Bacterial Strategies to Maintain Zinc Metallostasis at the Host-Pathogen Interface

Abstract

Among the biologically required first row, late d-block metals from Mn^II to Zn^II, the catalytic and structural reach of Zn^II ensures that this essential micronutrient touches nearly every major metabolic process or pathway in the cell. Zn is also toxic in excess, primarily because it is a highly competitive divalent metal and will displace more weakly bound transition metals in the active sites of metalloenzymes if left unregulated. The vertebrate innate immune system uses several strategies to exploit this "Achilles heel" of microbial physiology, but bacterial evolution has responded in kind. This review highlights recent insights into transcriptional, transport, and trafficking mechanisms that pathogens use to "win the fight" over zinc and thrive in an otherwise hostile environment.

Keywords: ABC transporter; allostery; antibiotics; bacteria; host-pathogen interaction; metal transporter; metallochaperone; metallophores; metalloprotein; metalloregulatory protein; metallostasis; zinc; zinc homeostasis.

FIGURE 1.

Schematic rendering of the bacterial pathogen response to host-induced Zn^II deficiency (top) and toxicity (bottom) for representative Gram-negative (GRAM −) (left) and Gram-positive (GRAM +) (right) organisms. Abbreviations used: OM, outer membrane; IM, inner (plasma) membrane; CM, cytoplasmic (plasma) membrane; ZIP, Zrt/Irt-like proteins found in bacteria (40, 41); MT, metallothionein; CDF, cation diffusion facilitator transporter; RND, resistance-nodulation-cell division transporter. Zn (yellow circles) homeostasis is governed by a pair of Zn-sensing regulators, an uptake regulator (exemplified by Zur or AdcR) and efflux regulator (by ZntR or pneumococcal SczA). The balance of Zn uptake and efflux activities establishes the total cytoplasmic Zn concentration (see text for details). The bioavailable or “buffered” pool of intracellular “free” Zn, defined here as that fraction of total Zn not tightly bound to protein and in rapid chemical exchange among small molecules (red circles), e.g. bacillithiol (94), metallothioneins in some cells (blue ribbon), and weakly bound proteome sites (not shown). These components collectively define Zn speciation in cells. Under Zn-limiting conditions, the shuttle of Zn to obligate Zn-requiring enzymes might require a specialized Zn chaperone (43) (orange symbol). In a Zn-sparing response under conditions of extreme Zn limitation, Zn-independent paralogs replace Zn-dependent ones, such as that which occurs with ribosomal proteins L31 and S14 (gray symbol) (78).

FIGURE 2.

A, graphical representation of the “set-point” model for metal homeostasis. In this model, the metal affinity for individual or pairs of metal sensor proteins (red and blue solid lines) defines the ability of the cytoplasm to buffer biologically required transitions metal ions. The K_Zn of the pair of regulators set the boundary of free Zn concentration in the cytoplasm, where 1/K_Zn ∼ [Zn^II]_free. The free metal concentration follows the Irving-Williams series (shaded areas). The total concentrations are represented in dashed lines (color coded in the graph). The metal affinity constants and concentrations were taken from Ref. . B, S. aureus CzrA in the Zn-bound form (Protein Data Bank (PDB): 2m30) with the Zn-binding site in the zoomed region. The apo form (PDB: 1r1u active repressor) is superimposed in red, showing minimal structural difference (31). C, the S. aureus CzrA in DNA-bound form (PDB: 2kjb) docked on a DNA operator (32). The apo form (PDB: 1r1u active repressor) is superimposed in red, representing the induced fit that the protein undergoes upon DNA binding. D, E. coli Zn₄-Zur₂-33-mer DNA complex (PDB: 4mte) (22). E, S. pneumoniae AdcR in the Zn state (PDB: 3tgn) docked on the DNA operator (34). F, the ZntR (PDB: 1q90) docked on the DNA operator based on the E. coli CueR structure (PDB: 4wls). The bent DNA conformation (PDB: 4wlw) is represented in red to show the conformational change at the level of DNA responsible of changes in gene expression upon Zn binding (23). In all the structures, the putative DNA-binding region is colored in purple and DNA operators are shown in orange. Red arrows indicate movement upon metal/DNA binding.

