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In view of the pressing need to identify new antibacterial agents able to combat multidrug-resistant bacteria, we investigated a series of fused selenazolinium derivatives (1-8) regarding their in vitro antimicrobial activities against 25 ESKAPE-pathogen strains. Ebselen was used as reference compound. Most of the selenocompounds demonstrated an excellent in vitro activity against all S. aureus strains, with activities comparable to or even exceeding the one of ebselen. In contrast to ebselen, some selenazolinium derivatives (1, 3, and 7) even displayed significant actions against all Gram-negative pathogens tested. The 3-bromo-2-(1-hydroxy-1-methylethyl)[1,2]selenazolo[2,3-a]pyridinium chloride (1) was particularly active (minimum inhibitory concentrations, MICs: 0.31-1.24 µg/mL for MRSA, and 0.31-2.48 µg/mL for Gram-negative bacteria) and devoid of any significant mutagenicity in the Ames assay. Our preliminary mechanistic studies in cell culture indicated that their mode of action is likely to be associated with an alteration of intracellular levels of glutathione and cysteine thiols of different proteins in the bacterial cells, hence supporting the idea that such compounds interact with the intracellular thiolstat. This alteration of pivotal cysteine residues is most likely the result of a direct or catalytic oxidative modification of such residues by the highly reactive selenium species (RSeS) employed.

The tricarboxylic acid cycle (TCA cycle) is a central metabolic pathway that provides energy, reducing potential, and biosynthetic intermediates. In Staphylococcus aureus, TCA cycle activity is controlled by several regulators (e.g. CcpA, CodY, and RpiRc) in response to the availability of sugars, amino acids, and environmental stress. Developing a bioinformatic search for additional carbon catabolite-responsive regulators in S. aureus, we identified a LysR-type regulator, catabolite control protein E (CcpE), with homology to the Bacillus subtilis CcpC regulator. Inactivation of ccpE in S. aureus strain Newman revealed that CcpE is a positive transcriptional effector of the first two enzymes of the TCA cycle, aconitase (citB) and to a lesser extent citrate synthase (citZ). Consistent with the transcriptional data, aconitase activity dramatically decreased in the ccpE mutant relative to the wild-type strain. The effect of ccpE inactivation on citB transcription and the lesser effect on citZ transcription were also reflected in electrophoretic mobility shift assays where CcpE bound to the citB promoter but not the citZ promoter. Metabolomic studies showed that inactivation of ccpE resulted in increased intracellular concentrations of acetate, citrate, lactate, and alanine, consistent with a redirection of carbon away from the TCA cycle. Taken together, our data suggest that CcpE is a major direct positive regulator of the TCA cycle gene citB.

In phagocytes, pathogen recognition is followed by Ca 2+ mobilization and NADPH oxidase 2 (NOX2)–mediated “oxidative burst,” which involves the rapid production of large amounts of reactive oxygen species (ROS). We showed that ORAI Ca 2+ channels control store-operated Ca 2+ entry, ROS production, and bacterial killing in primary human monocytes. ROS inactivate ORAI channels that lack an ORAI3 subunit. Staphylococcal infection of mice reduced the expression of the gene encoding the redox-sensitive Orai1 and increased the expression of the gene encoding the redox-insensitive Orai3 in the lungs or in bronchoalveolar lavages. A similar switch from ORAI1 to ORAI3 occurred in primary human monocytes exposed to bacterial peptides in culture. These alterations in ORAI1 and ORAI3 abundance shifted the channel assembly toward a more redox-insensitive configuration. Accordingly, silencing ORAI3 increased the redox sensitivity of the channel and enhanced oxidation-induced inhibition of NOX2. We generated a mathematical model that predicted additional features of the Ca 2+ -redox interplay. Our results identified the ORAI-NOX2 feedback loop as a determinant of monocyte immune responses.

Previous studies have demonstrated that various tricarboxylic acid (TCA) cycle genes, particularly the succinate dehydrogenase genes ( sdhCAB ), are upregulated in Staphylococcus aureus biofilms. To better study the role of this enzyme complex, an sdhCAB deletion mutant (Δ sdh ) was constructed. Compared to the wild type (wt) the mutant was impaired in planktonic growth under aerobic conditions, excreted acetic acid could not be reused and accumulated continuously, succinate was excreted and found in the culture supernatant, and metabolome analysis with cells grown in chemically defined medium revealed reduced uptake/metabolism of some amino acids from the growth medium. Moreover, the mutant was able to counteract the steadily decreasing extracellular pH by increased urease activity. The addition of fumarate to the growth medium restored the wt phenotype. The mutant showed a small-colony variant (SCV)-like phenotype, a slight increase in resistance to various aminoglycoside antibiotics, and decreased pigmentation. The decreased growth under aerobic conditions is due to the interruption of the TCA cycle (indicated by the accumulation of succinate and acetic acid) with the consequence that many fewer reduction equivalents (NADH and FADH 2 ) can fuel the respiratory chain. The results indicate that the TCA cycle is required for acetate and amino acid catabolism; its upregulation under biofilm conditions is advantageous under such nutrient- and oxygen-limited conditions.

