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Novel Riboswitch Ligand Analogs as Selective Inhibitors of Guanine-Related Metabolic Pathways Jérôme Mulhbacher1, Eric Brouillette1, Marianne Allard1, Louis-Charles Fortier2, François Malouin1*, Daniel A. Lafontaine1* 1 Département de biologie, Faculté des sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada, 2 Département de microbiologie et d’infectiologie, Faculté de médecine et sciences de la santé, Université de Sherbrooke, Sherbrooke, Québec, Canada Abstract Riboswitches are regulatory elements modulating gene expression in response to specific metabolite binding. It has been recently reported that riboswitch agonists may exhibit antimicrobial properties by binding to the riboswitch domain. Guanine riboswitches are involved in the regulation of transport and biosynthesis of purine metabolites, which are critical for the nucleotides cellular pool. Upon guanine binding, these riboswitches stabilize a 59-untranslated mRNA structure that causes transcription attenuation of the downstream open reading frame. In principle, any agonistic compound targeting a guanine riboswitch could cause gene repression even when the cell is starved for guanine. Antibiotics binding to riboswitches provide novel antimicrobial compounds that can be rationally designed from riboswitch crystal structures. Using this, we have identified a pyrimidine compound (PC1) binding guanine riboswitches that shows bactericidal activity against a subgroup of bacterial species including well-known nosocomial pathogens. This selective bacterial killing is only achieved when guaA, a gene coding for a GMP synthetase, is under the control of the riboswitch. Among the bacterial strains tested, several clinical strains exhibiting multiple drug resistance were inhibited suggesting that PC1 targets a different metabolic pathway. As a proof of principle, we have used a mouse model to show a direct correlation between the administration of PC1 and the reduction of Staphylococcus aureus infection in mammary glands. This work establishes the possibility of using existing structural knowledge to design novel guanine riboswitch-targeting antibiotics as powerful and selective antimicrobial compounds. Particularly, the finding of this new guanine riboswitch target is crucial as communityacquired bacterial infections have recently started to emerge. Citation: Mulhbacher J, Brouillette E, Allard M, Fortier L-C, Malouin F, et al. (2010) Novel Riboswitch Ligand Analogs as Selective Inhibitors of Guanine-Related Metabolic Pathways. PLoS Pathog 6(4): e1000865. doi:10.1371/journal.ppat.1000865 Editor: Michael R. Wessels, Children’s Hospital Boston, United States of America Received October 8, 2009; Accepted March 22, 2010; Published April 22, 2010 Copyright: ß 2010 Mulhbacher 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. Funding: This work was supported by the Canadian Institutes of Health Research (CIHR) to DAL and the National Sciences and Engineering Research Council of Canada (NSERC), Alberta Milk, Dairy Farmers of New Brunswick, Nova Scotia, Ontario and Prince Edward Island, Novalait Inc., Dairy Farmers of Canada, Canadian Dairy Network, AAFC, PHAC, Technology PEI Inc., Université de Montréal and University of Prince Edward Island through the Canadian Bovine Mastitis Research Network to FM. A post-doctoral fellowship from NSERC and studentship from the Faculté des sciences of the Université de Sherbrooke were also provided to JM and MA, respectively. DAL is a CIHR New Investigator scholar as well as a Chercheur-boursier Junior 2 from the Fonds de la recherche en Santé du Québec. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] (FM); [email protected] (DAL) 027 [4,5,6]. This particular strain is spreading in North America and Europe with currently little therapeutic solutions besides the use of metronidazole and vancomycin, which are increasingly associated with relapses and poor treatment outcome [7]. Previous attempts to discover alternative antibacterial drugs targeting RNA were mainly based on a fortuitous interaction between an exogenous ligand and its RNA target [1,8,9,10]. Metabolite-responsive riboswitches represent a novel solution to MDR since they could be considered as antimicrobial targets when agonistic ligands are employed as demonstrated for lysine, thiamine pyrophosphate (TPP), flavin mononucleotide (FMN) and guanine responsive riboswitches [1,11,12,13,14,15]. In the case of lysine and TPP riboswitches, previously described ligand analogs were reported to have a multitude of cellular effects in addition to inhibition of gene expression via riboswitch binding [16,17,18,19]. Pleiotropic effects were also observed for compounds targeting the guanine riboswitch and at least one analog was reported to be possibly incorporated in DNA during replication [15,20]. Thus, while it is of interest to select antibiotics that are chemically distinct Introduction Multiple drug resistance (MDR) has been a growing problem during the last decade, partly due to excessive use of antibiotics in human medicine and food animal production. MDR also stems from the fact that drug design has been largely based on limited chemical scaffolds leaving an opportunity for pathogens to circumvent antibiotic action mechanisms [1]. Staphylococcus aureus and Clostridium difficile are nosocomial pathogens responsible for a significant mortality rate in hospitals and increased health care costs [2]. Recently, community-acquired methicillin-resistant S. aureus (MRSA) infections have emerged and are commonly responsible for skin and soft-tissue infections that may rapidly evolve in severe and life-threatening infections [3,4]. Moreover, some emerging clones were shown to be resistant to vancomycin, which is considered as the last chance antibiotic [5]. The pathogen C. difficile has also dramatically increased the hospital-associated deaths in recent years due to the MDR emergence and spreading of the hypervirulent and high toxin-producing strain BI/NAP1/ PLoS Pathogens | www.plospathogens.org 1 April 2010 | Volume 6 | Issue 4 | e1000865 Riboswitch Analogs Targeting Guanine Metabolism fact that purine riboswitches efficiently bind pyrimidines, it may be possible to design novel antibiotics that bind to guanine riboswitches and therefore inhibit bacterial growth. Pyrimidine-based molecules that could fit into the guanine riboswitch aptamer binding site were selected based on molecular modeling of crystal structures [26,27] (Figure 2A). Using this approach, we identified two pyrimidine compounds 2,5,6triaminopyrimidin-4-one (PC1) and 2,6-diaminopyrimidin-4-one (PC2) that satisfied defined criteria such as geometrical constraint, hydrogen bonding pattern and molecule planarity (Figure 2B). As opposed to guanine, PC1 and PC2 lack one aromatic ring, making them electronically distinct from guanine despite their similarity to guanine in terms of H-bond donating and accepting potential. Next, using the established in-line probing assay [25,28], we monitored PC1/PC2-induced riboswitch conformational changes (Figure 2C). In absence of ligand, several cleavage products that map to previously reported single-stranded regions were observed [25,28]. However, a cleavage reduction consistent with a reorganization of the structure upon ligand binding was observed in the core domain in presence of guanine (Figure 2C). In-line probing assay with PC1 and PC2 instead of guanine showed an identical cleavage pattern for both pyrimidine compounds and guanine, suggesting that the core is reorganized similarly in presence of these compounds, consistent with the recently reported pyrimidine-bound riboswitch crystal structure [21]. To determine whether PC1 and PC2 repress gene expression, we transformed B. subtilis with transcriptional fusions in which a guanine riboswitch was fused to a lacZ reporter gene (Figure 2D). When cells were grown in minimal medium with increasing concentrations of PC1 and PC2, beta-galactosidase activity was clearly repressed in a dose-dependent manner suggesting a modulation of the guanine riboswitch gene regulation by both molecules. We also performed growth inhibition experiments using various concentrations of both PC1 and PC2 (Figure S2). While growth inhibition was observed in minimal medium, no such inhibition was observed using a richer medium such as cation-adjusted Muller-Hinton broth (CAMHB). This selective growth inhibition can be explained by PC1/PC2 inhibiting the biosynthesis or transport of essential metabolites, which are present in CAMHB but not in minimal medium. For instance, it was recently shown that guanine-related compounds can only inhibit B. subtilis growth in a minimal medium but not in Luria broth; the growth inhibition was partly attributed to the riboswitch-mediated repression of de novo purine synthesis [15]. Author Summary During the last 30 years, bacterial resistance to antibiotics has become a major problem. This situation is partly because today’s antibiotics are mainly based on a limited selection of chemical scaffolds, which makes it easier for bacterial pathogens to quickly develop resistance against new drug derivatives. This recurrent problem of multiple drug resistance implies a constant need to search for novel microbial targets and to modulate their activity using artificial molecules. Riboswitches are newly discovered gene regulatory elements that represent attractive targets for antimicrobial drugs. Riboswitches are RNA structures located in untranslated regions of messenger RNAs that regulate the expression of genes involved in the transport and metabolism of small metabolites. We have identified a new antibiotic specifically targeting riboswitches found in a subgroup of bacteria including Staphylococcus aureus and Clostridium difficile, which are nosocomial pathogens responsible for a significant mortality rate in hospitals, and increased health care costs. The riboswitch controls the expression of guaA that appears essential for virulence in the mammalian host. A murine model was used as a proof of principle to show that such an antibiotic could inhibit the growth of S. aureus in a mammal. Our work provides new insights into the discovery and design of novel antimicrobial agents against bacterial pathogens. from natural ligands to avoid cellular efflux or chemical modification, it is important to consider that these chemical differences will potentially help avoid patient toxicity due to offtarget binding. It is also important that the antibiotic provokes a bacteriostatic or bactericidal effect either by targeting a single gene, or a collection of genes, that is necessary for growth, or essential for bacterial survival or virulence. Thus, because modified pyrimidines can specifically bind the purine riboswitch with affinities in the low nanomolar range [21], they make excellent candidates to target purine riboswitches which are likely potent drug targets given their role in regulating purine metabolic pathways (Figure S1). For instance, the inactivation of the E. coli GMP synthetase guaA leads to guanine auxotrophy [22] whereas the inactivation of the B. subtilis IMP dehydrogenase guaB is lethal [23]. Here we show that the guanine riboswitch in S. aureus and C. difficile controls the expression of guaA and that this gene appears essential for virulence in a murine model. PC1-dependent bacterial growth inhibition requires guaA to be riboswitch-regulated Results Pyrimidine-based antibiotics modulate the guanine