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      Staphylococcus aureus Redirects Central Metabolism to Increase Iron Availability

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          Abstract

          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.

          Synopsis

          Virtually all bacterial pathogens require iron to successfully infect their human hosts. This presents a problem to invading bacteria because the majority of iron in humans is tightly bound by iron-binding proteins. To counteract this host defense, bacterial pathogens have developed elaborate mechanisms to acquire nutrient iron during infection. To gain insight into how the amount of available iron impacts the human pathogen Staphylococcus aureus, the authors identified proteins that increase or decrease abundance upon alterations in iron status. The authors found that under conditions of iron starvation, the Fur regulatory protein of S. aureus coordinates a redirection of the central metabolic pathways causing the bacteria to produce large amounts of acidic end-products. The accumulation of these acidic end-products facilitates the release of iron from host iron-binding proteins, in effect increasing the availability of this precious nutrient source. These findings provide a mechanistic explanation for how S. aureus alters its local microenvironment during infection to increase the availability of nutrient iron. Based on the well-established role for bacterial iron acquisition during pathogenesis, systems involved in iron acquisition represent excellent potential therapeutic targets against bacterial infection.

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          Most cited references39

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          A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli.

          A small RNA, RyhB, was found as part of a genomewide search for novel small RNAs in Escherichia coli. The RyhB 90-nt RNA down-regulates a set of iron-storage and iron-using proteins when iron is limiting; it is itself negatively regulated by the ferric uptake repressor protein, Fur (Ferric uptake regulator). RyhB RNA levels are inversely correlated with mRNA levels for the sdhCDAB operon, encoding succinate dehydrogenase, as well as five other genes previously shown to be positively regulated by Fur by an unknown mechanism. These include two other genes encoding enzymes in the tricarboxylic acid cycle, acnA and fumA, two ferritin genes, ftnA and bfr, and a gene for superoxide dismutase, sodB. Fur positive regulation of all these genes is fully reversed in an ryhB mutant. Our results explain the previously observed inability of fur mutants to grow on succinate. RyhB requires the RNA-binding protein, Hfq, for activity. Sequences within RyhB are complementary to regions within each of the target genes, suggesting that RyhB acts as an antisense RNA. In sdhCDAB, the complementary region is at the end of the first gene of the sdhCDAB operon; full-length sdhCDAB message disappears and a truncated message, equivalent in size to the region upstream of the complementarity, is detected when RyhB is expressed. RyhB provides a mechanism for the cell to down-regulate iron-storage proteins and nonessential iron-containing proteins when iron is limiting, thus modulating intracellular iron usage to supplement mechanisms for iron uptake directly regulated by Fur.
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            Identification of tandem duplicate regulatory small RNAs in Pseudomonas aeruginosa involved in iron homeostasis.

            In many bacteria, iron homeostasis is controlled primarily by the ferric uptake regulator (Fur), a transcriptional repressor. However, some genes, including those involved in iron storage, are positively regulated by Fur. A Fur-repressed regulatory small RNA (sRNA), RyhB, has been identified in Escherichia coli, and it has been demonstrated that negative regulation of genes by this sRNA is responsible for the positive regulation of some genes by Fur. No RyhB sequence homologs were found in Pseudomonas aeruginosa, despite the identification of genes positively regulated by its Fur homolog. A bioinformatics approach identified two tandem sRNAs in P. aeruginosa that were candidates for functional homologs of RyhB. These sRNAs (PrrF1 and PrrF2) are >95% identical to each other, and a functional Fur box precedes each. Their expression is induced under iron limitation. Deletion of both sRNAs is required to affect the iron-dependent regulation of an array of genes, including those involved in resistance to oxidative stress, iron storage, and intermediary metabolism. As in E. coli, induction of the PrrF sRNAs leads to the rapid loss of mRNAs for sodB (superoxide dismutase), sdh (succinate dehydrogenase), and a gene encoding a bacterioferritin. Thus, the PrrF sRNAs are the functional homologs of RyhB sRNA. At least one gene, bfrB, is positively regulated by Fur and Fe(2+), even in the absence of the PrrF sRNAs. This work suggests that the role of sRNAs in bacterial iron homeostasis may be broad, and approaches similar to those described here may identify these sRNAs in other organisms.
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              A novel experimental design for comparative two-dimensional gel analysis: two-dimensional difference gel electrophoresis incorporating a pooled internal standard.

              The comparison of two-dimensional (2-D) gel images from different samples is an established method used to study differences in protein expression. Conventional methods rely on comparing images from at least 2 different gels. Due to the high variation between gels, detection and quantification of protein differences can be problematic. Two-dimensional difference gel electrophoresis (Ettan trade mark DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. In the application of DIGE different samples are labelled with mass and charge matched spectrally resolvable fluorescent dyes and are then separated on the same 2-D gel. Using an Escherichia coli lysate "spiked" with varying amounts of four different known proteins, we have tested a novel experimental design that exploits the sample multiplexing capabilities of DIGE, by including a standard sample in each gel. The standard sample comprises equal amounts of each sample to be compared and was found to improve the accuracy of protein quantification between samples from different gels allowing accurate detection of small differences in protein levels between samples.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                ppat
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                August 2006
                25 August 2006
                : 2
                : 8
                : e87
                Affiliations
                [1 ] Mass Spectrometry Research Center, Department of Biochemistry, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America
                [2 ] Department of Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America
                Harvard Medical School, United States of America
                Author notes
                * To whom correspondence should be addressed. E-mail: eric.skaar@ 123456vanderbilt.edu
                Article
                06-PLPA-RA-0116R2 e87 plpa-02-08-07
                10.1371/journal.ppat.0020087
                1557832
                16933993
                d3572a92-b9b5-421a-8ffc-46b0ab5030a7
                Copyright: © 2006 Friedman et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 31 March 2006
                : 14 July 2006
                Page count
                Pages: 13
                Categories
                Research Article
                Biochemistry
                Infectious Diseases
                Microbiology
                Eubacteria
                Animals
                Custom metadata
                Friedman DB, Stauff DL, Pishchany G, Whitwell CW, Torres VJ, et al. (2006) Staphylococcus aureus redirects central metabolism to increase iron availability. PLoS Pathog 2(8): e87. DOI: 10.1371/journal.ppat.0020087

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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