Extracellular Zinc Competitively Inhibits Manganese Uptake and Compromises Oxidative Stress Management in <i>Streptococcus pneumoniae</i>

<div><p><i>Streptococcus pneumoniae</i> requires manganese for colonization of the human host, but the underlying molecular basis for this requirement has not been elucidated. Recently, it was shown that zinc could compromise manganese uptake and that zinc levels increased during infection by <i>S. pneumoniae</i> in all the niches that it colonized. Here we show, by quantitative means, that extracellular zinc acts in a dose dependent manner to competitively inhibit manganese uptake by <i>S. pneumoniae</i>, with an EC₅₀ of 30.2 µM for zinc in cation-defined media. By exploiting the ability to directly manipulate <i>S. pneumoniae</i> accumulation of manganese, we analyzed the connection between manganese and superoxide dismutase (SodA), a primary source of protection for <i>S. pneumoniae</i> against oxidative stress. We show that manganese starvation led to a decrease in <i>sodA</i> transcription indicating that expression of <i>sodA</i> was regulated through an unknown manganese responsive pathway. Intriguingly, examination of recombinant SodA revealed that the enzyme was potentially a cambialistic superoxide dismutase with an iron/manganese cofactor. SodA was also shown to provide the majority of protection against oxidative stress as a <i>S. pneumoniae</i> Δ<i>sodA</i> mutant strain was found to be hypersensitive to oxidative stress, despite having wild-type manganese levels, indicating that the metal ion alone was not sufficiently protective. Collectively, these results provide a quantitative assessment of the competitive effect of zinc upon manganese uptake and provide a molecular basis for how extracellular zinc exerts a ‘toxic’ effect on bacterial pathogens, such as <i>S. pneumoniae</i>.</p></div

<i>S. pneumoniae</i> response to oxidative stress.

<p>(<b>A</b>) Paraquat killing of the <i>S. pneumoniae</i> wild-type (D39) and Δ<i>sodA</i> mutant grown in CDM + 1 µM Mn(II) (white), and <i>S. pneumoniae</i> (D39) grown in 100 µM Zn(II):100 µM Mn(II) (black) or 100 µM Zn(II):1 µM Mn(II) (light gray) conditions. Survival was calculated as a percentage of c.f.u. after 30 minutes paraquat challenge compared to 30 minutes without challenge. The experiment was performed with 3 independent biological samples and data are the means (± SEM). The statistical significance of the differences in mean survival was determined by a two-tailed unpaired <i>t</i>-test (n.s. corresponds to not significant, * corresponds to <i>P</i> value < 0.05, and **** P value < 0.0001). (<b>B</b>) <i>S. pneumoniae</i> D39 mRNA transcription levels were examined after growth in CDM + 1 µM Mn(II) or 100 µM Zn(II):1 µM Mn(II). Real-time RT-PCR data for the indicated conditions were normalized against those obtained for the 16S rRNA control. Data are means (± SEM) of at least three biological replicates. The statistical significance of the differences in relative transcription level was determined by a two-tailed unpaired <i>t</i>-test (* corresponds to <i>P</i> value < 0.05, and ** to <i>P</i> value < 0.01). (<b>C</b>) <i>S. pneumoniae</i> D39 (filled) and Δ<i>sodA</i> (open) were grown in CDM supplemented with 1 µM Mn(II) until an A₆₀₀ of 0.3 was reached. Cells were washed in CDM and then inoculated to an A₆₀₀ of 0.05 in CDM consisting of in CDM + 1 µM Mn(II) (circle) or 300 µM Zn(II):1 µM Mn(II) (square). Data are means (± SEM) A₆₀₀ measurements from three independent biological experiments. Error bars, where not visible, are overlapped by the data points.</p

<i>In vitro S. pneumoniae</i> growth and metal ion accumulation.

<p>(<b>A</b>) Growth curves of <i>S. pneumoniae</i> grown in CDM with the following Zn(II):Mn(II) ratios (in μM): 300:1 (orange line, open diamond), 100:1 (purple, open triangle), 30:1 (red, open square), 10:1 (blue, open inverted triangle), and 1 µM Mn(II) (black, filled circle), respectively. Data are mean (± SEM) absorbance measurements from three independent biological experiments. Error bars, where not visible, are overlapped by the data points. (<b>B</b> and <b>C</b>) <i>S. pneumoniae</i> total cellular accumulation of Mn(II) (B) and Zn(II) (C) determined by ICP-MS of cells grown in following Zn(II):Mn(II) ratios (in μM): 100:1 (purple), 30:1 (red), 10:1 (blue), and 1 µM Mn(II) (black). Data are mean (± SEM) µg metal.g dry cell mass⁻¹ from duplicate measurements of at least 3 independent biological experiments. (D) Growth curves of <i>S. pneumoniae</i> grown in CDM with the following Zn(II):Mn(II) ratios (in µM): 300:300 (orange line, filled diamond), 100:100 (purple, filled triangle), 30:1 (red, filled square), 10:1 (blue, filled inverted triangle), and 1 µM Mn(II) (black, filled circle), respectively. Data are means (± SEM) A₆₀₀ measurements from three independent biological experiments. (<b>E</b> and <b>F</b>) <i>S. pneumoniae</i> total cellular accumulation of Mn(II) (E) and Zn(II) (F) determined by ICP-MS of cells grown in following Zn(II):Mn(II) ratios (in μM): 300:300 (orange), 100:100 (purple), 30:30 (red), 10:10 (blue), and CDM + 1 µM Mn(II) (black). Data are mean (± SEM) µg metal.g dry cell mass⁻¹ from duplicate measurements of at least 3 independent biological experiments. Statistical significance of the differences in the means was determined by a two-tailed unpaired <i>t</i>-test (n.s. corresponds to not significant and **** to <i>P</i> value < 0.0001).</p