FIGURE 3.

Membrane transporters for Zn^II efflux (top) and uptake (bottom). Abbreviations used: OM, outer membrane; IM, inner (plasma) membrane. Top, left: the P_1B-type ATPase is S. sonnei ZntA in the Zn-free “E2” ground state (PDB: 4umv) (37). Actuator (A, yellow), phosphorylation (P, blue), and nucleotide-binding (N, red) domains are shown. The transmembrane domain is shown in gray superimposed with the phosphorylation intermediate (PDB 4umw, transparent), representing the closure of an extracellular release pathway. A model of the intramembranous high-affinity Zn^II-binding site based on spectroscopic and functional studies (60) is shown in the inset. The red arrows indicate the putative metal pathway accompanied by protein rearrangements. Top, right: the CDF protein is E. coli YiiP (PDB: 3h90). The functional dimer is shown. The Zn-bound form of the cytoplasmic dimerization domain is shaded red. The Zn^II chelates shown represent the three distinct metal-binding sites (A, B, and C1/C2). Note that functional studies of S. pneumoniae CzcD are consistent with a single C-site metal site, not a binuclear cluster as indicated (50). The inward facing state (PDB 3j1z, transparent) is shown to represent the mechanism of metal release for this transporter. Bottom, left: the model for ZnuABC is based on the structure of BtuCDF (PDB: 4fi3) for cobalamin uptake. The detailed metal coordination displayed in the inset is taken from the crystal structure of ZnuA (PDB: 2osv) with the residues numbered according to the UniProt sequence. The solute-binding protein (ZnuA, red), permease (ZnuB dimer, gray), and ATPase (ZnuC dimer, blue) are shown. The model of ZnuD is from Neisseria meningitidis (PDB: 4rdr) (72). The “plug” domain (shaded blue), the β-barrel domain (shaded gray), and the metal-sensing extracellular loop 3 (shaded red), with the Cd^II-bound form transparent; PDB: 4rdt) are shown.

FIGURE 4.

Small molecules involved in Zn homeostasis and Zn speciation in cells. A, model of staphylopine biosynthesis and staphylopine-mediated transition metal acquisition by this broad-spectrum metallophore (77). Abbreviations used: SAM, S-adenosyl-l-methionine; MTA, 5′-methylthioadenosine; xNA, nicotianamine intermediate; PYR, pyruvate. Staphylopine is biosynthesized from l-His by the combined activities of CntK, a histidine racemase, CntL, a nicotianamine synthase-like (NAS-like) enzyme, and CntM, a staphylopine dehydrogenase. The metal-free metallophore is proposed to be exported through CntE, a major facilitator superfamily (MFS) transporter, and then imported as the metallated complex via a specific ABC transporter, CntABCDF. B, model of how histidine uptake and catabolism impact the labile histidine-Zn pool in A. baumannii under conditions of host-mediated Zn starvation (43). His is proposed to be taken up as His₂-Zn complex through HutT. ZigA is a Zn^II-activated GTPase that may directly activate the Zn-stimulated metalloenzyme HutH (histidine ammonia lyase), which converts l-His to trans-urocanic acid (UA) and ultimately glutamate (Glu) via the sequential activities of HutUIG, all encoded by the hut (histidine utilization) operon. Urocanic acid (43) and glutamate have lower affinities for Zn than histidine, thus potentially mobilizing Zn from the labile or rapidly exchanging pool for use in Zn-requiring processes. C, schematic model of bacillithiol (BSH) biosynthesis (99) and known role of bacillithiol in buffering cytoplasmic Zn (94). BshA, a glycosyltransferase that condenses N-acetyl glucosamine (GlcNAc) with l-malic acid (Mal); BshB (BshB1 and BshB2 are partially redundant), a deacetylase; BshC, a cysteine ligase.