Staphylococcus aureus Redirects Central Metabolism to Increase Iron Availability

Staphylococcus aureus pathogenesis is significantly influenced by the iron status of the host. However, the regulatory impact of host iron sources on S. aureus gene expression remains unknown. In this study, we combine multivariable difference gel electrophoresis and mass spectrometry with multivariate statistical analyses to systematically cluster cellular protein response across distinct iron-exposure conditions. Quadruplicate samples were simultaneously analyzed for alterations in protein abundance and/or post-translational modification state in response to environmental (iron chelation, hemin treatment) or genetic (Δfur) alterations in bacterial iron exposure. We identified 120 proteins representing several coordinated biochemical pathways that are affected by changes in iron-exposure status. Highlighted in these experiments is the identification of the heme-regulated transport system (HrtAB), a novel transport system which plays a critical role in staphylococcal heme metabolism. Further, we show that regulated overproduction of acidic end-products brought on by iron starvation decreases local pH resulting in the release of iron from the host iron-sequestering protein transferrin. These findings reveal novel strategies used by S. aureus to acquire scarce nutrients in the hostile host environment and begin to define the iron and heme-dependent regulons of S. aureus

The Hrt System Is Required for Staphyloccocal Growth in Hemin

<p>S. aureus Newman (WT), SAV2359 mutant <i>(hrtA),</i> SAV2360 mutant <i>(hrtB),</i> transduced SAV2359 mutant <i>(</i>t-<i>hrtA),</i> and transduced SAV2360 mutant <i>(</i>t-<i>hrtB)</i> strains were grown in iron-free medium supplemented with iron (Fe) or with hemin (hemin). Bacterial growth was determined by measuring the optical density (O.D.₆₀₀) of cultures. Results represent the mean ± SD from triplicate determinations. Asterisks denote statistically significant differences from wild-type as determined by a Student's <i>t</i>-test (<i>p</i> < 0.05).</p

Lactate Is a Primary End-Product of Carbohydrate Metabolism by Iron-Starved S. aureus

<p>A subset of the predicted central metabolic reactions of S. aureus is shown. Proteins shown in red are up-regulated in the absence of iron or upon inactivation of <i>fur</i>. Proteins shown in blue are down-regulated in the absence of iron or upon inactivation of <i>fur</i>. Proteins shown in green are down-regulated in the absence of Fur independent of iron status. The red arrows predict the direction of reactions upon iron starvation, while the blue arrows predict reactions that are inhibited upon iron starvation.</p

Proteome Analysis Using 2D DIGE

<div><p>(A) False–colored representative gel from the eight-gel set containing three differentially labeled samples as described in Materials and Methods. Cy2-labeled internal standard (blue), Cy3-labeled control No. 1 (green), and Cy5-labeled iron-starved No. 1 (red) are overlaid for illustrative purposes.</p><p>(B) Unsupervised PCA properly groups the 16 individual DIGE expression maps differentiated by two principle components (PC1 and PC2) and demonstrates high reproducibility between the replicate samples within each group.</p><p>(C) Composites of DIGE expression patterns representing the five proteins that increase abundance in the presence of hemin (Class I, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020087#ppat-0020087-t001" target="_blank">Table 1</a>), the 29 Class II protein features negatively affected by Fur and iron (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020087#ppat-0020087-t002" target="_blank">Table 2</a>), and the 30 Class III protein features positively affected by Fur and iron (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020087#ppat-0020087-t003" target="_blank">Table 3</a>).</p><p>(D) Unsupervised hierarchical clustering of the 16 individual DIGE expression maps (groups, shown along top) and of individual proteins (shown on the left), with relative expression values for each protein displayed as a heat map using a relative scale ranging from −0.5 (green) to +0.5 (red). The gel number (1 through 8), samples (A, TSB; B, iron-starved; C, hemin; and D, <i>Δfur</i>), and Cy3/5 dye labeling for each sample are listed below.</p></div

Design, Implementation and Multisite Evaluation of a System Suitability Protocol for the Quantitative Assessment of Instrument Performance in Liquid Chromatography-Multiple Reaction Monitoring-MS (LC-MRM-MS)*

Multiple reaction monitoring (MRM) mass spectrometry coupled with stable isotope dilution (SID) and liquid chromatography (LC) is increasingly used in biological and clinical studies for precise and reproducible quantification of peptides and proteins in complex sample matrices. Robust LC-SID-MRM-MS-based assays that can be replicated across laboratories and ultimately in clinical laboratory settings require standardized protocols to demonstrate that the analysis platforms are performing adequately. We developed a system suitability protocol (SSP), which employs a predigested mixture of six proteins, to facilitate performance evaluation of LC-SID-MRM-MS instrument platforms, configured with nanoflow-LC systems interfaced to triple quadrupole mass spectrometers. The SSP was designed for use with low multiplex analyses as well as high multiplex approaches when software-driven scheduling of data acquisition is required. Performance was assessed by monitoring of a range of chromatographic and mass spectrometric metrics including peak width, chromatographic resolution, peak capacity, and the variability in peak area and analyte retention time (RT) stability. The SSP, which was evaluated in 11 laboratories on a total of 15 different instruments, enabled early diagnoses of LC and MS anomalies that indicated suboptimal LC-MRM-MS performance. The observed range in variation of each of the metrics scrutinized serves to define the criteria for optimized LC-SID-MRM-MS platforms for routine use, with pass/fail criteria for system suitability performance measures defined as peak area coefficient of variation &lt;0.15, peak width coefficient of variation &lt;0.15, standard deviation of RT &lt;0.15 min (9 s), and the RT drift &lt;0.5min (30 s). The deleterious effect of a marginally performing LC-SID-MRM-MS system on the limit of quantification (LOQ) in targeted quantitative assays illustrates the use and need for a SSP to establish robust and reliable system performance. Use of a SSP helps to ensure that analyte quantification measurements can be replicated with good precision within and across multiple laboratories and should facilitate more widespread use of MRM-MS technology by the basic biomedical and clinical laboratory research communities

Large-scale interlaboratory study to develop, analytically validate and apply highly multiplexed, quantitative peptide assays to measure cancer-relevant proteins in plasma

There is an increasing need in biology and clinical medicine to robustly and reliably measure tens-to-hundreds of peptides and proteins in clinical and biological samples with high sensitivity, specificity, reproducibility and repeatability. Previously, we demonstrated that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma, and that the resulting assays can be transferred across laboratories while maintaining high reproducibility and quantitative precision. Here we significantly extend that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems can develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and 7 control proteins to precisely and reproducibly measure the analytes in human plasma. To ensure consistent generation of high quality data we incorporated a system suitability protocol (SSP) into our experimental design. The SSP enabled real-time monitoring of LC-MRM-MS performance during assay development and implementation, facilitating early detection and correction of chromatographic and instrumental problems. Low to sub-nanogram/mL sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and inter-laboratory reproducibility was <20%, sufficient for most biological studies and candidate protein biomarker verification. Digestion recovery of peptides was assessed and quantitative accuracy improved using heavy isotope labeled versions of the proteins as internal standards. Using the highly multiplexed assay, participating laboratories were able to precisely and reproducibly determine the levels of a series of analytes in blinded samples used to simulate an inter-laboratory clinical study of patient samples. Our study further establishes that LC-MRM-MS using stable isotope dilution, with appropriate attention to analytical validation and appropriate quality c`ontrol measures, enables sensitive, specific, reproducible and quantitative measurements of proteins and peptides in complex biological matrices such as plasma