riboswitch activity The S. aureus ATCC 29213 genome contains a unique guanine riboswitch located immediately upstream of the xpt gene (Figure S3). Very interestingly, RT-PCR experiments identified that the riboswitch controls a four-gene operon consisting of xpt, pbuX, guaB and guaA, thus placing guaA and guaB under the control of a riboswitch in S. aureus (Figure S3). To determine if PC1 and PC2 have antibiotic activities by targeting the guanine riboswitch in S. aureus, we performed antibiograms with PC1 and PC2 as well as with three additional molecules having similar structures (compounds 3, 4 and 5). While compounds 3 and 5 are structurally very close to PC1 and PC2, compound 4 is a guanine analog (Figure 3A). Surprisingly, among the five compounds tested, only PC1 inhibited bacterial growth in Muller-Hinton agar, which is consistent with its ability to modulate riboswitch gene expression in B. subtilis (Figure 2D). The absence of PC2 antibiotic activity is consistent with the ,5-fold lower PC2-mediated gene expression modulation in B. subtilis, which may result from the lower number of riboswitch- Guanine-sensing riboswitches are members of the purine riboswitch class, which also comprises adenine and 29-deoxyguanosine [24]. The guanine riboswitch negatively regulates transcription elongation at high guanine concentration in Bacillus subtilis [25] (Figure 1A). The guanine aptamer is organized around a three-way junction connecting three helices, in which a critically important nucleotide is involved in a Watson-Crick base pair interaction with the bound ligand [25] (Figure 1B). The ligand binding site contains a cavity in which the metabolite is completely surrounded by RNA contacts suggesting that most atomic positions are important for the formation of the native ligandRNA complex [26,27]. By using appropriate aminopyrimidines, it is also possible to recreate the correct network of hydrogen bonds required to ensure proper complex formation as previously shown for the adenine riboswitch [21]. Thus, by taking advantage of the PLoS Pathogens | www.plospathogens.org 2 April 2010 | Volume 6 | Issue 4 | e1000865 Riboswitch Analogs Targeting Guanine Metabolism Figure 1. The structure of the guanine riboswitch. (A) Scheme representing the guanine riboswitch secondary structures in absence (ON state) or in presence (OFF state) of guanine. The formation of a guanine-riboswitch complex results in the adoption of an intrinsic terminator element that prematurely stops transcription. (B) Consensus sequence and secondary structure of the guanine riboswitch aptamer. Nucleotides indicated in blue, orange and green represent nucleotides that are conserved .90% and those colored in black are conserved .80% [28]. Nucleotides and lines in blue and in green indicate interactions with the ligand via hydrogen bonding and base stacking, respectively. The cytosine 74 which confers ligand binding specificity is shown in orange and the bound guanine is shown as a red rounded rectangle. doi:10.1371/journal.ppat.1000865.g001 ligand interactions (Figure 2B). The binding affinity of PC1 suggests that the guanine riboswitch can tolerate modifications on the ligand pyrimidine ring that are not strongly deleterious for complex formation (,100 nM vs ,5 nM for PC1 and guanine, respectively). The binding affinity of PC1 is very similar to that of hypoxanthine, which is a naturally occurring guanine analog [25]. To explore the antibacterial activity spectrum of PC1, we used several Gram-positive bacterial species which are potential human pathogens containing guanine riboswitches. Of the 15 species tested, 9 showed marked cellular growth inhibition, including MDR strains and the C. difficile CD6 isolate representing the hypervirulent NAP1/027 strain (Figure 3B). Interestingly, when analyzing guanine riboswitch-regulated genes, we observed that all PC1-responsive strains had guaA under riboswitch control whereas the PC1-unresponsive ones did not employ riboswitch regulation to control guaA. The best example of this correlation is that while 16S rDNA sequence analysis indicates that B. subtilis and Bacillus halodurans are very closely related species [29], B. halodurans has a guaA-controlled riboswitch and is sensitive to PC1 whereas B. subtilis lacks a guaA-controlled riboswitch and is resistant to PC1. Antibiogram results also showed that strains exhibiting pronounced MDR phenotypes are sensitive to PC1 suggesting that the antimicrobial activity does not involve action mechanisms common to other known antibiotics. PLoS Pathogens | www.plospathogens.org Because our data suggest that PC1 acts by repressing the GMP synthetase guaA, we reasoned that the PC1 inhibitory activity should be reduced by GMP supplementation. S. aureus cells were thus grown with or without supplemented GMP, and colony forming units (CFU) were determined following serial microdilutions (Figure 3C). As predicted, bacterial growth inhibition was relieved when GMP was provided to cells grown in presence of PC1 for 2 h or 4 h, supporting the hypothesis that bacterial growth inhibition is caused by the riboswitch-mediated guaA gene repression that results in GMP cellular depletion. The PC1 specificity was also confirmed using the Gramnegative bacterium Escherichia coli ATCC 35695, a strain that does not contain guanine riboswitches. As expected, E. coli showed no growth inhibition in presence of PC1 even when using strains deficient for the AcrAB efflux system or having increased membrane permeability (Figure S4). These results suggest that the inability of PC1 to inhibit E. coli most probably results from guaA not being under the control of a guanine riboswitch in E. coli. Bactericidal activity and specificity of PC1 To further characterize the riboswitch inhibitory action mechanism of PC1, S. aureus cells were grown in CAMHB in presence of various ligand concentrations. We obtained a PC1 dose-dependent growth inhibition response characterized by a 3 April 2010 | Volume 6 | Issue 4 | e1000865 Riboswitch Analogs Targeting Guanine Metabolism Figure 2. Guanine riboswitch agonists can be used to modulate gene expression. (A) Hydrogen bonds (left panel) and stacking interactions (right panel) formed between the bound guanine and the guanine riboswitch [26,27]. Oxygen, nitrogen, and phosphorus atoms are in red, blue, and yellow, respectively (left panel). Nucleotides follow the color scheme used in Figure 1B. Figures were prepared using PyMol (DeLano Scientific, San Francisco, CA, USA). (B) Molecular recognition features for guanine (G) and predicted ones for PC1 and PC2. Blue and red arrows represent hydrogen bond acceptors and donors, respectively. (C) In-line probing assays of the B. subtilis xpt riboswitch in the absence (2) or in the presence (+) of 1 mM guanine (G), and 1 mM or 10 mM for both PC1 and PC2. Sites of substantial ligand-induced protections (positions 49–54) are assigned on the right by a vertical bracket. Lanes NR, L and T1 correspond to molecules that were not reacted or that were partially digested by alkali or by RNase T1, respectively. Guanines are identified on the left as molecular weight markers. (D) The beta-galactosidase activity of a xpt-lacZ transcriptional fusion construct integrated in the genome of B. subtilis by recombination was assayed after 4 h of growth at 37uC in minimal medium in presence of the indicated ligand concentrations. Each experiment was performed three times and the average as well as standard deviations are shown. doi:10.1371/journal.ppat.1000865.g002 To analyze the PC1-mediated riboswitch inhibition on S. aureus gene expression, we performed a transcriptomic microarray analysis containing a selection of S. aureus genes involved in different cellular processes such as virulence, secretion, general stress responses, sensory/regulatory systems, antibiotic resistance, iron transport and general biosynthesis [30] (Figure 4D). Among the 468 genes analyzed, 72% were repressed by at least two folds when S. aureus was treated with PC1 where the 16S rRNA gene was the most repressed (Table S1). This result is consistent with a riboswitch-mediated guaA gene expression inhibition leading to GMP cellular depletion and RNA synthesis inhibition. This is supported by the low expression of the guanine riboswitch operon (xpt, pbuX, guaB and guaA) as well as the two DNA gyrase subunits MIC of 0.625 mg/mL (Figure 4A). PC2 was also used and its antibiotic activity was found to be less efficient than PC1, as observed in B. subtilis (Figure S2). When compared to known antibiotics, PC1 was found to have an extremely rapid bactericidal activity similar to ciprofloxacin, one of the most bactericidal antibiotics (Figure 4B). For instance, a 4 h treatment with PC1 led to 6.6760.58 and 5.4261.02 log reductions in CFU/mL compared to the untreated control for cultures of S. aureus ATCC 29213 and C. difficile CD6, respectively. When the same experiment was repeated by adding either GMP or AMP to the culture for 8 hours, bacterial growth was restored by a factor of 103 only in presence of GMP (Figure 4C), suggesting that PC1 growth inhibition activity is specific to guanine metabolism. PLoS Pathogens | www.plospathogens.org 4 April 2010 | Volume 6 | Issue 4 | e1000865 Riboswitch Analogs Targeting Guanine Metabolism Figure 3. S. aureus growth inhibition requires guaA to be under a riboswitch control. (A) Antibiograms performed on the S. aureus ATCC 29213 strain using 75 mg of PC1 (1), PC2 (2), 2-amino-4-hydroxy-6-methylpyrimidine (3), 9-methyl guanine (4), 5-bromo-6-methyl pyrimidine (5) and PBS as control (6). Chemical structures of PC1 and PC2 are shown in Figure 2B. (B) PC1 antibiograms using bacterial strains with guanine riboswitches. While PC1-insensitive strains do not have guaA under a riboswitch control (Bs: Bacillus subtilis, Ef: Enterococcus feacium, Lm: Listeria monocytogenes, STRpy: Streptococcus pyogenes, STRd: Streptococcus dysgalactiae and STRu: Streptococcus uberis), PC1-sensitive strains control the guaA gene expression via a riboswitch mechanism (Bh: Bacillus halodurans, STAa: S. aureus ATCC 29213, STAh: S. haemolyticus, SA228a: S. aureus resistant to betalactam, erythromycin, ciprofloxacin, gentamicin and tetracycline but susceptible to vancomycin, MRSA COL: methicilin resistant S. aureus COL, STAe: S. epidermidis, Cb: Clostridium botulinum, CD630: C. difficile strain 630, CD6: C. difficile representing the hypervirulent NAP1/027 strain). (C) Influence of GMP on PC1 bacterial growth inhibition. Spots from serial dilutions of S. aureus cultures in cation-adjusted Muller-Hinton broth (CAMHB) in absence or presence of 600 mg/mL PC1 or 600 mg/mL PC1 supplemented with 100 mM GMP. doi:10.1371/journal.ppat.1000865.g003 treated cells in a dose-dependent manner (data not shown) but its low solubility prevents full recovery at higher doses. It is also probable that GMP-related feedback inhibitory mechanisms were responsible for some of the gene repression observed (as in the case of the guanylate kinase gmk). Taken together, these results are consistent with PC1 mainly acting through a riboswitch inhibition mechanism that ultimately results in GMP cellular deprivation and S. aureus growth inhibition. (gyrA and gyrB), which were used as housekeeping gene controls (Figure 4D). Of all the monitored genes in the microarray analysis, only ahpF and ahpC, two genes involved in stress response mechanisms, were activated by the PC1 treatment. However, when S. aureus was treated with PC1 and GMP, the microarray data showed an expression profile in which only 21% of the genes surveyed were repressed. Whereas the housekeeping gyrase genes were no longer repressed, the expression of the guanine riboswitch operon was still reduced, consistent with PC1 binding the riboswitch operon and inhibiting gene expression. The other repressed genes mainly comprised those involved in virulence and cell wall synthesis suggesting that the GMP supplemented cells were still under stress [31], which is in agreement with the partial growth rescue observed in Figure 4C. GMP is able to rescue PC1PLoS Pathogens | www.plospathogens.org PC1 inhibits S. aureus growth in a murine model Because our data showed that the growth repression activity of PC1 is influenced by the presence of GMP, we decided to assess the bactericidal activity of PC1 in a murine mastitis model of S. aureus infection, which adequately represents the clinical context. 