The family of SOD enzymes in microbial pathogens.

<p>In a gram-negative bacteria such as <i>E</i>. <i>coli</i> (left), the Mn-SodA and Fe-SodA are intracellular/cytosolic where Mn and Fe metals ions are generally bioavailable. Cu is extruded into the periplasmic space, driving the evolution of Cu/Zn-containing SodC in this extra-cytosolic compartment. In eukaryotes (right), the mitochondria thought to evolve from a gram-negative bacteria exhibits the equivalent partitioning of a Mn-Sod2 to the mitochondrial matrix and Cu/Zn Sod1 to the mitochondrial IMS and cytosol. Some pathogenic microbes have acquired additional SODs (lined in red), including the highly stable prophage-derived SodC-F in pathogenic <i>E</i>. <i>coli</i> (left) and in <i>Candida albicans</i> (right), the cytosolic Mn-Sod3 as a backup for Cu/Zn Sod1, and the Cu-only extracellular Sod4, Sod5, and Sod6.</p

Model for Cu-only fungal SOD5 at the macrophage–pathogen interface.

<p>Activated macrophages may attack invading microbes with toxic levels of Cu and superoxide (O₂^-). With its open Cu site, Sod5 on the surface of the fungal pathogen may be able to capture this host Cu, fueling the SOD enzyme to remove the superoxide in counterattack.</p

Effect of inhibiting AOX respiration on mitochondrial superoxide under copper starvation.

<p>MitoSOX Red fluorescence was measured in the designated strains. Where indicated, cells were treated with 5 mM SHAM for 1 hour to inhibit AOX respiration prior to MitoSOX Red incubation. Results represent the averages of two independent cultures with error bars representing standard deviation.</p

Effects of copper starvation and <i>sod</i> gene mutations on mitochondrial superoxide and <i>AOX2</i> expression.

<p>(A-C) MitoSOX Red fluorescence was measured spectrophotometrically at 580 nm where fluorescence intensity is the signal obtained with 2x10⁷ cells as described in <i>Materials and Methods</i>. (A-B) A comparison of the effects of <i>sod1Δ/Δ</i> versus <i>sod2Δ/Δ</i> mutations on MitoSOX Red fluorescence where results represent the averages of eight and four independent samples respectively, and statistical significance was determined using a paired two-tailed Student’s t-test. Asterisks indicate statistical significance with P values < 0.05. Error bars represent standard deviation. The baseline MitoSOX Red fluorescence of non-stressed wild type cells typically varies between 20–40 fluorescent units, but regardless of this baseline, <i>sod1Δ/Δ</i> and <i>sod2Δ/Δ</i> mutants exhibited enhanced fluorescence. (C) The indicated strains were grown in the presence or absence of 800 μM BCS as designated. Results represent the averages of three independent cultures where error bars are standard deviation. The difference between minus and plus BCS with the wild type strain is not statistically significant (p = 0.2063). (D) <i>AOX2</i> and <i>SOD3</i> mRNA were quantified by qRT-PCR from wild type (SN152) and the isogenic <i>mac1Δ/Δ</i> strain as described in <i>Materials and Methods</i>. Values are normalized to that obtained with wild type SN152 <i>C</i>. <i>albicans</i> cells without BCS. Shown are averages of two biological replicates where error bars are standard deviation.</p

AOX respiration contributes to oxygen consumption during copper starvation.

<p><i>C</i>. <i>albicans</i> strain CA-IF100 grown in the presence or absence of BCS was subjected to measurements of oxygen consumption using a Clark electrode. (A,B) Shown are representative profiles of oxygen consumption as monitored by percent oxygen saturation in solution. At the indicated time points, cells were treated with 5 mM SHAM to inhibit AOX respiration followed by 1 mM KCN to inhibit COX respiration. (C,D) The rates of oxygen consumption were derived from oxygen saturation curves. Plotted are the percentages of oxygen consumption inhibited by SHAM and attributed to AOX (C) and the oxygen consumption inhibited by KCN and attributed to COX (D). Results represent the averages of three independent samples with error bars representing standard deviation. Statistical significance was determined using a paired two-tailed Student’s t-test. Asterisks indicate statistical significance with P values < 0.05.</p

Constitutive expression of SOD1 inhibits COX repression during copper starvation.