rSodA purification and characterization.

<p>(<b>A</b>) Purified rSodA electrophoretically separated on a 12.5% SDS polyacrylamide gel, with the major band stained by PAGE Blue. (<b>B</b>) Determination of the apparent molecular mass of the purified rSodA by gel-permeation chromatography on a Superdex 200 10/300 column. Inset is the linear regression of the protein molecular mass standards used to calibrate the column (carbonic anhydrase  = 29 kDa, bovine serum albumin  = 66 kDa, yeast alcohol dehydrogenase  =  150 kDa, sweet potato β-Amylase  =  200kDa). rSodA eluted with a calculated molecular mass of 60.8 kDa. (<b>C</b>) The SOD activity of apo-rSodA (white) and rSodA loaded with Mn(II) (black) or Fe(II) (light gray) was measured. The data are presented as SOD activity units (U) per µM protein. Data are means (± SEM) of three biological replicates.</p

Competitive effect of Zn(II) on metal ion accumulation.

<p>(<b>A</b>) The concentration response curve fitting data for Mn(II) accumulation in <i>S. pneumoniae</i> D39 under extracellular Zn(II) stress. Data were normalized by comparison with non-competitive growth conditions [CDM + 1 µM Mn(II)]. Curve fitting was performed in Graphpad Prism version 5.0d (Graphpad). (<b>B</b>, <b>C</b>, <b>D</b>, and <b>E</b>) <i>S. pneumoniae</i> total cellular accumulation of Fe(II/III) (B), Co(II) (C), Ni(II) (D), and Cu(II) (E), determined by ICP-MS, when grown in CDM supplemented with 1 µM Mn(II), 10 µM Zn(II):1 µM Mn(II), 30 µM Zn(II):1 µM Mn(II), and 100 µM Zn:1 µM Mn. Data are mean (± SEM) µg metal.g dry cell mass⁻¹ measurements from duplicate measurements of at least 3 independent biological experiments. The statistical significance of the differences in concentrations was determined by a two-tailed unpaired <i>t</i>-test (n.s. corresponds to not significant, * to <i>P</i> value < 0.05, and ** to <i>P</i> value < 0.01).</p

Image_1_Arachidonic Acid Stress Impacts Pneumococcal Fatty Acid Homeostasis.tiff

<p>Free fatty acids hold dual roles during infection, serving to modulate the host immune response while also functioning directly as antimicrobials. Of particular importance are the long chain polyunsaturated fatty acids, which are not commonly found in bacterial organisms, that have been proposed to have antibacterial roles. Arachidonic acid (AA) is a highly abundant long chain polyunsaturated fatty acid and we examined its effect upon Streptococcus pneumoniae. Here, we observed that in a murine model of S. pneumoniae infection the concentration of AA significantly increases in the blood. The impact of AA stress upon the pathogen was then assessed by a combination of biochemical, biophysical and microbiological assays. In vitro bacterial growth and intra-macrophage survival assays revealed that AA has detrimental effects on pneumococcal fitness. Subsequent analyses demonstrated that AA exerts antimicrobial activity via insertion into the pneumococcal membrane, although this did not increase the susceptibility of the bacterium to antibiotic, oxidative or metal ion stress. Transcriptomic profiling showed that AA treatment also resulted in a dramatic down-regulation of the genes involved in fatty acid biosynthesis, in addition to impacts on other metabolic processes, such as carbon-source utilization. Hence, these data reveal that AA has two distinct mechanisms of perturbing the pneumococcal membrane composition. Collectively, this work provides a molecular basis for the antimicrobial contribution of AA to combat pneumococcal infections.</p