5 April 2010 | Volume 6 | Issue 4 | e1000865 Riboswitch Analogs Targeting Guanine Metabolism Figure 4. PC1 shows bactericidal activity through cellular GMP depletion. (A) Minimal inhibitory concentrations (MIC) determination of PC1 and PC2 on S. aureus strain ATCC 29213 in CAMHB. MIC values of 600 mg/mL and 5000 mg/mL were obtained for PC1 and PC2, respectively. (B) Bactericidal activity of PC1 and other known antibiotics against S. aureus as a function of time. For determination of the bactericidal effect of PC1, bacteria were inoculated at 105 CFU/mL in absence (cont) or presence of 0.5 mg/mL ciprofloxacin (cipro), 0.5 mg/mL erythromycin (erythro), 1 mg/mL vancomycin (vanco) and 600 mg/mL PC1. The concentration of each antibiotic corresponds to their MIC. (C) Bactericidal activity of PC1 against S. aureus as a function of time in absence (cont) or presence of 600 mg/mL PC1, and in presence of PC1 with 100 mM GMP or AMP. (D) Relative expression of S. aureus genes under the control of a guanine riboswitch when grown in presence of PC1 or PC1 with GMP. Results obtained in presence of PC1 are normalized using xpt gene expression. Bacteria were inoculated at 108 CFU/mL in CAMHB in absence or presence of 600 mg/mL PC1 with or without 100 mM GMP. Each experiment was performed three times and the average as well as standard deviations are shown. doi:10.1371/journal.ppat.1000865.g004 Indeed, in addition to morbid nosocomial infections caused by S. aureus, this bacterium is one of the major pathogen leading to bovine mastitis, which is the most frequent and costly disease for dairy producers with current antibiotic therapies usually failing to eliminate infections from dairy herds [32]. The antimicrobial activity of PC1 was therefore first tested on several S. aureus isolates from mastitic cows, some of which having persisting chronic infections (Figure 5A). A bactericidal effect of at least 4 orders of magnitude was observed after a 4 h treatment with PC1. Next, to ascertain that guaA was expressed in vivo and that this gene may be important during infection, we monitored the expression level of guaA by real-time PCR. When strain 1290 was grown either in broth culture in vitro or when it was directly isolated from the PLoS Pathogens | www.plospathogens.org mastitic milk of infected cows (M. Allard and F. Malouin, in preparation), very similar expression levels were found for guaA and the essential gene gyrB in both environments. This suggests that PC1 could have an impact on guaA expression in vivo and thus be used to treat S. aureus infections. The proof of concept for the therapeutic efficacy of PC1 was established in our murine model of S. aureus-induced mastitis [33]. At 4 h post-infection, different concentrations of PC1 were administered to infected mice that were sacrificed 6 h later (Figure 5B). When compared to mice that were not treated with PC1, viable bacterial counts in the mammary gland were drastically reduced in a dose-dependent manner. This strong therapeutic effect was highly comparable to what we observed 6 April 2010 | Volume 6 | Issue 4 | e1000865 Riboswitch Analogs Targeting Guanine Metabolism Figure 5. PC1 inhibits S. aureus clinical isolates in vitro and in vivo. (A) Fold reduction in viable counts (log10 CFU/mL) for the reference S. aureus strain ATCC 29213 and for selected bovine isolates after a 4 h exposure to PC1 as compared to the untreated culture. Newbould 305 (ATCC 29740) and SHY97-3906 are bovine isolates from typical mastitis cases and isolates 3, 557 and 1290 were from cows with persisting intra-mammary infections and chronic mastitis. (B) Bacterial counts (CFU) obtained from mice mammary glands 10 h post-infection with S. aureus. Mice mammary glands were treated (intra-mammary administration) 4 h after infection with PBS with or without PC1 at 10, 50 or 100 mg/gland. Each dot represents the CFU of each individual gland (n = 6–12) and the median value for each group is indicated by the bar. Statistical differences (P,0.05) between CFU recovered from treated and untreated animals are shown by asterisks (non-parametric Kruskal-Wallis ANOVA with Dunn’s post test). doi:10.1371/journal.ppat.1000865.g005 indicate that guaA is an important contributor to the survival of S. aureus during infection and that it can be used as an antibiotic target. The major limitation to validate an antibiotic that targets riboswitches is to evaluate the antibiotic specificity of action. In this particular case, PC1 is not a broad-spectrum antibacterial drug given that it does not target all bacteria containing guanine riboswitches, but only those in which guaA is under the control of a riboswitch. It is not excluded that other riboswitch-controlled genes may participate in the PC1-dependent bacterial growth inhibition (e.g., guaB), or that PC1 may bind other cellular targets, which alone or in combination with the riboswitch-controlled gene repression, would repress bacterial growth. Nevertheless, the restricted nature of growth inhibition likely indicates that PC1 inhibits bacterial growth through riboswitch binding and not via an alternative mechanism such as DNA incorporation (Figure 6). For instance, when performing antibiograms using the guanine analog 6-thioguanine, a general growth inhibition was observed in E. coli and S. aureus (Figure S7), consistent with its incorporation into DNA that perturbs the epigenetic pathway of gene regulation [40]. The selective antibacterial activity of PC1 toward S. aureus was also supported by the lack of apparent toxicity for mice treated with the experimental compound at concentrations as high as 100 mg/gland with no sign of discomfort including vocalizations, curved back, piloerection and hypothermia. There was also no apparent cytotoxicity upon histological observations of mammary tissues in PC1-treated mice compared to PBS-treated glands (Figure S8). Recently, the Breaker group used purine derivatives modified in position 2 or 4 to target guanine riboswitches and found three molecules that inhibited bacterial growth [15]. Among these molecules, only one was able to repress the expression of a guanine riboswitch-controlled reporter gene suggesting that at least two molecules inhibited cell growth through a different mechanism of action. It is possible that the mode of action of these two molecules involves nucleic acids incorporation following ribosylation at with known antibiotics. For example, amoxicillin decreased the bacterial load in the mammary gland to a log10 median value of 3.97 CFU/g of gland at a dose of 50 mg/gland. Noteworthy, a dose of 50 mg of amoxicillin would represent 100xMIC/g of gland, whereas a similar dose would only represent a twelfth of the MIC/ g of gland for PC1. This result is consistent with the idea that PC1 is most efficient in the mammary gland environment suggesting that the microaerobic condition (i.e., low oxidative environment) of the mastitic milk[34] helps PC1 therapeutic efficacy. Consistent with these results, we found that the potency of PC1 was significantly increased by preventing its oxidation using a reductive agent such as DTT in susceptibility tests in vitro (Figure S5). Discussion Despite previous large scale screen data suggesting that guaA is not essential for S. aureus growth [23,35] in relatively rich media, we show here that blocking guaA expression can lead to bactericidal activity in various bacterial species. In support of guaA for cell viability, it has been recently reported that mutations occurring in guaA prevent Streptococcus suis [36] and Salmonella thyphimurium [37] from properly infecting porcine and murine models, respectively, suggesting that GMP bioavailability may be reduced during host infection and that guaA is likely to be crucial for bacterial infection in mammals. Thus, together with studies showing the importance of guaA for bacterial growth in urine or blood [38,39], our data suggest that mammalian infection sites may significantly differ in their nutrient compositions from those used in large scale screens [23,35], and that care should be taken when assessing the ‘‘essentiality’’ of a gene. Furthermore, when assessing whether S. aureus could develop resistance to PC1, no resistant bacteria were obtained after more than 30 passages suggesting that maintaining a functional guaA-regulated riboswitch is a vital process (Figure S6). Taken together, the demonstration that guaA expression is normally maintained in S. aureus grown in vivo and the strong therapeutic effect resulting from PC1 treatment PLoS Pathogens | www.plospathogens.org 7 April 2010 | Volume 6 | Issue 4 | e1000865 Riboswitch Analogs Targeting Guanine Metabolism Figure 6. Scheme representing the action mechanism of PC1 on S. aureus guanine pathway. Genes highlighted in blue correspond to those of the operon xpt-pbuX-guaB-guaA that is controlled by the guanine riboswitch. Red lines indicate genes that are inhibited by PC1 via its binding to the guanine riboswitch. Of these four inhibited genes, guaA and guaB are known to be critical for guanine nucleotide biosynthesis [22,23,36,37,38,39] and their inhibition very likely lead to the repression of GMP synthesis and most probably of RNA and DNA production. doi:10.1371/journal.ppat.1000865.g006 Canadian Council on Animal Care were respected during all the procedures. position 9 of the purine analog, as demonstrated for 6-thioguanine. It is also interesting to mention that the antibiotic activity was only observed using B. subtilis strains cultivated in minimal medium whereas no growth inhibition was detected in rich media. In our study, we selected guanine riboswitch ligands that cannot be ribosylated to prevent alternative modes of action, and favored a pyrimidine compound (PC1) that retained most of the key functional groups. By testing various bacterial species, we observed that the bactericidal activity of PC1 was only seen against bacteria using a guanine riboswitch to control guaA expression, and this even when bacteria were grown in a rich medium. This demonstrates that analog binding to riboswitch aptamers is not the only determinant to achieve selective and efficient bactericidal effects. This study shows for the first time that antibiotics targeting riboswitches may be efficient to kill bacterial pathogens in vitro as well as in mammalian infection models. We found here that the selective bacterial killing of PC1 is only achieved when guaA, a GMP synthetase, is under the control of the riboswitch and when the antimicrobial agent cannot be ribosylated. The narrow spectrum of activity we demonstrated here for PC1 is very interesting since two of the target bacteria, S. aureus and C. difficile, are among the most problematic nosocomial pathogens. The spread of MDR in those bacterial species also stresses the importance to develop new antibiotics that avoid current mechanisms of resistance. The use of narrow spectrum drugs should be encouraged whenever possible to reduce any selective pressure for resistance in non-targeted bacteria. Here, we also showed that the development of resistance toward PC1 is likely to be infrequent. Reagents 4-hydroxy-2,5,6-triaminopyrimidine (PC1), 2,4-diamino-6-hydroxypirimidine (PC2) and 9-methylguanine were purchased from Fluka. 