15 research outputs found
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 EC<sub>50</sub> 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 A<sub>600</sub> of 0.3 was reached. Cells were washed in CDM and then inoculated to an A<sub>600</sub> 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) A<sub>600</sub> 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<sup>−1</sup> 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) A<sub>600</sub> 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<sup>−1</sup> 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<sup>−1</sup> 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
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
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
The catabolic phenome of <i>Acinetobacter baumannii</i>.
<p>Strengths of carbon utilisation phenotypes of <i>A. baumannii</i> strains D1279779, ACICU, AYE and ATCC 17978 were determined using Biolog Phenotype Microarray plates PM1 and PM2. The maximal kinetic curve height was expressed as a greyscale ranging from 101 (light grey) to 320 OmniLog units (black). Phenotypes are arranged from strongest to weakest relative to <i>A. baumannii</i> D1279779. Phenotypes <101 OmniLog units (white) were considered negative.</p
Genome map of <i>Acinetobacter baumannii</i> D1279779.
<p>The two outermost circles denote positions of protein coding sequences (CDSs) on the positive (circle 1) and negative (circle 2) strands coloured according to clusters of orthologous groups (COGs) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058628#pone.0058628-Tatusov1" target="_blank">[99]</a> functional category: A (lavender), B (apricot), C (olive), D (light brown), E (dark green), F (electric pink), G (electric green), H (peach), I (red), J (dark red), K (midnight blue), L (plum), M (teal), N (blue), O (aquamarine), P (orange), Q (yellow), R (dark grey), S (grey), T (light purple), U (light green), V (light yellow), and unknown COG (black). Circle 3 represents positions of identified regions of genomic plasticity (red) and ISAb13s (black) ordered clockwise from the origin of chromosomal replication as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058628#pone-0058628-t002" target="_blank">Table 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058628#pone-0058628-t004" target="_blank">Table 4</a>, respectively. Circle 4 denotes the calculated chi-squared values based on the trinucleotide composition of the DNA sequence. Circles 5-14 show DNA conservation between D1279779 and other sequenced <i>A. baumannii</i> strains based on pairwise BLASTN alignments (evalue threshold 1e-10). Strain comparisons (outermost to innermost): 1656-2 (teal), ACICU (aquamarine), MDR-ZJ06 (orange), MDR-TJ (light purple), TCDC-AB0715 (red), AB307-0294 (blue), AB0057 (olive), AYE (green-brown), ATCC 17978 (teal) and SDF (aquamarine). The innermost circle denotes positive (green) and negative (purple) GC-skew and the scale in kilobase pairs. The CGView software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058628#pone.0058628-Stothard1" target="_blank">[100]</a> was utilised to construct the genome map.</p
50kb inversion in the genome of <i>Acinetobacter baumannii</i> D1279779.
<p>The DNA region between rRNA operons one and six in <i>A. baumannii</i> D1279779 is conserved (≥96% nucleotide identity) but is inverted relative to other <i>A. baumannii</i> genomes (represented by ACICU), as depicted by the grey shading. Genes on positive and negative strands are depicted on the top and bottom row of rectangles, respectively. Conserved regions (≥99% nucleotide identity) in the same orientation are depicted by black shading. Locations of several conserved genes and the origin of chromosomal replication (<i>oriC</i>) are indicated. This figure was generated using the combined outputs of MAUVE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058628#pone.0058628-Darling1" target="_blank">[42]</a> and the Artemis Comparison Tool <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058628#pone.0058628-Carver1" target="_blank">[102]</a>.</p