Table_1_Arachidonic Acid Stress Impacts Pneumococcal Fatty Acid Homeostasis.docx

<p>Free fatty acids hold dual roles during infection, serving to modulate the host immune response while also functioning directly as antimicrobials. Of particular importance are the long chain polyunsaturated fatty acids, which are not commonly found in bacterial organisms, that have been proposed to have antibacterial roles. Arachidonic acid (AA) is a highly abundant long chain polyunsaturated fatty acid and we examined its effect upon Streptococcus pneumoniae. Here, we observed that in a murine model of S. pneumoniae infection the concentration of AA significantly increases in the blood. The impact of AA stress upon the pathogen was then assessed by a combination of biochemical, biophysical and microbiological assays. In vitro bacterial growth and intra-macrophage survival assays revealed that AA has detrimental effects on pneumococcal fitness. Subsequent analyses demonstrated that AA exerts antimicrobial activity via insertion into the pneumococcal membrane, although this did not increase the susceptibility of the bacterium to antibiotic, oxidative or metal ion stress. Transcriptomic profiling showed that AA treatment also resulted in a dramatic down-regulation of the genes involved in fatty acid biosynthesis, in addition to impacts on other metabolic processes, such as carbon-source utilization. Hence, these data reveal that AA has two distinct mechanisms of perturbing the pneumococcal membrane composition. Collectively, this work provides a molecular basis for the antimicrobial contribution of AA to combat pneumococcal infections.</p

Microstructured Optical Fibers and Live Cells: A Water-Soluble, Photochromic Zinc Sensor

A new biologically compatible Zn­(II) sensor was fabricated by embedding a Zn­(II) sensing spiropyran within the surface of a liposome derived from Escherichia coli lipids (<b>LSP2</b>). Solution-based experiments with increasing Zn­(II) concentrations show improved aqueous solubility and sensitivity compared to the isolated spiropyran molecule (<b>SP2</b>). <b>LSP2</b> is capable of sensing Zn­(II) efflux from dying cells with preliminary data indicating that sensing is localized near the surface membrane of HEK 293 cells. Finally, <b>LSP2</b> is suitable for development into a nanoliter-scale dip-sensor for Zn­(II) using microstructured optical fiber as the sensing platform to detect Zn­(II) in the range of 100 ρM with minimal photobleaching. Existing spiropyran based sensing molecules can thus be made biologically compatible, with an ability to operate with improved sensitivity using nanoscale liquid sample volumes. This work represents the first instance where photochromic spiropyran molecules and liposomes are combined to create a new and multifunctional sensing entity for Zn­(II)

Table_1_Zinc acquisition and its contribution to Klebsiella pneumoniae virulence.xlsx

Klebsiella pneumoniae is a World Health Organization priority pathogen and a significant clinical concern for infections of the respiratory and urinary tracts due to widespread and increasing resistance to antimicrobials. In the absence of a vaccine, there is an urgent need to identify novel targets for therapeutic development. Bacterial pathogens, including K. pneumoniae, require the d-block metal ion zinc as an essential micronutrient, which serves as a cofactor for ~6% of the proteome. During infection, zinc acquisition necessitates the use of high affinity uptake systems to overcome niche-specific zinc limitation and host-mediated nutritional immunity. Here, we report the identification of ZnuCBA and ZniCBA, two ATP-binding cassette permeases that are highly conserved in Klebsiella species and contribute to K. pneumoniae AJ218 zinc homeostasis, and the high-resolution structure of the zinc-recruiting solute-binding protein ZniA. The Znu and Zni permeases appear functionally redundant with abrogation of both systems required to reduce K. pneumoniae zinc accumulation. Disruption of both systems also exerted pleiotropic effects on the homeostasis of other d-block elements. Zinc limitation perturbed K. pneumoniae cell morphology and compromised resistance to stressors, such as salt and oxidative stress. The mutant strain lacking both systems showed significantly impaired virulence in acute lung infection models, highlighting the necessity of zinc acquisition in the virulence and pathogenicity of K. pneumoniae.</p

DataSheet_1_Zinc acquisition and its contribution to Klebsiella pneumoniae virulence.docx

Klebsiella pneumoniae is a World Health Organization priority pathogen and a significant clinical concern for infections of the respiratory and urinary tracts due to widespread and increasing resistance to antimicrobials. In the absence of a vaccine, there is an urgent need to identify novel targets for therapeutic development. Bacterial pathogens, including K. pneumoniae, require the d-block metal ion zinc as an essential micronutrient, which serves as a cofactor for ~6% of the proteome. During infection, zinc acquisition necessitates the use of high affinity uptake systems to overcome niche-specific zinc limitation and host-mediated nutritional immunity. Here, we report the identification of ZnuCBA and ZniCBA, two ATP-binding cassette permeases that are highly conserved in Klebsiella species and contribute to K. pneumoniae AJ218 zinc homeostasis, and the high-resolution structure of the zinc-recruiting solute-binding protein ZniA. The Znu and Zni permeases appear functionally redundant with abrogation of both systems required to reduce K. pneumoniae zinc accumulation. Disruption of both systems also exerted pleiotropic effects on the homeostasis of other d-block elements. Zinc limitation perturbed K. pneumoniae cell morphology and compromised resistance to stressors, such as salt and oxidative stress. The mutant strain lacking both systems showed significantly impaired virulence in acute lung infection models, highlighting the necessity of zinc acquisition in the virulence and pathogenicity of K. pneumoniae.</p