2-amino-5-bromo-6-methyl-4-pyrimidol and 2-amino-4hydroxy-6-methylpyrimidine were purchased from Aldrich. Strategy of ligand selection Our ligand selection took into account guanine binding requirements [25] and crystal structure interactions [21,26,27]. Planar molecules were selected to preserve stacking interactions with adenines 21 and 52 in the guanine aptamer binding site. One of our important selection criteria was to avoid the presence of functional groups that could serve as a ribosylation site in purine or pyrimidine analogs that would allow subsequent nucleic acid incorporation and non-specific antibiotic effect. Successfully identified molecules were drawn using Chem3D Pro (CambridgeSoft) and docked onto the guanine aptamer crystal structure (PDB 1U8D). The in silico procedure was important to validate aptamerligand interactions and to avoid sterical obstructions that would perturb ligand binding. Transcription of RNA For the production of guanine riboswitch aptamers, DNA templates were prepared from partial duplexes and transcribed using T7 RNA polymerase as previously described [28]. The aptamer sequences used in this study are based on the genomic sequence to which a GCG sequence is added to the 59 side to allow high transcription yield and to minimize the 59 heterogeneity [28]. Methods Ethics statement In-line probing assays The institutional ethics committee on animal experimentation of the Faculté des sciences of the Université de Sherbrooke (QC, Canada) approved these experiments and the guidelines of the [59-32P] RNA molecules were incubated for 96 h at 25uC in 50 mM Tris-HCl buffer, pH 8.5, 20 mM MgCl2 and 100 mM KCl in absence or in presence of indicated ligand concentrations. PLoS Pathogens | www.plospathogens.org 8 April 2010 | Volume 6 | Issue 4 | e1000865 Riboswitch Analogs Targeting Guanine Metabolism 4 h after infection with PBS or PBS with 10, 50 and 100 mg/gland of PC1 and mice were sacrified 6 h later for mammary gland sampling and homogenization. The tissues used for CFU counts were homogenized in 2 mL of PBS and the bacterial content was evaluated by serial logarithmic dilutions on agar. The detection limit was 100 CFU/g of gland. The reactions were stopped with a 97% formamide solution containing 10 mM EDTA and samples were purified by electrophoresis in 10% polyacrylamide gels (acrylamide:bisacrylamide; 19:1) containing 8 M urea. Gel were dried and exposed to Phosphor Imager screens. beta-galactosidase assays Supporting Information Regulation of the beta-galactosidase reporter gene expression in presence of PC1 or PC2 was determined using an xpt-lacZ transcriptional fusion construct integrated in the genome of B. subtilis by recombination. The beta-galactosidase activity was measured after 4 h of growth at 37uC in minimal medium in absence or presence of the indicated ligand concentrations [41]. Figure S1 Guanine and guanine-related metabolic pathways in different bacterial species. Guanine riboswitch-regulated genes are shown using a color scheme where Bacillus subtilis, Clostridium difficile and Staphylococcus aureus are indicated in green, orange and blue, respectively. Dashed lines represent metabolite membrane transporters. The synthesis of GMP is highly dependent upon guaA and guaB, which respectively encodes a GMP synthetase and an IMP dehydrogenase. While guaB has been shown to be essential in B. subtilis [23], mutations in guaA have been reported to make cells auxotroph for guanine [22]. The importance of guaA and guaB has also been observed during bacterial infections in murine and porcine models [36,37]. Thus, guaA and guaB encode two enzymes critical for guanine nucleotide biosynthesis and reduction of their expression via riboswitch action is very likely to produce bacterial growth inhibition. Found at: doi:10.1371/journal.ppat.1000865.s001 (0.32 MB TIF) Antibiogram assays Bacteria were inoculated at 105 CFU/mL in melted MullerHinton agar. After agar medium was solidified, six wells of 4 mm in diameter were made and filled with 10 mL of the tested molecules (5 mg/mL). Plates were incubated for 16 h at 37uC. Antibiotic minimal inhibitory concentrations The minimal inhibitory concentration (MIC) of PC1 and PC2 against S. aureus strain ATCC 29213 was determined using a microdilution method in 96-well plates [30]. Bacteria were inoculated at 105 CFU/mL and incubated at 37uC for 24 h in cation-adjusted Muller-Hinton broth (CAMHB). Bacterial growth was detected by measuring the OD at 595 nm on a microplate reader. Figure S2 B. subtilis growth is inhibited by PC1 acting as a guanine riboswitch antibiotic. (A) Minimal inhibitory concentrations (MIC) of PC1 and PC2 against B. subtilis. The MICs were determined using a microdilution method in 96-well plates. Bacteria were inoculated at 105 CFU/mL in cation-adjusted Muller-Hinton broth (CAMHB) or in minimal media (MM) and incubated at 37uC for 24 h. MIC values of 1250 mg/mL and 5000 mg/mL were respectively obtained for PC1 and PC2 in MM, but no growth inhibition was observed in CAMHB. (B) PC1 modulates riboswitch activity in CAMHB in a dose-dependent manner. Beta-galactosidase activity of a xpt riboswitch-lacZ transcriptional fusion construct integrated by recombination in the genome of B. subtilis was assayed after 4 h of growth at 37uC in CAMHB in the absence or presence of the indicated ligand concentrations. This confirms that PC1 can modulate riboswitch activity in a relatively rich media such as CAMHB and not only in a minimal media (Figure 2D), where growth inhibition is observed. Each experiment was performed three times and the average as well as the SD are shown. Found at: doi:10.1371/journal.ppat.1000865.s002 (0.39 MB TIF) Antibiotic bactericidal activity Time-kill experiments were performed for the determination of the bactericidal effect of test antibiotics. Bacteria were inoculated at 105 CFU/mL in CAMHB in absence or presence of the antibiotic at its MIC with or without 100 mM GMP. Bacterial permeability to GMP was increased by adding 0.002% Triton X100. At several time points, bacteria were sampled and serially diluted before spreading on tryptic soya agar (TSA) plates for CFU determinations. Plates were incubated for 24 h at 37uC. Transcriptomic microarray Bacteria were inoculated at 108 CFU/mL in CAMHB in absence or presence of 600 mg/mL PC1 or 600 mg/mL PC1 supplemented with 100 mM GMP. After 30 min of growth, RNA was extracted and 2.5 mg of RNA were submitted to reverse transcription to generate fluorescent probes through an aminoallyl cDNA labeling procedure before being hybridized on the microarray [30]. Figure S3 Schematic representation of the S. aureus xpt-pbuXguaB-guaA operon controlled by the guanine riboswitch. (A) The predicted number of nucleotides for each intergenic region is shown. (B) RT-PCR of intergenic regions performed using total RNA. Note that an amplification product is obtained in all cases indicating that xpt, pbuX, guaB and guaA are included in an operon controlled by the guanine riboswitch. RNA was extracted from lysate using a Quiagen RNeasy kit and treated with DNase I in presence of RNase inhibitors. Following this, 1 mg was used for reverse transcription with 200 units of SuperScript II (Invitrogen), using 100 pmoles of a DNA oligonucleotide used as a primer. The reaction was performed at 42uC for 1 h and used in a PCR reaction using an appropriate forward primer. Lane L represents a 100 bp ladder with the number of base pairs indicated for each band. Lanes xpt-pbuX, pbuX-guaB and guaB-guaA represent PCR reactions amplifying corresponding intergenic regions. Found at: doi:10.1371/journal.ppat.1000865.s003 (1.02 MB TIF) Murine mastitis model Experimental conditions used here for the mastitis model were previously optimized for S. aureus Newbould and antibiotic treatment [42]. CD-1 lactating mice (Charles River, St. Constant, Canada) were used 12 to 14 days after offspring birth and typically weighed 35 to 40 g. Pups were removed 1 h before bacterial inoculation of mammary glands and a mixture of ketamine/ xylazine at 87 and 13 mg/kg of weight, respectively, was used for anesthesia of lactating mice. A 100 ml syringe with a 33-gauge blunt needle was used to inoculate both L4 (on the left) and R4 (on the right) abdominal mammary glands. These large glands constitute the fourth pair found from head to tail. Each udder canal was exposed by a small cut at the near end of the teat under a binocular and 100 mL of bacterial suspension (1 CFU/mL) was injected through the orifice. Mice mammary glands were treated PLoS Pathogens | www.plospathogens.org 9 April 2010 | Volume 6 | Issue 4 | e1000865 Riboswitch Analogs Targeting Guanine Metabolism Figure S4 PC1 does not inhibit the growth of the Gram negative bacterium E. coli, which does not naturally contain guanine riboswitches. The MIC of PC1 and other known antibiotics against E. coli ATCC 35695 were determined using a broth microdilution method. As expected, PC1 does not show any antibiotic activity toward E. coli ATCC 35695, most probably because of the absence of a guanine riboswitch. However, to exclude the possibility that the lack of inhibitory activity is due to a poor cell penetration of PC1 into E. coli or to an active efflux of this compound out of the cells, we also tested PC1 activity against two isogenic E. coli mutants. While E. coli ATCC 35695 is a standard strain, AcrAB is deficient for the multidrug efflux pump AcrAB [43] and Imp has an increased membrane permeability [44]. Our results show that there is still no inhibitory activity of PC1 against any of the mutants. Please note that the Imp increased membrane permeability is confirmed by the antibiotic activity of vancomycin, which is a large glycopeptide molecule for which Gram negative bacteria are normally impermeable. Besides, the reduced efflux activity of AcrAB is verified from the ability of erythromycin to inhibit bacterial growth. Erythromycin is also able to inhibit Imp bacterial growth due to the increased membrane permeability. These results indicate that the lack of PC1 antibiotic activity toward E. coli is not due to its active efflux by the bacteria or its inability to pass through the cell membrane. All concentrations are in mg/mL and the chemical structure for PC1 is shown in Figure 2B. Found at: doi:10.1371/journal.ppat.1000865.s004 (0.12 MB TIF) antibiotics, ciprofloxacin and rifampicin, were added to the histogram. High level resistance to ciprofloxacin and rifampicin is rapidly selected (within 5 daily passages) in S. aureus. Such rapid development of resistance for the traditional drugs is consistent with the selection of known single point mutations each able to provide a decrease in drug affinity for the bacterial cell target. There are at least 2 known point mutations in GyrA conferring resistance to ciprofloxacin in