A molecular mechanism for bacterial susceptibility to zinc

Abstract

Transition row metal ions are both essential and toxic to microorganisms. Zinc in excess has significant toxicity to bacteria, and host release of Zn(II) at mucosal surfaces is an important innate defence mechanism. However, the molecular mechanisms by which Zn(II) affords protection have not been defined. We show that in Streptococcus pneumoniae extracellular Zn(II) inhibits the acquisition of the essential metal Mn(II) by competing for binding to the solute binding protein PsaA. We show that, although Mn(II) is the high-affinity substrate for PsaA, Zn(II) can still bind, albeit with a difference in affinity of nearly two orders of magnitude. Despite the difference in metal ion affinities, high-resolution structures of PsaA in complex with Mn(II) or Zn(II) showed almost no difference. However, Zn(II)-PsaA is significantly more thermally stable than Mn(II)-PsaA, suggesting that Zn(II) binding may be irreversible. In vitro growth analyses show that extracellular Zn(II) is able to inhibit Mn(II) intracellular accumulation with little effect on intracellular Zn(II). The phenotype of S. pneumoniae grown at high Zn(II):Mn(II) ratios, i.e. induced Mn(II) starvation, closely mimicked a ΔpsaA mutant, which is unable to accumulate Mn(II). S. pneumoniae infection in vivo elicits massive elevation of the Zn(II):Mn(II) ratio and, in vitro, these Zn(II):Mn(II) ratios inhibited growth due to Mn(II) starvation, resulting in heightened sensitivity to oxidative stress and polymorphonuclear leucocyte killing. These results demonstrate that microbial susceptibility to Zn(II) toxicity is mediated by extracellular cation competition and that this can be harnessed by the innate immune response.

Figure 1. Biophysical characterization of purified PsaA.

(A) Representative ITC measurements for titration of 4.5 µM PsaA with 40 µM Mn(II). (B) Titration of 20 µM PsaA with 250 µM Zn(II). For each experiment the rates of heat release are shown above the corresponding plots of integrated heat. Both of the curves were fitted to a single site (n = 1) model and the K_D calculated from replicate experiments (± SEM). (C) The overall fold of PsaA with the metal ion shown in purple between the two domains. (D) The metal binding site. The 2F_O-F_c electron density map (contoured at the 1.0σ level) is shown in blue for the coordinating residues and the metal ion. The Mn(II) is shown as a purple sphere and the residues are in ball-and-stick representation (carbon atoms in green, oxygen in red and nitrogen in blue). Also shown in orange is the anomalous difference Fourier map contoured at the 5.0 σ level, computed using the Bijvoet differences collected at the manganese K-edge peak wavelength. (E) Thermal stability of PsaA. The sets of curves show the thermal transition of 10 µM PsaA incubated with 10 µM Mn(II) or 10 µM Zn(II). The curves are representative of three independent experiments (n = 3). (F) Thermal stability of PsaA with increasing Mn(II) concentrations as indicated.

Figure 2. In vitro metal competition.

In vitro growth measurements of S. pneumoniae wild-type (D39) and ΔpsaA. (A) Bacteria were grown in C+Y medium consisting of the following Zn(II):Mn(II) ratios (in µM): 1000∶1, 250∶1, 100∶1, 50∶1 10∶1, 1∶1, and C+Y with 1 µM MnSO₄, respectively. Data are mean (± SEM) A ₆₀₀ measurements from seven independent biological experiments (n = 7). (B) Bacteria were grown in C+Y medium consisting of the following Zn(II):Mn(II) ratios (in µM): 100∶1, 250∶1, 100∶100, 250∶250, and C+Y+1 µM MnSO₄ supplementation, respectively. Data are means (±SEM) A ₆₀₀ measurements from seven independent biological experiments (n = 7). (C) Bacteria were grown in C+Y medium supplemented with 1 µM MnSO₄ until an A ₆₀₀ of 0.3 was reached. Cells were washed in C+Y medium and then inoculated to an A ₆₀₀ of 0.2 in C+Y medium consisting of the following Zn(II):Mn(II) ratios (in µM): 100∶1, 300∶1, and 1000∶1. Data are means (±SEM) A ₆₀₀ measurements from seven independent biological experiments (n = 7). (D) In vitro growth measurements of the ΔpsaA mutant. Bacteria were grown in C+Y medium consisting of the following Zn(II):Mn(II) ratios (in µM): 100∶1, 10∶1, and C+Y+1 µM Mn supplementation, respectively. The wild-type S. pneumoniae D39 grown in C+Y+1 µM Mn supplementation is shown for reference. Data are means (±SEM) A ₆₀₀ measurements from seven independent biological experiments (n = 7). (E) S. pneumoniae intracellular Zn(II) accumulation determined by ICPMS. Data are mean (± SEM) ng Zn(II)/g cell measurements from duplicate measurements of 2 independent biological experiments. (F) Intracellular Mn(II) accumulation determined by ICPMS. Data are mean (± SEM) ng Mn(II)/g cell measurements from duplicate measurements 2 independent biological experiments. The statistical significance of the differences in concentrations was determined by a two-tailed unpaired t-test. P-values of <0.05, <0.005 and <0.0005 are denoted by *, ** or ***, respectively.

Figure 3. In vitro effects on bacterial survival and in vivo metal concentrations.

(A) Paraquat killing of the S. pneumoniae wild-type (D39) and ΔpsaA mutant grown in C+Y+1 µM Mn, and S. pneumoniae grown in 100 µM Zn(II)∶1 µM Mn(II) (D39 100∶1). Survival was calculated as a percentage of colonies at 30 minutes compared to 0 minutes. The experiment was performed with 3 independent biological samples (n = 3) and data are the means (±SEM). (B) PMN killing of S. pneumoniae D39 and ΔpsaA mutant grown in C+Y+1 µM MnSO₄, and S. pneumoniae grown in 100 µM Zn(II):1 µM Mn(II) (D39 100∶1). The experiment was performed in triplicate (n = 3) and shown data are means (±SEM). (C) In vivo niche Zn(II) comparisons. Zn(II) accumulation determined by ICPMS from mouse tissues of naïve (n = 5) and S. pneumoniae-infected mice (n = 10). The statistical significance of the differences in the in vivo mouse tissue Zn(II) concentrations was determined by a two-tailed unpaired t-test. (D) In vivo niche Mn(II) comparisons. Mn(II) accumulation determined by ICPMS from mouse tissues of naïve (n = 5) and S. pneumoniae-infected mice (n = 10). The statistical significance of the differences was determined by a two-tailed unpaired t-test. P-values of <0.05, <0.005 and <0.0005 are denoted by *, ** or ***, respectively.