Metallo-β-lactamases and a tug-of-war for the available zinc at the host-pathogen interface

Abstract

Metallo-β-lactamases (MBLs) are zinc-dependent hydrolases that inactivate virtually all β-lactam antibiotics. The expression of MBLs by Gram-negative bacteria severely limits the therapeutic options to treat infections. MBLs bind the essential metal ions in the bacterial periplasm, and their activity is challenged upon the zinc starvation conditions elicited by the native immune response. Metal depletion compromises both the enzyme activity and stability in the periplasm, impacting on the resistance profile in vivo. Thus, novel inhibitory approaches involve the use of chelating agents or metal-based drugs that displace the native metal ion. However, newer MBL variants incorporate mutations that improve their metal binding abilities or stabilize the metal-depleted form, revealing that metal starvation is a driving force acting on MBL evolution. Future challenges require addressing the gap between in cell and in vitro studies, dissecting the mechanism for MBL metalation and determining the metal content in situ.

Keywords: Antibiotic resistance; Metallo-β-lactamases; Periplasmic zinc homeostasis; Protein evolution; Zinc.

Figure 1.

Active site Zn(II) coordination spheres for B1, B2 and B3 MBLs. Zn(II) ions and water/hydroxide ions are represented as grey and red spheres, respectively. PDB codes for the structures used are: 5N5G (B1, VIM-1), 3SD9 (B2, Sfh-I) and 3LVZ (B3, BJP-1).

Figure 2.

Metabolism of NDMs in the periplasmic space of Gram-negative bacteria. Once exported, processed and lipidated, NDM enzymes are folded and metalated, and then translocated to the inner-face of the outer membrane. Metal loss from the active site of NDM-1 under extracellular Zn(II) restriction generates a degradation-prone apo-enzyme [8] due to an increased flexibility in the C-terminal region (colored in red), which is targeted by periplasmic proteases [1]. Clinical variants of NDM-1 circumvent periplasmic degradation by accumulating mutations that either increase the metal binding affinities (M154L) or stabilize the apo-enzyme structure (A223V or E152K) [17,36].

Figure 3.

Mechanisms of Zn(II) internalization in Gram-negative bacteria. In Zn(II)-replete conditions (left), Zn(II) ions enter the periplasmic space through non-specific porins and are transported to the cytoplasm by constitutively expressed antiporters, such as ZupT (in E. coli) [56]. Upon Zn(II) limitation (right), the Zur regulon is de-repressed and additional importers and accessory proteins are produced (highlighted in yellow). These include the high-affinity importer ZnuABC [10], widely-conserved in bacteria, the periplasmic zinc-binding protein ZinT from Enterobacterales [57], which delivers Zn(II) ions to ZnuABC, the TonB-dependent ZnuD importers from Neisseria meningitidis and non-fermenters [10], the capture of extracellular Zn(II) through secretion and incorporation of zincophores, such as TseZ from Burkholderia thailandensis [58], and receptors specific for Zn(II)-bound calprotectin, such as CbpA from N. meningitidis [59] or TdfH from Neisseria gonorrhoeae [60], that strip Zn(II) from it. IM: inner membrane; OM: outer membrane.

Figure 4.

a) Structures of MBL inhibitors that act by metal chelation. b) Scheme showing the structure of the prochelator cephalosporin PcephPT, and the release of a pyrithione group from the drug upon hydrolysis of its β-lactam ring [43] c) Structures of metal-based MBL inhibitors. d) Active sites in NDM-1 bound to Bi(III) (left) and Au(I) (right), resulting from treatment with CBS [44] and AUR [45], respectively.

Figure 5.

a) Structure of fluorogenic carbapenem developed by Mao et al. [51] b) Structures of covalent-binding fluorescent probes, developed by Chen et al. [53] (right) and Singha et al. [54] (left). c) Structure of the reversible fluorescent probe developed by Que et al. [55]. In all cases, the fluorescent moiety is shown in blue.