addition to possible over-expression of the NorA efflux pump system also occurring through mutations (at least 3 possible mutations) [46] and at least 17 possible different mutations in RpoB enabling resistance to rifampicin have been documented [47]. The absence of resistance observed in presence of PC1 is probably because reestablishing guaA gene expression in the presence of PC1 requires multiple mutational steps thus reducing the frequency of resistance development and/or that maintaining a functional riboswitch is a vital process that does not allow bacteria to bypass PC1 antibiotic action. PC1 experiments were performed three times and the average as well as the SD are shown. Found at: doi:10.1371/journal.ppat.1000865.s006 (0.46 MB TIF) Figure S7 Antibiograms performed on strains of E. coli ATCC 35695 and Methicilin Resistant S. aureus strain COL. E. coli ATCC 35695 (A) and S. aureus strain COL (B) were grown in absence (well #1) or in presence of 7.5 mg (well #2) or 15 mg 6-thioguanine (well #3). Please note that 6-thioguanine is able to be ribosylated (C) and incorporated in DNA[40], which probably explains its riboswitch-independent antibiotic activity toward both E. coli and S. aureus. Found at: doi:10.1371/journal.ppat.1000865.s007 (3.01 MB TIF) DTT increases the antibiotic activity of PC1 probably by preventing its oxidative self-condensation. (A) MICs were determined using a microdilution method in 96-well microplates in absence or presence of DTT at the concentrations shown. Bacteria were inoculated at 105 CFU/mL and incubated at 37uC for 24 h in CAMHB. Please note that the MIC is decreased by a factor of ,40x in the presence of 0.05% DTT. At low PC1 concentrations, it can be observed that DTT does not inhibit bacterial growth by itself. (B) Schematic representation of the probable oxidative selfcondensation of PC1 that could be prevented by DTT. Previous studies have shown that 4,5-diaminopyrimidines can produce insoluble and deeply-colored orange substances such as pyrimido[5,4-g]- and pyrimido[4,5-g]pteridines by oxidative selfcondensation [45]. Given that PC1 is structurally very similar to 4,5-diaminopyrimidines and that it produces an orange precipitate over time, it is likely that PC1 also self-condenses by air oxidation. This is consistent with the observation that a reductive agent such as DTT can slow down the formation of the precipitate (data not shown). Found at: doi:10.1371/journal.ppat.1000865.s005 (0.37 MB TIF) Figure S5 Figure S8 Histology of mice mammary glands treated with PC1. Mice were either injected with (A) 100 mL PBS or with (B) 100 mL PBS containing 100 mg of PC1. The treatment was allowed for 6 h and mammary glands were excised, fixed in 4% formaldehyde for 24 h at room temperature and embedded in paraffin wax. Hematoxylin-eosin staining was done on sections of 5 mm thickness. Magnifications on the pictures are 2006. The histology study reveals that there is no observable damage to the gland following PC1 injection. Found at: doi:10.1371/journal.ppat.1000865.s008 (5.90 MB TIF) Table S1 Transcriptomic microarray showing the relative expression of S. aureus genes as a function of PC1 and GMP. Found at: doi:10.1371/journal.ppat.1000865.s009 (0.08 MB XLS) Acknowledgments We thank members of the Lafontaine laboratory and Eric Marsault for helpful discussions and Dr. Alain Lavigueur for critical reading of the manuscript and Gilles Grondin for the histological studies. Figure S6 Serial passages in the presence of sub-inhibitory concentrations of test antibiotics demonstrating the inability of S. aureus to develop resistance toward PC1. The MIC of test compounds against S. aureus strain ATCC 29213 recovered from broth cultures containing sub-inhibitory concentrations of antibiotics (0.25X of MIC) was determined every 5 passages for up to 30 passages. As a comparison, results obtained with two known Author Contributions Conceived and designed the experiments: JM LCF FM DAL. 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J Antimicrob Chemother 47: 153–156. 11 April 2010 | Volume 6 | Issue 4 | e1000865 SUPPLEMENTARY TABLE 1 Experimental conditions (Test vs control Cy3 vs Cy5, PC1 600!g/mL alone or with 100 !M GMP, incubation time 30min, S aureus ATCC29213 inoculation 10e8 CFU/mL) ID SACOL Gene General Function Gene Description 1254 1159 1160 672 1158 272 18 144 149 2025 2026 2218 1371 2242 2213 1449 145 1734 2340 2688 140 1908 150 2694 936 1490 142 1396 20 938 2363 2271 1194 1026 1373 1195 1079 2347 2270 457 141 1448 1385 1173 276 22 950 2718 935 2272 700 195 1963 679 306 148 494 689 1742 2023 1320 1196 766 860 762 151 701 1812 5 136 2287 2577 799 690 96 1066 2054 992 2362 163 765 2413 154 277 699 2712 95 1637 743 703 2394 1272 2067 2293 86 758 541 127 Control Energy metabolism Energy metabolism Virulence regulator Energy metabolism Stress response Nucleic acid metabolism Virulence capsule Virulence capsule Virulence regulator Virulence regulator Nucleic acid metabolism Nucleic acid metabolism Nucleic acid metabolism Nucleic acid metabolism Energy metabolism Virulence capsule Energy metabolism Membrane transport Virulence biofilm Virulence capsule Energy metabolism Virulence capsule Virulence Cell wall/Cell division Cell wall/Cell division Virulence capsule Cell wall/Cell division Regulation Cell wall/Cell division Energy metabolism Membrane transport Cell wall/Cell division Stress response Stress response Cell wall/Cell division Nucleic acid metabolism Membrane transport Membrane transport Stress response Virulence capsule Energy metabolism Energy metabolism Virulence enzyme/toxin Virulence enzyme/toxin Regulation Membrane transport Membrane transport Cell wall/Cell division Membrane transport Membrane transport Membrane transport Membrane transport Membrane transport Membrane transport Virulence capsule Energy metabolism Membrane transport Energy metabolism Virulence regulator Energy metabolism Cell wall/Cell division Virulence regulator Virulence enzyme/toxin Virulence enzyme/toxin Virulence capsule Nucleic acid metabolism Virulence regulator Nucleic acid metabolism Virulence capsule Virulence regulator Stress response Membrane transport Membrane transport Virulence regulator Cell wall/Cell division Stress response Membrane transport Energy metabolism Membrane transport Virulence regulator Membrane transport Energy metabolism Stress response Cell wall/Cell division Stress response Virulence adhesin Stress response Cell wall/Cell division Nucleic acid metabolism Energy metabolism Membrane transport Membrane transport Stress response Membrane transport Energy metabolism Virulence regulator Membrane transport Ribosomal RNA 16S Succinate dehydrogenase Succinate dehydrogenase Staphylococcal accessory regulator A Succinate dehydrogenase Hypothetical protein Adenylsuccinate synthetase Capsular polysaccharide biosynthesis Capsular polysaccharide biosynthesis Accessory gene regulator protein C Accessory gene regulator protein A Adenylate kinase GMP reductase Xanthine/uracil permease DNA-directed RNA polymerase, alpha subunit 2-oxoglutarate dehydrogenase Capsular polysaccharide biosynthesis Glyceraldehyde 3P deshydrogenase Sodium/glutamate symporter Intercellular adhesion regulator Capsular polysaccharide biosynthesis Fumarate hydratase Capsular polysaccharide biosynthesis Triacylglycerol lipase DltB protein, lipoteichoic acid synthesis Penicillin-binding protein 2 Capsular polysaccharide biosynthesis, UDP-N-acetylglucosamine 2-epimerase Membrane protein, putative (methicillin/oxacillin resistance-related protein) Sensory box histidine kinase, two component system DltD protein, lipoteichoic acid synthesis L-lactate permease Molybdenum ABC transporter, permease protein Penicillin-binding protein 1 Conserved hypothetical protein (toxic anion resistance) Conserved protein Phospho-N-acetylmuramoyl-pentapeptide-transferase Amidophosphoribosyltransferase Drug resistance transporter, EmrB/QacA Molybdenum ABC transporter, ATP-binding protein Conserved protein Capsular polysaccharide biosynthesis 2-oxoglutarate dehydrogenase Aconitate hydratase Alpha-hemolysin Diarrhoeal toxin Unknown function, two component system Na/H antiporter Anion transporter family protein D-alanine-activating enzyme / D-alanine-D-alanyl carrier protein ligase Molybdenum ABC transporter, molybdenum-binding protein ABC transporter Maltose ABC transporter, permease protein Proline permease Na+/H+ antiporter component, MnhA component putative ABC transporter, ATP-binding protein Capsular polysaccharide biosynthesis, galactosyltransferase NADH dehydrogenase I, F subunit Manganese ABC transporter, ATP-binding protein Citrate synthase Accessory gene regulator protein B Glycerol kinase UDP-N-acetylmuramoylalanine-D-glutamate ligase Two-component response regulator systems involved in virulence Thermonuclease Probable hemolysin Capsular polysaccharide biosynthesis, UDP-N-acetylglucosamine 2-epimerase Nucleoside permease NupC putative Regulator of toxin, Rot DNA gyrase, B subunit Capsular polysaccharide biosynthesis Staphylococcal accessory regulator R Dehydrosqualene synthase Possible ferrichrome ABC transporter, lipoprotein Manganese ABC transporter, ATP-binding protein Staphylococcal accessory regulator S involved in virulence Autolysis and methicillin resistant-related protein RNA polymerase sigma factor B Oligopeptide ABC transporter, permease protein Malate:quinone oxidoreductase Drug transporter, putative Two-component response regulator systems involved in virulence Drug resistance transporter, EmrB/QacA subfamily Aldehyde dehydrogenase Hypothetical protein Penicillin-binding protein 4 drp35 protein, induced by detergents that perturb membrane integrity Immunoglobulin G binding protein A Chaperone protein bacitracin resistance protein Conserved hypothetical protein Nitrate reductase Dipeptide transport operon repressor Potassium-transporting ATPase, B subunit NAD/NADP octopine/nopaline dehydrogenase, Csb22 Drug transporter, putative 1-phosphofructokinase, PTS system StageV sporulation protein G homolog Phosphonate ABC transporter, ATP-binding protein ARNr16S sdhA sdhB sarA sdhC purA cap5I cap5N agrC agrA adk guaC rpoA sucA cap5J gapA2 gltS icaR cap5E fumC cap5O lip dltB pbp2 cap5G fmtC yycG dltD pbpA mraY purF Csb12 cap5F sucB hla yukA yycl mnhF dltA putP cap5M nuoF mntB gltA agrB glpK murD saeR nuc cap5P rot gyrB cap5A sarR crtM sstD mntA sarS fmt sigB oppC mqo saeS pbp4 drp35 spa dnaK bacA narH codY kdpB Csb22 fruK spoVG PC1 conditions PC1+GMP Expression mean Expression mean (log2 test vs control) (log2 test vs control) -2,276315179 0,124166548 -2,266763775 0,209533678 -2,205044554 0,100398981 -2,139859562 -1,153239324 -1,971835092 0,249345701 -1,907698397 -1,606319039 -1,846259849 0,04037557 -1,815654842 -2,889059757 -1,784005645 -2,821112981 -1,777667869 -1,382458034 -1,774362501 -1,490145879 -1,76442508 0,589716533 -1,760380429 -2,238302653 -1,751125689 -2,552369633 -1,741631903 0,447415688 -1,71330638 -0,192127347 -1,688078618 -2,441351897 -1,660960927 -0,140416383 -1,654312419 0,171927784 -1,645879568 -1,451540589 -1,641499835 -3,194995211 -1,623326637 -0,820555424 -1,609147137 -2,661417701 -1,578259669 -1,2430572 -1,570719399 -0,449325579 -1,552597598 0,220384095 -1,547451826 -2,918010858 -1,543381403 -1,35858805 -1,521387703 -0,862262076 -1,51945954 -0,338891055 -1,503772748 3,021915309 -1,503506466 -1,023331258 -1,491845527 -0,378792682 -1,487932833 -0,020179165 -1,487592464 -1,758507247 -1,477075831 -0,325974743 -1,471858151 -2,874759702 -1,471456632 -0,080242866 -1,46874895 -1,013976036 -1,462406194 0,632822 -1,460616418 -3,008400868 -1,456317648 -0,443859366 -1,45481507 0,237683636 -1,444455408 -0,918134001 -1,405294647 -1,140579126 -1,404933905 -0,857612223 -1,394796477 -0,43906089 -1,387438492 -0,962162731 -1,386853591 -0,815893941 -1,383249994 -0,93269218 -1,378179532 -1,354195229 -1,369364431 -0,401189163 -1,36871877 -0,595243894 -1,365938987 -4,194934517 -1,346763512 -0,378267351 -1,344548654 -1,98088575 -1,332516284 -0,221305303 -1,327342135 -6,616648898 -1,322817966 0,082200023 -1,301299694 -1,169153088 -1,298185926 0,326744321 -1,292737977 -0,40174189 -1,29208997 -0,684152776 -1,290659635 -1,861763669 -1,284205984 -1,247008645 -1,27167011 -2,605659184 -1,268461061 -1,416809677 -1,266421525 -0,6142797 -1,266320558 -0,239525421 -1,265093242 -3,141989055 -1,264155803 -1,029776045 -1,260161111 -0,348047043 -1,256011572 0,444778626 -1,251266351 -6,496446924 -1,244756232 -0,697355349 -1,240243621 -0,750287521 -1,240003729 -0,407054575 -1,239779053 -1,282216578 -1,222197975 0,016302743 -1,221760292 -0,899877807 -1,20563678 -0,440077857 -1,197896449 -1,190868881 -1,19078249 -0,06355122 -1,189078032 -1,002982146 -1,18858172 -1,0959026 -1,187461263 0,021739335 -1,183902588 -2,689550181 -1,181735612 0,130788454 -1,18064911 -0,441889572 -1,18004176 -1,239969327 -1,180035641 -0,324282691 -1,17453011 0,147534267 -1,172527125 -2,091974307 -1,170002475 -1,246505442 -1,163177463 -0,527297627 -1,162133852 0,233116892 -1,161574661 -0,299397213 -1,156375005 -0,463504769 1197 2266 2125 1609 1639 1114 1970 754 2291 2352 809 2582 2430 1427 2022 1664 1193 1328 1657 1103 2576 1790 2055 1638 2068 2392 1994 1198 1912 608 1105 1262 968 1898 1414 78 1680 1142 2708 1184 1535 2691 2572 620 209 121 757 29 2006 459 2656 1534 1199 2151 248 969 2134 1613 1424 1825 2066 660 1715 695 2581 2004 1357 2016 2505 1055 162 2566 1741 1969 1021 2712 810 544 1944 1777 2676 2342 98 2325 245 2399 1085 2422 1398 1421 246 1468 1743 1716 1054 2659 2421 2564 416 2057 688 1057 609 712 2431 divlB moeA pbp3 hrcA sspB2 norA tcaA hld ftsL glnR pdhB crtN murC rsbW grpE kdpA narl ftsA Csb33 sdrC pdhD sucC spsA cbf1 plc csbD/Csb8 isdD ext srrA icaB copA proP coa deoD pbuX arcB srrB ftsZ femD lrgB spsB kdpC adh hemB tagG groEL sasG sspC mmpL icd purB drp35 prsA htrA sasA/sdrD corA sirB lyt nirR hlgB lytR hemD menB aur hlgC feoB rsbU mntC sspA sdrD Cell wall/Cell division Membrane transport Stress response Cell wall/Cell division Stress response Membrane transport Virulence Membrane transport Virulence Cell wall/Cell division Regulation Cell wall/Cell division Membrane transport Membrane transport Virulence enzyme/toxin Stress response Cell wall/Cell division Cell wall/Cell division Virulence enzyme/toxin Energy metabolism Stress response Cell wall/Cell division Stress response Stress response Membrane transport Energy metabolism Membrane transport Cell wall/Cell division Stress response Virulence adhesin Energy metabolism Energy metabolism Membrane transport Nucleic acid metabolism Membrane transport Virulence enzyme/toxin Stress response Membrane transport Membrane transport Virulence enzyme/toxin Virulence regulator Virulence biofilm Membrane transport Stress response Virulence enzyme/toxin Nucleic acid metabolism Virulence regulator Energy metabolism Virulence enzyme/toxin Nucleic acid metabolism Energy metabolism Virulence regulator Cell wall/Cell division Cell wall /Cell division Cell wall/Cell division Membrane transport Membrane transport Membrane transport Membrane transport Cell wall/Cell division Membrane transport Energy metabolism Energy metabolism Cell wall/Cell division Stress response Virulence enzyme/toxin Virulence enzyme/toxin Stress response Virulence adhesin Virulence enzyme/toxin Energy metabolism Membrane transport Energy metabolism Nucleic acid metabolism Membrane transport Stress response Cell wall/Cell division Energy metabolism Stress response Stress response Virulence adhesin Membrane transport Membrane transport Virulence regulator Cell wall/Cell division Energy metabolism Membrane transport Virulence enzyme/toxin Cell wall/Cell division Membrane transport Cell wall/Cell division Membrane transport Membrane transport Energy metabolism Energy metabolism Virulence enzyme/toxin Virulence enzyme/toxin Membrane transport Stress response Stress response Membrane transport Virulence enzyme/toxin Virulence adhesin Lipid metabolism Membrane transport Cell division protein Molybdopterin biosynthesis MoeA protein, putative Peptidase, M20/M25/M40 family Penicillin-binding protein 3 Heat-inductible transcription repressor Mn2+/Fe2+ transporter, NRAMP family Cysteine protease Drug resistance transporter SsaA-like protein TcaA protein, cell wall, teicoplanin and MRSA associated resistance GGDEF Domain protein, c-di-GMP membrane protein, similar to acyltransferase in N315 ABC transporter, permease/ATP-binding protein ABC transporter, ATP-binding protein, not identified Delta-hemolysin, RNAIII Conserved hypothetical protein TIGR00370, homolog mazE E.coli FtsL protein, putative Glutamine synthetase repressor Probable enterotoxin Pyruvate dehydrogenase component, beta subunit Squalene desaturase UDP-N-acetylmuramate--alanine ligase Anti-sigma factor B Adaptations to atypical conditions Potassium-transporting ATPase, A subunit Respiratory nitrate reductase, gamma subunit ABC transporter, ATP-binding protein, not identified Cell division protein FtsA Glucosamine-6-phosphate isomerase, putative, Csb33 Cell adhesion Pyruvate dehydrogenase component, lipoamide dehydrogenase Succinyl-CoA synthase Homolog N315 sa 0825, type-I signal peptidase, spsA Cmp-binding-factor 1 (mobile and extrachromosomal element functions) ABC transporter, ATP-binding protein, not identified Phospholipase, Phosphatidylinositol phosphodiesterase Conserved protein Heme-iron transport, heme-iron permease system ABC transporter, ATP-binding protein Exfoliatine Toxin production Two-component response regulator systems Biosynthesis and degradation of surface polysaccharides and lipopolysaccharides Copper translocating ATPase Osmoprotectant proline transporter Coagulase Purine nucleoside phosphorylase Transcriptional regulator, DeoR family, PTS system Biosynthesis of cofactors, prosthetic groups, and carriers Aerolysin/leukocidin family Xanthine permease Aspartate / ornithine carbamoyltransferase Two-component response regulator systems Cell division protein FtsZ FemD protein, phosphoglucosamine mutase Holin-like LrgB protein, murein hydrolase Homolog N315 sa 0826 type-I signal peptidase 1B, spsB Conserved hypothetical protein ABC transporter, ATP-binding protein, not identified Phosphate ABC transporter N-acetylmuramoyl-L-alanine amidase Potassium-transporting ATPase, C subunit Alcool deshydrogenase, Zn containing Delta-aminolevulinic acid dehydratase Teichoic acid ABC transporter protein, putative Homolog to staphyloxanthine biosynthesis proteins Leukocidin Thermonuclease Chaperonine Homolog to Pls, a putative MRSA adhesin Cysteine protease SspC Formate dehydrogenase, NAD-dependent MmpL efflux pump putative Isocitrate dehydrogenase, NADP-dependent Adenylsuccinate lyase Possible multi-drug efflux protein drp35 protein, induced by detergents that perturb membrane integrity Undecaprenyl-phosphate alpha-N-acetylglucosaminyltransferase Ribose-phosphate pyrophosphokinase hypothetical protein Serine protease HtrA Protein with cell sorting signals Magnesium and cobalt transport protein CorA Possible staphylobactin ABC transporter transcriptional regulator, LysR family Two-component response regulator systems Transcriptional regulator NirR ABC transporter, ATP-binding protein, not identified Cellular processes: Toxin production and resistance Transcriptional regulator, homolog peptide methionine sulfoxide reductase regulator MsrR Phosphate ABC transporter Response regulator Membrane protein Aminoacid permease Uroporphyrinogen-III synthase HemD Enoyl-CoA hydratase/isomerase family protein Aureolysin, zinc metalloproteinase Gamma hemolysin, component C FeoB, Possible iron-tranport protein Conserved domain protein Sigma factor B regulator protein Manganese ABC transporter, ATP-binding protein Serine protease V8 Putative cell-surface protein, unknown ligand Lipase/esterase ABC transporter, ATP binding/permease with Fur box -1,155378858 -1,154041159 -1,152830291 -1,148274663 -1,145810131 -1,141526507 -1,137845849 -1,131170203 -1,126525981 -1,125399717 -1,119435983 -1,117380663 -1,109815973 -1,106538915 -1,105256208 -1,102788025 -1,099762321 -1,098983521 -1,098939551 -1,095181631 -1,095082539 -1,092782922 -1,091214578 -1,090803584 -1,087411847 -1,086633388 -1,084961854 -1,084442746 -1,079173447 -1,078454614 -1,078257133 -1,076701208 -1,074700261 -1,074615504 -1,070706657 -1,069319769 -1,060242128 -1,05800892 -1,056143216 -1,055190699 -1,055004122 -1,054776924 -1,052270206 -1,046660509 -1,044740464 -1,044097442 -1,042779709 -1,04197603 -1,038085784 -1,036446371 -1,035365026 -1,029851824 -1,026784746 -1,021836199 -1,020516577 -1,020250282 -1,019000782 -1,018581883 -1,017094716 -1,016957085 -1,015784055 -1,011646805 -1,009182129 -1,008277401 -1,007118268 -1,006773567 -1,004825137 -1,004713741 -1,000671136 -0,99884969 -0,998814736 -0,996237494 -0,99422194 -0,992308737 -0,99026907 -0,986839296 -0,986137644 -0,986093492 -0,983642177 -0,983279917 -0,983094163 -0,982352891 -0,982297092 -0,982060913 -0,981742657 -0,978663393 -0,977835316 -0,977670076 -0,977357053 -0,977200022 -0,975699638 -0,973497384 -0,972583332 -0,968538018 -0,960944667 -0,957356672 -0,956318573 -0,955742087 -0,954826738 -0,954535176 -0,952743826 -0,951004845 -0,946542089 -0,945208408 -0,944790781 -0,232539035 -0,942980474 -0,846596146 -0,756625075 0,280069062 -0,189935182 -1,061718274 -0,431114173 1,234373938 -0,657359359 -0,284886989 -0,141703051 -0,528461199 -0,25488132 -3,162784462 -0,130763434 -0,838468985 -0,788601399 0,440227961 0,298374754 -0,405756695 -0,048382015 -0,422816977 0,027589959 -2,33715196 -0,124831259 -0,462714288 -0,767580958 -0,609535549 -0,471609817 -0,152755368 -0,143017236 -0,395108212 -0,750341923 -0,415126621 -0,81778672 -0,204965887 -0,249137201 -0,479052356 -0,626879935 0,444315544 -0,668160713 -0,673321728 -0,472584861 2,38003833 0,53102718 -0,122194871 -0,48387509 0,264432495 -4,412188978 -0,199509574 -0,131150572 -0,502283548 -0,709757377 -0,626151479 -0,107786935 -0,60189808 -0,286725966 0,967036501 0,070754225 -1,979233966 1,122692946 -0,608593522 -1,327670884 0,085546164 0,349642672 -0,698655039 0,25924396 -0,996701121 -0,56482643 -0,591206989 -0,645195874 0,332808915 -1,974674379 -0,808308747 -0,75885511 -0,436232916 -0,481878119 -0,167161916 -0,654387568 -0,840510969 -0,838902046 -0,559539909 -0,198417571 -1,123536138 0,141090995 -0,390232158 -0,263422646 -0,489618129 0,246220675 -1,144128005 -0,274302888 -0,562543677 -0,216460125 0,107575867 -0,549651197 1,06146974 -0,318323634 -0,378450992 -1,371437013 -6,289586915 -0,790955298 -0,576852991 -0,598108707 -0,387481364 539 718 798 1450 2408 2060 1013 415 638 31 505 2056 203 1933 1604 1640 1943 2395 1612 808 966 2509 50 2573 665 1739 1415 1611 118 2171 2194 1932 159 473 266 636 691 1072 2198 1719 2652 1164 2506 458 1263 1028 2416 2075 1423 97 1636 6 1329 1869 2657 944 666 247 551 856 2689 2017 2365 1806 422 1422 470 217 978 922 474 2580 2690 2511 2150 696 698 787 1352 472 2397 1416 2321 442 1040 1411 1864 2036 2419 504 460 1271 907 1353 264 1222 1270 1823 1180 842 1045 2024 2565 244 186 purR sstC arlS alr rsbV Csb3 glkA vraS narG pgi