<p>(A) Schematic showing the coding region (black) and intron (white) of the <i>SOD1</i> gene with sequence of the wild type and mutated MAC1 sites in SOD1^rep (<i>SOD1</i> repressed by copper) and SOD1^con (<i>SOD1</i> constitutively expressed). (B) Strains expressing a single copy of either SOD1^rep or SOD1^con were grown in the presence or absence of BCS and whole cell lysates were (top) immunoblotted for SOD1 and SOD3 as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168400#pone.0168400.g001" target="_blank">Fig 1A</a> and (bottom) subjected to SOD enzymatic activity analysis by the native gel assay. Numbers on left of immunoblot represent molecular weight markers and the positions of SOD1 and SOD2 migration on the native gel are indicated on the right. Densitometric tracings show that the intensity of SOD1 activity in SOD1^con +BCS is 38% that of the corresponding–BCS sample. (C) Cytochrome c oxidase respiration was measured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168400#pone.0168400.g005" target="_blank">Fig 5D</a> as a function of KCN inhibitable oxygen consumption. Results represent the averages of two independent experiments with a total of four biological replicates with error bars representing standard deviation. Statistical significance was determined using a paired two-tailed Student’s t-test. Asterisks indicate statistical significance with P values < 0.005.</p

Adaption to copper in <i>Candida albicans</i> involving SOD enzymes and the mode of mitochondrial respiration.

<p>Cartoon to illustrate <i>C</i>. <i>albicans</i> adaptation to low copper by alternating SOD enzymes and inducing AOX respiration. When copper is abundant (left), <i>C</i>. <i>albicans</i> utilizes COX respiration and SOD1 is active in both the IMS and the cytosol. During copper starvation (right), cells switch to expressing SOD3 in the cytosol, but the IMS becomes devoid of a SOD enzyme. AOX respiration is induced which helps to lower IMS superoxide. Regardless of copper conditions, COX respiration remains active and mitochondrial matrix superoxide is managed by SOD2.</p

A fraction of <i>C</i>. <i>albicans</i> SOD1 but not SOD3 localizes to the mitochondria IMS.

<p><i>C</i>. <i>albicans</i> strain CAIF-100 was grown in enriched medium in either the presence or absence of 800 μM of the extracellular Cu(I) chelator BCS as indicated. (A) Lysates were prepared from spheroplasts and were analyzed for SOD1 and SOD3 by immunoblot. (B) Total cellular copper was measured by atomic absorption spectroscopy. (C-D) Spheroplast cell lysates (Total) were fractionated into the largely cytosolic post-mitochondrial supernatant (PMS) and crude mitochondria (mito) by differential centrifugation. 70 μg of total lysate protein and the same cell equivalents of PMS and mitochondria were analyzed by immunoblot for SOD2 (marker of mitochondrial matrix), PGK1 (marker of cytosol) and either SOD1 (C) or SOD3 (D). MW = molecular weight markers. SOD1 often appears as two bands, both of which are down-regulated by copper starvation.</p

<i>C</i>. <i>albicans</i> SOD1 localizes to the mitochondrial IMS and requires its copper chaperone CCS1 for mitochondrial localization.

<p>(A) Cell lysates from wild type (CAIF-100) and the isogenic <i>ccs1Δ/Δ</i> strain were fractionated into PMS and mitochondria and analyzed by immunoblot for SOD1 and SOD2. (B) The mitochondrial fraction derived from 70 μg total lysate protein was further fractionated into IMS and matrix as described in <i>Materials and Methods</i> and analyzed for SOD1 and SOD2 by immunoblot. Numbers on left indicate molecular weight markers.</p

MitoSOX Red as a probe for mitochondrial superoxide in <i>C</i>. <i>albicans</i>.

<p>Log phase CA-1F100 <i>C</i>. <i>albicans</i> wild type cells (A, B) or the isogenic <i>sod1Δ/Δ</i> strain (C) grown under non-stress conditions were incubated with 5 mM MitoSOX Red as a marker for mitochondrial superoxide and imaged by fluorescence microscopy at 63X. (A) “DIC” = light microscopy images and “MitoTracker” = cells treated with both MitoSox Red and 500 μM MitoTracker Green as a marker for mitochondria. (B,C) Wild type and <i>sod1Δ/Δ</i> cells were examined in parallel where the top and bottom rows show representative MitoSox Red fluorescence and DIC images, respectively.</p