Here, we investigate the functionality of the oxygen-responsive nitrogen regulation system NreABC in the human pathogen Staphylococcus aureus and evaluate its role in anaerobic gene regulation and virulence factor expression. Deletion of nreABC resulted in severe impairment of dissimilatory nitrate and nitrite reduction and led to a small-colony phenotype in the presence of nitrate during anaerobic growth. For characterization of the NreABC regulon, comparative DNA microarray and proteomic analyses between the wild type and nreABC mutant were performed under anoxic conditions in the absence and presence of nitrate. A reduced expression of virulence factors was not observed in the mutant. However, both the transcription of genes involved in nitrate and nitrite reduction and the accumulation of corresponding proteins were highly decreased in the nreABC mutant, which was unable to utilize nitrate as a respiratory oxidant and, hence, was forced to use fermentative pathways. These data were corroborated by the quantification of the extracellular metabolites lactate and acetate. Using an Escherichia coli -compatible two-plasmid system, the activation of the promoters of the nitrate and nitrite reductase operons and of the putative nitrate/nitrite transporter gene narK by NreBC was confirmed. Overall, our data indicate that NreABC is very likely a specific regulation system that is essential for the transcriptional activation of genes involved in dissimilatory reduction and transport of nitrate and nitrite. The study underscores the importance of NreABC as a fitness factor for S. aureus in anoxic environments.

Carbon metabolism and virulence determinant production are often linked in pathogenic bacteria, and several regulatory elements have been reported to mediate this linkage in Staphylococcus aureus. Previously, we described a novel protein, catabolite control protein E (CcpE) that functions as a regulator of the tricarboxylic acid cycle. Here we demonstrate that CcpE also regulates virulence determinant biosynthesis and pathogenesis. Specifically, deletion of ccpE in S. aureus strain Newman revealed that CcpE affects transcription of virulence factors such as capA, the first gene in the capsule biosynthetic operon; hla, encoding α-toxin; and psmα, encoding the phenol-soluble modulin cluster α. Electrophoretic mobility shift assays demonstrated that CcpE binds to the hla promoter. Mice challenged with S. aureus strain Newman or its isogenic ΔccpE derivative revealed increased disease severity in the ΔccpE mutant using two animal models; an acute lung infection model and a skin infection model. Complementation of the mutant with the ccpE wild-type allele restored all phenotypes, demonstrating that CcpE is negative regulator of virulence in S. aureus.

Over the past decade, metabolomics has emerged as an important technique for systems biology. Measuring all the metabolites in a biological system provides an invaluable source of information to explore various cellular processes, and to investigate the impact of environmental factors and genetic modifications. Nuclear magnetic resonance (NMR) spectroscopy is an important method routinely employed in metabolomics. NMR provides comprehensive structural and quantitative information useful for metabolomics fingerprinting, chemometric analysis, metabolite identification and metabolic pathway construction. A successful metabolomics study relies on proper experimental protocols for the collection, handling, processing and analysis of metabolomics data. Critically, these protocols should eliminate or avoid biologically-irrelevant changes to the metabolome. We provide a comprehensive description of our NMR-based metabolomics procedures optimized for the analysis of bacterial metabolomes. The technical details described within this manuscript should provide a useful guide to reliably apply our NMR-based metabolomics methodology to systems biology studies.

Staphylococcus aureus is a medically important pathogen that synthesizes a wide range of virulence determinants. The synthesis of many staphylococcal virulence determinants is regulated in part by stress-induced changes in the activity of the tricarboxylic acid (TCA) cycle. One metabolic change associated with TCA cycle stress is an increased concentration of ribose, leading us to hypothesize that a pentose phosphate pathway (PPP)-responsive regulator mediates some of the TCA cycle-dependent regulatory effects. Using bioinformatics, we identified three potential ribose-responsive regulators that belong to the RpiR family of transcriptional regulators. To determine whether these RpiR homologues affect PPP activity and virulence determinant synthesis, the rpiR homologues were inactivated, and the effects on PPP activity and virulence factor synthesis were assessed. Two of the three homologues (RpiRB and RpiRC) positively influence the transcription of the PPP genes rpiA and zwf , while the third homologue (RpiRA) is slightly antagonistic to the other homologues. In addition, inactivation of RpiRC altered the temporal transcription of RNAIII, the effector molecule of the agr quorum-sensing system. These data confirm the close linkage of central metabolism and virulence determinant synthesis, and they establish a metabolic override for quorum-sensing-dependent regulation of RNAIII transcription.