fnbB pls copP pho sodA hysA sgtB mvk sirR folD budA1 hemA clfB sarT xpt sucD sirC dnaJ gyrA femC splA arcA lrgA ftsH cflA icaA groES icaD fnbA fmtB/sasB tagB tagD Csb29 nasE Csb28 femB splF hlgA guaB hsIU ent2 rpoZ hsIV arsB eno agrD feoA scdA Nucleic acid metabolism Membrane transport Membrane transport Virulence regulator Stress response Cell wall/Cell division Membrane transport Stress response Cell wall/Cell division Cell wall/Cell division Membrane transport Stress response Membrane transport Stress response Energy metabolism Energy metabolism Regulation Energy metabolism Membrane transport Regulation Energy metabolism Virulence adhesin Virulence adhesin Membrane transport Membrane transport Stress response Membrane transport Regulation Stress response Membrane transport Virulence enzyme/toxin Cell wall/Cell division Membrane transport Virulence enzyme/toxin Stress response Cell wall/Cell division Regulation Energy metabolism Energy metabolism Energy metabolism Virulence adhesin Virulence adhesin Virulence regulator Nucleic acid metabolism Energy metabolism Virulence enzyme/toxin Membrane transport Virulence adhesin Membrane transport Membrane transport Stress response Nucleic acid metabolism Cell wall/Cell division Virulence enzyme/toxin Energy metabolism Energy metabolism Membrane transport Cell wall/Cell division Cell wall/Cell division Virulence adhesin Virulence biofilm Stress response Stress response Virulence adhesin Membrane transport Membrane transport Virulence enzyme/toxin Membrane transport Membrane transport Stress response Virulence enzyme/toxin Stress response Virulence biofilm Virulence adhesin Virulence adhesin Cell wall/Cell division Cell wall/Cell division Stress response Membrane transport Virulence enzyme/toxin Energy metabolism Membrane transport Stress response Virulence enzyme/toxin Membrane transport Cell wall/Cell division Virulence enzyme/toxin Membrane transport Virulence enzyme/toxin Membrane transport Nucleic acid metabolism Stress response Virulence enzyme/toxin Membrane transport Membrane transport Nucleic acid metabolism Stress response Membrane transport Virulence enzyme/toxin Energy metabolism Membrane transport Virulence regulator Membrane transport Cell wall/Cell division Membrane transport Pur operon repressor ABC transporter, ATP-binding protein, not identified Possible ferrichrome ABC transporter ATP-binding protein Sensor histidine kinase Lipoprotein, putative, Homolog mazE E.coli Alanine racemase Magnesium transporter Dyp-type peroxidase family protein Phosphomevalonate kinase Glycerophosphoryl diester phosphodiesterase, putative ABC transporter, ATP-binding protein, not identified Anti-anti-sigma factor Possible iron-binding protein ThiJ/PfpI family protein, Csb3 Glucokinase Oxygen-independent coproporphyrinogen III oxidase Two-component response regulator systems Nitrate reductase ABC transporter, permease protein membrane protein putative, related to c-di-GMP Glucose-6-phosphate isomerase Fibronectin-binding protein, FNBPs Methicillin resistant surface protein Copper-ion-binding protein Possible ferrichrome ABC transporter permease Two-component response regulator systems ABC transporter, ATP-binding protein, not identified Transcription regulator Fur family homolog Superoxide dismutase Aerobactin biosynthesis protein, IucA/IucC family Hyaluronidase Transglycosylase domain protein ABC transporter, permease Exotoxin 5, putative Hypothetical protein mevalonate kinase Iron dependent repressor Methylenetetrahydrofolate dehydrogenase/cyclohydrolase Alpha-acetolactate decarboxylase Glutamyl-tRNA reductase Ser-Asp rich FNBP Protein with cell wall sorting signal, fibrinogen-binding protein Staphylococcal accessory regulator T Xanthine phosphoribosyltransferase Succinyl-CoA synthase Hypothetical protein, probable serine protease Cation efflux familiy protein Proteins with cell sorting signals Phosphate ABC transporter Possible staphylobactin ABC transporter Protein folding and stabilization DNA gyrase, A subunit Biosynthesis of murein sacculus and peptidoglycan Protease Arginine deiminase NADH dehydrogenase Possible ferrichrome ABC transporter permease Holin-like LrgA protein, murein hydrolase Cell-division initiation protein, putative Clumping factor A Biosynthesis and degradation of surface polysaccharides and lipopolysaccharides Chaperonine Conserved hypothetical protein Proteins with cell sorting signals ABC transporter, ATP-binding protein, not identified Phosphate ABC transporter Exotoxin, putative ABC transporter, ATP-binding protein, not identified Membrane protein putative Conserved hypothetical protein Exotoxin 4, putative Hypothetical protein Biosynthesis and degradation of surface polysaccharides and lipopolysaccharides Fibronectin-binding protein A FmtB, proteins with cell sorting signals, associated to MRSA Teichoic acid biosynthesis protein B Cholinephosphate cytidylyltransferase, putative Conserved protein, Csb29 ABC transporter, ATP-binding protein, not identified Exotoxin, putative Nitrate reductase possible ABC transporter, ATP-binding protein, not identified Oxidoreductase dehydrogenase/reductase, Csb28 Possible exotoxin ABC transporter, ATP-binding protein, not identified Biosynthesis of murein sacculus and peptidoglycan Probable serine protease ABC transporter, ATP-binding protein, not identified Gamma-hemolysin, component A ABC transporter, ATP-binding protein, not identified Inosine-5-monophosphate dehydrogenase Heat shock protein HslVU Enterotoxin ABC transporter, ATP-binding protein, not identified ABC transporter, ATP-binding protein, not identified DNA-directed RNA polymerase, omega subunit Heat shock protein HslV Arsenical pump membrane protein Exotoxin 3 Enolase Possible ferrichrome/Iron ABC transp. ATP-binding Accessory gene regulator protein D FeoA domain protein Cell division and morphogenesis-related protein ABC transporter, ATP-binding protein, not identified -0,943206542 -0,943103062 -0,942560914 -0,941664906 -0,938503954 -0,937938769 -0,936239218 -0,935914836 -0,935847213 -0,935739178 -0,935353765 -0,93417832 -0,933851786 -0,928053783 -0,927100342 -0,927026923 -0,92616882 -0,926014218 -0,925613496 -0,925360957 -0,922687183 -0,92252976 -0,920535838 -0,920522342 -0,92014351 -0,919055966 -0,91848836 -0,91725191 -0,917145401 -0,916490194 -0,915049447 -0,912530921 -0,912372608 -0,912287495 -0,911800931 -0,909751895 -0,909561018 -0,909418847 -0,909314822 -0,908633068 -0,907974879 -0,90737888 -0,90722258 -0,906945603 -0,906894932 -0,90148633 -0,89914569 -0,898599994 -0,897450712 -0,893922548 -0,891120101 -0,890050408 -0,889083262 -0,886436759 -0,885214346 -0,884903449 -0,883922982 -0,883389547 -0,882921168 -0,881845259 -0,88068519 -0,878464038 -0,874680916 -0,874518035 -0,870716298 -0,865475702 -0,865463273 -0,857755052 -0,857686158 -0,856916759 -0,855032359 -0,850852564 -0,850025692 -0,843984307 -0,841417048 -0,837470195 -0,836930643 -0,836830937 -0,835867255 -0,832656056 -0,824953414 -0,823811792 -0,823468007 -0,819300791 -0,817806848 -0,8158206 -0,808175076 -0,804051787 -0,804043559 -0,803727008 -0,803407808 -0,803065041 -0,80179727 -0,800420926 -0,797668718 -0,791217995 -0,790529428 -0,790005898 -0,784577079 -0,784383846 -0,783604891 -0,78206127 -0,778001598 -0,777811457 -0,771905967 -0,471992655 -0,558095287 -0,012882906 -1,504898743 -1,131541865 -1,184134172 -0,764846957 -0,444192227 -0,279172678 -0,306020306 -0,600585053 -0,310575981 -0,655407595 -0,40580854 -0,09478348 -0,332159724 -0,620808027 0,285723525 -1,17526222 -0,478838979 -0,80252837 0,704385477 -0,423932838 -0,601541761 -1,069459833 -0,363601453 -0,185669479 -0,083744712 -1,17035484 -0,531344197 -0,266496225 -0,299870355 -0,391238945 -0,289822481 -0,355388873 0,125679077 -0,226882444 -1,197035114 -0,457846131 0,090519796 -0,744246872 0,959801133 -0,344876927 -4,945077803 0,000591939 -1,25436124 0,243143703 -0,257376636 -0,089906991 0,040848432 -0,124611712 -0,021619811 -0,852260145 -0,33098825 0,004037635 -0,252300069 -1,077052165 -0,490228774 -0,365075662 0,247179289 -0,360292956 0,28816261 -0,169352827 -0,829089786 -0,452710662 -0,079960744 -0,283076649 -0,578146766 -0,653529842 -0,434718902 -0,535679779 0,127470741 -0,249587101 -0,244323048 -0,405383007 -0,412496594 -0,678837573 0,212835513 -0,392595363 -0,385059426 -0,008254679 -0,333608743 -0,628858711 -1,063035687 -0,369254199 -0,197605097 -0,357876873 -0,982220764 1,363990325 -0,454821648 -1,740076261 0,096424683 -0,261649507 -0,515260978 -0,35113764 -1,997058748 0,080987877 -0,487158134 -0,320329591 -0,02838933 -0,543279555 -0,268434416 -0,4459861 -0,146122213 -0,358414874 155 2375 979 369 185 1881 1717 2073 317 1390 506 2253 941 1921 2398 1056 478 693 1714 2002 1461 2379 2010 2144 2426 697 2256 1327 220 797 1023 1417 2683 2599 884 2003 841 1459 839 184 1868 2189 1169 723 1867 1451 706 1942 1781 779 705 1880 1721 1618 1441 1052 1929 2166 2168 2531 1178 921 1453 840 2138 1168 882 461 2074 2692 2385 1389 2136 2554 418 1146 977 1951 1221 1866 567 976 2563 1745 1143 1893 2165 1410 1905 2173 556 2211 2092 2584 1179 2549 301 1397 780 1144 1145 2116 796 2553 99 clpB clpP lukE hemC murF geh parC femX bcp nirB sspB tagA hemL map dfrA Csb19 bioB tagX hml sstB murE hlb pgm msrA pgk splB ssaA splC arlR fhuG vraR sasI/isdH fhuB lukD clpX rpoD menD htsB murG tpiA fib/efb guaA ddlA icaC parE Csb9 cidB isdG frpA gmk splD ctsR clp pyk isdE htsA femA vraR asp23 murA isaA msrA recQ isdF srtB murZ sstA cidC sirA Membrane transport Membrane transport Stress response Stress response Membrane transport Virulence enzyme/toxin Energy metabolism Cell wall/Cell division Virulence enzyme/toxin Nucleic acid metabolism Membrane transport Cell wall/Cell division Energy metabolism Iron metabolism Energy metabolism Virulence enzyme/toxin Virulence enzyme/toxin Cell wall/Cell division Energy metabolism Stress response Nucleic acid metabolism Stress response Membrane transport Membrane transport Energy metabolism Cell wall/Cell division Stress response Stress response Energy metabolism Membrane transport Cell wall/Cell division Membrane transport Cell wall /Cell division Membrane transport Membrane transport Virulence enzyme/toxin Energy metabolism Cell wall/Cell division Energy metabolism Membrane transport Virulence enzyme/toxin Regulation Virulence adhesin Cell wall/Cell division Virulence enzyme/toxin Virulence regulator Iron metabolism Regulation Membrane transport Membrane transport Membrane transport Virulence enzyme/toxin Stress response Regulation Membrane transport Energy metabolism Membrane transport Membrane transport Membrane transport Stress response Virulence enzyme/toxin Virulence enzyme/toxin Cell wall/Cell division Energy metabolism Membrane transport Virulence adhesin Membrane transport Nucleic acid metabolism Cell wall/Cell division Virulence biofilm Stress response Nucleic acid metabolism Stress response Cell wall/Cell division Membrane transport Membrane transport Membrane transport Cell wall/Cell division Energy metabolism Virulence enzyme/toxin Stress response Membrane transport Stress response Energy metabolism Membrane transport Membrane transport Membrane transport Cell wall/Cell division Regulation Stress response Stress Response Membrane transport Cell wall/Cell division Virulence adhesin Virulence enzyme/toxin Lipid metabolism Energy metabolism Cell wall/Cell division Nucleic acid metabolism Membrane transport Virulence adhesin Cell wall/Cell division Membrane transport Cell wall/Cell division Iron metabolism Cation efflux familiy protein Membrane transporter