Lactamases, the enzymes that hydrolyze b-lactam antibiotics, remain the greatest threat to the usage of these agents. In this review, the mechanism of hydrolysis is discussed for both those enzymes that use serine at the active site and those that require divalent zinc ions for hydrolysis. The b-lactamases now include .2000 unique, naturally occurring amino acid sequences. Some of the clinically most important of these are the class A penicillinases, the extended-spectrum b-lactamases (ESBLs), the AmpC cephalosporinases, and the carbapenem-hydrolyzing enzymes in both the serine and metalloenzyme groups. Because of the versatility of these enzymes to evolve as new b-lactams are used therapeutically, new approaches to antimicrobial therapy may be required. espite the tremendous advancements in biomedicine, the production of b-lactam hydrolyzing enzymes, b-lactamases, by Gramnegative and -positive bacteria still remains one of the most significant threats to human health (Hauck et al. 2016). With the introduction of every new class of antibiotics, bacteria have continued to evolve resistance, as they are amazingly capable of responding to environmental pressure via selection of existing mutations and acquisition of new genes. The most significant threat has been faced by b-lactam antibiotics. The rapid evolution of b-lactamases, especially carbapenem hydrolyzing enzymes, makes each new drug obsolete in a very short D period of time (Bush 2010a,b, 2014; Drawz and Bonomo 2010). MECHANISM OF b-LACTAM ACTION To properly appreciate the mechanisms by which b-lactamases have changed the status of b-lactams in our therapeutic armamentarium, it is important to briefly review how b-lactams kill bacteria. b-Lactam antibiotics show their bactericidal effects by inhibiting enzymes involved in cell-wall synthesis, that is, penicillinbinding proteins (PBPs). The integrity of the bacterial cell wall is essential to maintaining cell shape in a hypertonic and hostile environ- Editors: Lynn L. Silver and Karen Bush Additional Perspectives on Antibiotics and Antibiotic Resistance available at www.perspectivesinmedicine.org Copyright # 2017 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a025239 Cite this article as Cold Spring Harb Perspect Med 2017;7:a025239 1 www.perspectivesinmedicine.org R.A. Bonomo ment such as serum, urine, lung mucus, or gastrointestinal tract. Osmotic stability is preserved by a rigid cell wall comprised of alternating Nacetylmuramic acid (NAM) and N-acetylglucosamine (NAG) units. These glycosidic units are linked by a transglycosidases. A pentapeptide is attached to each NAM unit; the PBPs act as transpeptidases to catalyze the cross-linking of two D-alanine-D-alanine NAM pentapeptides. This cross-linking of adjacent glycan strands confers the rigidity of the cell wall. In the 1960s, Strominger realized that the b-lactam ring is sterically similar to the D-alanine-D-alanine of the NAM pentapeptide (Drawz and Bonomo 2010; Fisher and Mobashery 2014; Fishovitz et al. 2015). As a result, PBPs “mistakenly” use the b-lactam as a substrate “building block” during cell-wall synthesis. This “error” results in acylation of the PBP, which renders the enzyme unable to catalyze further carry out transpeptidation reactions. As cell-wall synthesis slows, constitutive peptidoglycan autolysis continues as a result of amidases, bacterial autolytic enzymes. The breakdown of the murein sacculus, the peptidoglycan net that surrounds the bacterium, leads to cell-wall compromise and increased permeability. In this way, the b-lactam-mediated inhibition of transpeptidation causes cell lysis. MECHANISMS OF RESISTANCE TO b-LACTAMS There are four primary mechanisms by which bacteria can overcome b-lactam antibiotics (Drawz and Bonomo 2010; Papp-Wallace et al. 2011). First, changes in the active site of PBPs can lower the affinity for b-lactam antibiotics and subsequently increase resistance to these agents, such as in PBP2x of Streptococcus pneumoniae. In a similar manner, penicillin resistance in Streptococcus sanguis, Streptococcus oralis, and Streptococcus mitis developed from horizontal transfer of a PBP2b gene from S. pneumoniae. Methicillin resistance in Staphylococcus spp. is another example of an altered PBP. Although the cause for this resistance is heterogeneous, it is often conferred by acquisition of the mec el2 ement, the mecA gene, which encodes PBP2a (also denoted PBP20 ). This low-affinity transpeptidase can assemble new cell wall in the presence of high concentration of penicillins (i.e., methicillin) and cephalosporins. Second, to access PBPs on the surface of the inner membrane, b-lactams must either diffuse through or directly traverse porin channels in the outer membrane of Gram-negative bacterial cell walls. Resistance to b-lactams can occur when these porin proteins are modified such that they are not produced in a fully active form. Some Gram-negative bacteria show resistance to carbapenems based on loss and or reduction of these outer membrane proteins, such as the loss of OprD, which is associated with resistance to imipenem and reduced susceptibility to meropenem in Pseudomonas aeruginosa (Papp-Wallace et al. 2011). Third, multicomponent drug efflux pump systems (mex), as part of either an acquired or intrinsic resistance repertoire, are capable of exporting a wide-range of substrates from the periplasm of Gram-negative bacteria to the surrounding environment (Papp-Wallace et al. 2011). These pumps are an important determinant of multidrug resistance in many Gramnegative pathogens, particularly notable in P. aeruginosa and Acinetobacter spp. Other pumps are found in the enteric bacteria but will not be discussed in detail, as this is beyond the scope of this review. As an example of the role played by efflux pumps, increased production of the MexA– MexB system, in combination with the low intrinsic permeability of P. aeruginosa, can contribute to decreased susceptibility to penicillins, cephalosporins, carbapenems, as well as quinolones, tetracycline, and chloramphenicol. Last, b-lactamases hydrolyze b-lactams. This is the most common and important mechanism of resistance in Gram-negative bacteria and will be the focus of this review. The description of b-lactamases conferring resistance to penicillins and cephalosporins has been extensively detailed (Drawz and Bonomo 2010; PappWallace et al. 2011). This work will build on those reviews and highligh (...truncated)