Recent developments in copper and zinc homeostasis in bacterial pathogens

Abstract

Copper and zinc homeostasis systems in pathogenic bacteria are required to resist host efforts to manipulate the availability and toxicity of these metal ions. Central to this microbial adaptive response is the involvement of metal-trafficking and metal-sensing proteins that ultimately exercise control of metal speciation in the cell. Cu-specific and Zn-specific metalloregulatory proteins regulate the transcription of metal-responsive genes while metallochaperones and related proteins ensure that these metals are appropriately buffered by the intracellular milieu and delivered to correct intracellular targets. In this review, we summarize recent findings on how bacterial pathogens mount a metal-specific response to derail host efforts to win the 'fight over metals.'

Figure 1

Bioinorganic chemistry at the host-pathogen interface. Cu(II) has the highest affinity for a given ligand compared to other first row transition metals as exemplified by the height of the red bars which depict the NIST approved log K values relative to the Cu(II)-aspartic acid complex [Cu(II)-aspartic acid, log K = 8.9]. This empirical relationship is described as the Irving-Williams series and relates to the relative competitiveness of first-row transition metals in a cellular environment [18]. Cu(I) predominates in the cytoplasm and like Zn(II), forms high-affinity complexes with softer acids (histidine, cysteine, methionine), and is therefore also considered a highly competitive metal. Bioavailability is inversely proportional to competitiveness and has been roughly approximated on the basis of the relative binding affinities of metal-dependent transcriptional regulators [16]. Metal-centric nutritional immunity is defined as the host’s attempt to both sequester metal ions from cells (upward-facing blue arrows) and/or bombard the bacterial cytoplasm with metal-ion stress (downward-facing blue arrows). Roles of Co and Ni in nutritional immunity are not yet known (black bars).

Figure 2

Overview of copper sensing and trafficking within the bacterial cytoplasm. Copper enters the cytoplasm through largely unknown mechanisms. Copper speciation within the cell depends on the relative concentrations of Cu(I) bound to the bioavailable pool, e.g., copper bound to low-molecular-weight thiols, cytoplasmic binding proteins, e.g., MymT [28] and CutC [29]), chaperones, and Cu(I) sensors. The thermodynamics and kinetics of Cu(I) speciation remain incompletely understood and may be dictated by the concentrations at which copper homeostasis proteins become saturated. Importantly, Cu(I) overload must ultimately be sensed by Cu(I)-dependent metalloregulators (light green calipers) causing transcriptional derepression as a result of dissociation from the DNA operator-promoter region (white rectangle, opr) (or transcriptional activation) and expression of Cu(I) resistance genes (pink rectangle, labeled genes). It is these upregulated copper resistance proteins that ultimately function in Cu(I) resistance, either via sequestration or export through P-type ATPases.

Figure 3

Structural insights into the bioinorganic chemistry of proteins operating at the host-pathogen interface. Top panel, Cu(I)-trafficking proteins: (a) Cu(I)-bound CopZ [PDB 1K0V]; (b) binuclear Cu(I)-bound soluble CupA (sCupA) [PDB 4F2E] transfers Cu(I) from S1 Cu site to S2 Cu site of CopA_MBD [PDB 4F2F]. Inset, Cu(I) binding sites of sCupA [33••]. Bottom panel, crystal structures of (c) Cu(I)-bound M. tuberculosis CsoR (PDB 2HH7) [36,39], (d) Mn(II)-bound calprotectin S100 A8/A9 heterodimer [51••] (Ca ions not shown for clarity) (PDB 4GGF), (e) Zn(II)-bound AdcR homodimer [PDB 3TGN] [60], and (f) Zn(II)-bound Streptomyces Zur homodimer [PDB 3MWM] [42•]. In each case, protomers are shaded blue and grey with known or probable DNA binding domains/residues shaded purple. Labels for metal-binding sites are in italics. Insets, metal-binding sites with dashed lines indicating either first-coordination sphere or second-sphere hydrogen-bonding interactions.

Figure 4

Cellular response to either limited (left) or toxic (right) Zn(II) concentrations is mediated by the coordinate action of zinc uptake (dark green calipers) and efflux (light green calipers) transcriptional regulators that control the expression of import (grey boxes) and efflux (pink boxes) genes, respectively, as a result of a Zn(II)-regulated binding (activation or inhibition, respectively) to their DNA operators (white boxes, opr). Left panel, Zn(II) uptake regulators have low affinity for their DNA operator sequence in the apo state under zinc limiting conditions which allows for the expression of import genes. Zn(II) efflux regulators have high affinity for their DNA operator in the absence of Zn(II) and repress efflux. This response allows the cell to maintain a bioavailable concentration of Zn(II) that is sufficient for cellular needs. Trafficking of Zn(II) to selected proteins may involve the action of zinc chaperones, for which there is no definitive evidence. Right panel, under conditions of high extracellular zinc, the uptake regulators are metallated and bind with high affinity for their DNA operator, thereby repressing import. In the presence of Zn(II), efflux regulators dissociate from their operator sequence (shown), or become transcriptional activators, in the Zn(II)-bound state, resulting in the transcription of export genes. Zinc speciation in the cytoplasm is projected to involve small molecules, Zn-requiring metalloproteins, and possibly zinc chaperones, as indicated. There is some evidence that zinc-uptake and -efflux regulation occurs at distinct zinc concentrations added to cells, with repression of uptake genes occurring at lower total zinc relative to derepression/activation of export genes [22].