Protease SigA-dependant stress genes / TIGR 2007= ATP-dependent Clp protease, ATP-binding subunit ClpB Protease SigA-dependant stress genes / TIGR 2007=Prophage L54a, Clp protease, putative ABC transporter, ATP-binding protein, not identified Leukotoxin Porphobilinogen deaminase UDP-N-acetylmuramoyl-tripeptide-D-alanyl-D-alanine ligase Lipase, glycerol ester hydrolase DNA topoisomerase IV, A subunit ABC transporter, ATP-binding protein, not identified FemX protein, peptidyl transferase, putative FmhB NADH dehydrogenase Bacterioferritin comigratory protein Nitrate reductase, large subunit Cysteine protease SspB Exotoxin 3, putative UDP-N-acetyl-D-mannosamine transferase Glutamate-1-semialdehyde-2,1-aminomutase Protein modification and repair Dihydrofolate reductase Conserved protein, Csb19 (biosynthesis/metabolism-associated gene in Moisan) Iron-compound ABC transporter, subst. binding ABC transporter, ATP-binding protein, not identified Biotin synthetase Teichoic acid biosynthesis protein X Transcriptional regulator, MarR family Aluminum resistance protein Flavohemoprotein Possible ferrichrome ABC transporter permease UDP-N-acetylmuramoylalanyl-D-glutamate--2,6-diaminopimelate ligase ABC transporter, ATP-binding protein, not identified Peptide methionine sulfoxide reductase, putative Conserved domain protein ABC transporter, ATP-binding protein, not identified beta hemolysin Phosphoglycerate mutase (mal, gal to G1-P to G6-P) Methionine sulfoxide reductase Phosphoglycerate kinase ABC transporter, ATP-binding protein, not identified Probable serine protease Transcriptionnal regulator, Sir 2 family Fibrinogen-binding protein Homolog to streptococcal secretory antigen A, LysM domain and Antigenic surface protein Probable serine protease DNA-binding response regulator Ferrichrome transport permease DNA-binding response regulator, VraR Cell wall surface anchor family protein, harA N315 homolog ABC transporter, ATP-binding protein, not identified Ferrichrome transport permease Leukotoxin ATP-dependent Clp protease, ATP-binding subunit ClpX RNA polymerase sigma factor, homolog sigA Conserved hypothetical protein, similar to tellurite resistance prot Carboxylate synthase ABC transporter, ATP-binding protein, not identified Possible heme-iron transport system, ABC transporter permease Conserved hypothetical protein Transcriptional regulator, MarR family Exotoxin 1 Probable hemolysin UDP-GlucNac-MurNac-(pentapeptide) PP-C55-GlucNac transferase Triosephosphate isomerase Cation efflux familiy protein Fibrinogen-binding protein ABC transporter, ATP-binding protein, not identified GMP synthetase D-alanine-D-alanine ligase Biosynthesis and degradation of surface polysaccharides and lipopolysaccharides Heat shock protein, Hsp20 family DNA topoisomerase IV, B subunit Conserved protein, Csb9 (biosynthesis/metabolism-associated genes in Moisan) Membrane protein, putative, linked to LrgAB MttA/Hcf 106 family protein with Fur box Heme-iron transport, cytoplasmic protein Fur-regulated protein A, possible membrane protein (Allard frp operon, Morissey 2002 IAI) Mur ligase family protein guanylate kinase Probable serine protease Transcriptional regulator Hydrolase, haloacid dehalogenase like familly ATP-dependant protease Pyruvate kinase Heme-iron transport, heme-iron permease system ABC transporter, ATP-binding protein, not identified Possible heme-iron transport system, ABC transporter permease Biosynthesis of murein sacculus and peptidoglycan DNA-binding response regulator, VraR Alkaline shock protein 23 Chaperonine, Hsp33 homolog ABC transporter, ATP-binding protein UDP-N-acetylglucosamine 1-carboxyvinyltransferase Immunodominant protein A, lytic transglycosylase Exotoxin 2 Esterase, putative Formate / nitrite transporter protein Methionine sulfoxide reductase ATP-dependent DNA helicase Heme-iron transport, heme-iron permease system Sortase B UDP-N-acetylglucosamine 1-carboxyvinyltransferase Possible ferrichrome ABC transporter permease Pyruvate oxidase, linked to LrgAB Possible staphylobactin ABC transporter -0,768143394 -0,766861519 -0,764265876 -0,762749026 -0,756513723 -0,748803699 -0,748542827 -0,739690799 -0,725724572 -0,722002876 -0,715387049 -0,71490505 -0,71086179 -0,703987286 -0,699084988 -0,693435424 -0,692515714 -0,690842417 -0,689428109 -0,68838679 -0,687400348 -0,683059458 -0,682411402 -0,679458061 -0,67939942 -0,679382568 -0,672568715 -0,672217932 -0,671439084 -0,662487706 -0,662435175 -0,660888497 -0,65820488 -0,654324976 -0,65125332 -0,646444332 -0,646020719 -0,640561017 -0,635067291 -0,631696813 -0,627785234 -0,625914494 -0,610822735 -0,609791654 -0,606036692 -0,59988448 -0,596443362 -0,596350395 -0,5962317 -0,593874764 -0,585925486 -0,581911764 -0,571372073 -0,567150787 -0,564374034 -0,563448791 -0,560001835 -0,558845823 -0,553427574 -0,548557225 -0,546812966 -0,546000271 -0,544131545 -0,542117397 -0,537130955 -0,536728793 -0,531532615 -0,521961389 -0,512642298 -0,501443227 -0,501300337 -0,498343732 -0,490032201 -0,484544005 -0,481746482 -0,481485171 -0,475642463 -0,468031201 -0,465149598 -0,440698698 -0,432562533 -0,425857928 -0,420092125 -0,419470966 -0,416225977 -0,414214275 -0,410047718 -0,404265772 -0,40389511 -0,402682756 -0,395398997 -0,395212014 -0,394821319 -0,393106072 -0,391951846 -0,387754389 -0,381274143 -0,37977698 -0,379563316 -0,37928743 -0,372220844 -0,364536938 -0,330102334 -0,326950201 -0,324193739 -0,380517377 -0,670224644 0,879255482 -0,248117208 -0,344838803 -0,31096108 0,036186801 -0,204015309 -1,832117496 0,188981176 -0,719865502 -1,678047543 -0,349664431 -0,757401463 -0,132129483 -0,861435483 0,472855402 -1,376789308 -0,470425232 -0,328957716 -0,272902112 -0,226112211 -0,27455695 -0,344631569 0,257238378 -0,838534669 -0,734782772 -0,028873252 -0,266022815 0,071398809 -1,265048124 -0,406760917 -0,754252227 -0,530327719 -0,898640558 -0,2184672 0,22992182 -0,017199382 0,227281434 -0,453737182 -0,336740498 -0,277545059 0,258617461 -0,198324808 -0,41878155 -1,521129989 -0,477577942 -0,44371612 -0,708029618 -0,636593048 -0,510911908 -0,433423112 0,203020638 -0,149912775 -0,112167132 -0,042229077 -0,69396101 -0,321298883 -0,761001575 -0,391155341 -0,369603961 -0,202288294 -0,901223832 0,343197672 -1,410895793 0,273391486 -1,143280387 -1,231757361 -0,797704001 -1,004588498 -0,606156165 -0,330256468 -0,394333239 -0,885682767 -0,937958972 -0,874986936 -0,805343957 -0,374642707 -2,094146605 -0,49022983 -0,035488553 -0,575828747 0,050904427 -0,670363028 -1,15506456 -0,547092356 -0,323735069 -0,346680934 -0,678291588 0,084054844 -0,354103121 -1,099195772 -0,106897565 -0,914943217 -0,330865902 -0,515575362 1,826110802 -1,074154225 -0,407813618 -1,156200689 -1,051411791 -0,532242656 -0,309802519 0,819756111 -0,805022489 2555 1887 507 2169 107 2210 1511 1687 104 106 1984 1141 1062 617 105 857 1161 108 694 2277 1889 2114 1919 102 101 1888 222 823 2369 1138 1746 2088 2534 2117 161 204 2167 838 453 2170 100 263 829 914 824 704 205 1368 2539 1541 1140 1952 451 452 cidR hemY sbnH gerCB lytH sbnE sbnG aldA isdC atl Csb4 sbnF murI sbnI tagH fhuD2 hemE ald/Csb24 sbnC sbnB hemH ldh uvrB isdB pfk sceD fba pflB htsC gapA1 sbnA lytM trxB uvrA fhuA pflA katA srtA isdA ahpF ahpC Cell wall/Cell division Energy metabolism Membrane transport Membrane transport Membrane transport Membrane transport Stress response Cell wall/Cell division Membrane transport Membrane transport Energy metabolism Membrane transport Cell wall/Cell division Energy metabolism Membrane transport Virulence enzyme/toxin Cell wall/Cell division Membrane transport Cell wall/Cell division Membrane transport Energy metabolism Stress response Regulation Membrane transport Membrane transport Energy metabolism Energy metabolism Stress response Stress response Membrane transport Energy metabolism Virulence enzyme/toxin Energy metabolism Energy metabolism Membrane transport Energy metabolism Membrane transport Energy metabolism Energy metabolism Membrane transport Membrane transport Cell wall/Cell division Stress response Membrane transport Stress response Membrane transport Energy metabolism Stress response Virulence adhesin Regulation Membrane transport Stress response Stress response Stress response Transcriptional regulator, LysR family, putative, linked to LrgAB Porphyrin biosynthesis LysM domain protein Aerobactin biosynthesis protein, IucA/IucC family Staphylobactin biosynthesis ABC transporter, ATP-binding protein, not identified Methlytransferase, UbiE/COQ5 family N-acetylmuramoyl-L-alanine amidase, putative Staphylobactin biosynthesis Staphylobactin biosynthesis Aldehyde dehydrogenase Heme-iron transport, NPQTN cell wall surface anchored protein Bifunctionnal autolysin Hexulose-6P synthase, Csb4 (biosynthesis/metabolism in Moisan et al) Staphylobactin biosynthesis Staphylocoagulase, putative Glutamate racemase Staphylobactin biosynthesis Teichoic acid ABC transporter protein, putative Ferrichrome transport, receptor Porphyrin biosynthesis Aldehyde dehydrogenase, Csb24 (biosynthesis/metabolism-associated genes in Moisan) Transcription regulator Fur family homolog Staphylobactin biosynthesis Staphylobactin biosynthesis Porphyrin biosynthesis L-lactate deshydrogenase Excinuclease ABC, B subunit Disulfide reductase with Fur box Heme-iron transport, LPXTG cell wall surface anchored protein Phosphofructokinase Exotoxin, SceD like NAD(P)H-flavin oxidoreductase Fructose-bisphosphate aldolase, class II Conserved hypothetical protein with Fur box Formate acetyltransferase Possible heme-iron transport system, ABC transporter, ATP-binding Glyceraldehyde 3P deshydrogenase NAD(P)H-flavin oxidoreductase Putative transporter with Fur box Staphylobactin biosynthesis Peptidoglycan hydrolase Thioredoxin reductase (TIGR function class= electron transport, nitrofurantoin resistance) ABC transporter, ATP-binding protein, not identified Excinuclease ABC, A subunit Ferrichrome transport ATP-binding protein Formate-lyase activating enzyme Catalase SigA dependant gene / TIGR 2007=Catalase Sortase A Transcription regulator Fur family homolog Heme-iron transport, LPXTG cell wall surface anchored protein Possible ferritin Alkyl hydroperoxide reductase, subunit F Alkyl hydroperoxide reductase, subunit C -0,313110991 -0,307274001 -0,298971801 -0,281471681 -0,24064555 -0,239605945 -0,230354965 -0,224896241 -0,211359697 -0,210855278 -0,208133864 -0,203222064 -0,182867036 -0,146422417 -0,119042594 -0,099920266 -0,052965657 -0,030064786 -0,027145434 -0,023880714 -0,008349284 -0,003087953 0,009741377 0,013211987 0,025775588 0,048484607 0,06538663 0,075490791 0,080741937 0,099114147 0,104578944 0,113578697 0,137619491 0,154406674 0,154967622 0,160268762 0,189299191 0,195618132 0,206161855 0,276822384 0,305522302 0,329512894 0,339969956 0,449018214 0,465082723 0,478879651 0,544383132 0,622188191 0,62989621 0,650908345 0,945305109 1,245132061 2,421742835 2,781390482 -0,920923172 0,029810662 -0,500451514 -0,4828043 -0,121733613 -0,847719808 -0,390197058 -1,240728036 -0,218290965 -0,196577033 -0,583157583 -1,186225189 -0,023445846 -0,383670442 -0,040558377 0,810263105 -1,219944185 -0,169398254 -1,647250143 -0,329746023 -0,10551987 0,360060297 0,116783865 0,257270993 -0,250521798 0,171279651 5,44162314 1,314080714 -0,271516781 -0,808806945 -1,287033354 2,205689824 -0,545136306 -0,08453544 -1,513413045 2,23013755 0,456378173 0,562624807 0,117231162 -0,643914351 -0,31931345 1,314461326 -0,373023245 1,049368599 1,517744316 -0,019215495 1,513805381 0,096512641 -0,810575454 0,47767602 -1,742332797 0,471733164 1,980538629 1,87988606