The high resolution 2-D protein gel electrophoresis technique combined with MALDI-TOF MS and a recently developed fluorescence-based thiol modification assay were used to investigate the cellular response of Staphylococcus aureus to oxidative stress. Addition of hydrogen peroxide, diamide, and the superoxide generating agent paraquat to exponentially growing cells revealed complex changes in the protein expression pattern. In particular, proteins involved in detoxification, repair systems, and intermediary metabolism were found to be up-regulated. Interestingly, there is only a small overlap of proteins induced by all these stressors. Exposure to hydrogen peroxide mediated a significant increase of DNA repair enzymes, whereas treatment with diamide affected proteins involved in protein repair and degradation. The activity of proteins under oxidative stress conditions can be modulated by oxidation of thiol groups. In growing cells, protein thiols were found to be mainly present in the reduced state. Diamide mediated a strong increase of reversibly oxidized thiols in a variety of metabolic enzymes. By contrast, hydrogen peroxide resulted in the reversible oxidation especially of proteins with active site cysteines. Moreover, high levels of hydrogen peroxide influenced the pI of three proteins containing cysteines within their active sites (GapA1, AhpC, and HchA) indicating the generation of sulfinic or sulfonic acid by irreversible oxidation of thiols.
The nonpathogenic Bacillus subtilis and the pathogen Staphylococcus aureus are gram-positive model organisms that have to cope with the radical nitric oxide (NO) generated by nitrite reductases of denitrifying bacteria and by the inducible NO synthases of immune cells of the host, respectively. The response of both microorganisms to NO was analyzed by using a two-dimensional gel approach. Metabolic labeling of the proteins revealed major changes in the synthesis pattern of cytosolic proteins after the addition of the NO donor MAHMA NONOate. Whereas B. subtilis induced several oxidative stress-responsive regulons controlled by Fur, PerR, OhrR, and Spx, as well as the general stress response controlled by the alternative sigma factor SigB, the more resistant S. aureus showed an increased synthesis rate of proteins involved in anaerobic metabolism. These data were confirmed by nuclear magnetic resonance analyses indicating that NO causes a drastically higher increase in the formation of lactate and butanediol in S. aureus than in B. subtilis. Monitoring the intracellular protein thiol state, we observed no increase in reversible or irreversible protein thiol modifications after NO stress in either organism. Obviously, NO itself does not cause general protein thiol oxidations. In contrast, exposure of cells to NO prior to peroxide stress diminished the irreversible thiol oxidation caused by hydrogen peroxide.Bacillus subtilis and Staphylococcus aureus are gram-positive model bacteria. Whereas B. subtilis is considered to be harmless, S. aureus is a facultative pathogen and the leading cause of nosocomial and community-acquired infections. Both microorganisms have to cope with high amounts of nitric oxide (NO) (up to the M range) generated from coexisting denitrifying bacteria or from the innate immune response of the host, respectively (17,31,55,68). Hence, the ability of B. subtilis and S. aureus to protect themselves against NO might be crucial for survival in their respective natural habitats.The cytotoxic properties of the small, lipophilic, and freely diffusible radical NO are attributed to its high reactivity. Indirect effects of NO are caused by the reaction of the radical with oxygen or superoxide, resulting in the formation of a number of additional reactive nitrogen species, including nitrogen dioxide, peroxynitrite, and dinitrogen trioxide. These nitrogen species differ in reactivity, stability, and biological activity but result in a broad spectrum of antimicrobial activity (25). In general, reactive oxygen and nitrogen species can interact with numerous targets, including thiols, metal centers, tyrosine residues in proteins, nucleotide bases, and lipids (20,69). NO directly affects the activity of enzymes by the reaction with bound free radicals or with metal centers (51, 72, 100). For example, the formation of metal-nitrosyl complexes in respiratory enzymes was shown to inhibit bacterial respiration (8,13,71,88) and the formation of a dinitrosyl-iron complex of a protein essential for branched-chain am...
Staphylococcus aureus colonizes and infects humans as well as animals. In the present study, 17 S. aureus strains isolated from cows suffering from mastitis were characterized. The well-established multilocus sequence typing (MLST) technique and a diagnostic microarray covering 185 S. aureus virulence and resistance genes were used for genetic and epidemiological analyses. Virulence gene expression studies were performed by analyzing the extracellular protein pattern of each isolate on 2-D gels. By this way, a pronounced heterogeneity of the extracellular proteome between the bovine isolates has been observed which was attributed to genome plasticity and variation of gene expression. Merely 12 proteins were expressed in at least 80% of the isolates, i.e. Atl, Aur, GlpQ, Hla, LtaS, Nuc, PdhB, SAB0846, SAB2176, SAB0566, SspA, and SspB forming the core exoproteome. Fifteen extracellular proteins were highly variably expressed and only present in less than 20% of the isolates. This includes the serine proteases SplB, C, and F, and the superantigens SEC-bov, SEL and TSST-1. Compared to human isolates we identified at least six proteins with significantly different expression frequencies. While SAB0846 was expressed more frequently in bovine isolates, LytM, EbpS, Spa, Geh, and LukL1 were seen less frequently in these isolates.
Gel-based proteomics is a powerful approach to study the physiology of Staphylococcus aureus under various growth restricting conditions. We analyzed 679 protein spots from a reference 2-dimensional gel of cytosolic proteins of S. aureus COL by mass spectrometry resulting in 521 different proteins. 4,692 time dependent protein synthesis profiles were generated by exposing S. aureus to nine infection-related stress and starvation stimuli (H2O2, diamide, paraquat, NO, fermentation, nitrate respiration, heat shock, puromycin, mupirocin). These expression profiles are stored in an online resource called Aureolib (http://www.aureolib.de). Moreover, information on target genes of 75 regulators and regulatory elements were included in the database. Cross-comparisons of this extensive data collection of protein synthesis profiles using the tools implemented in Aureolib lead to the identification of stress and starvation specific marker proteins. Altogether, 226 protein synthesis profiles showed induction ratios of 2.5-fold or higher under at least one of the tested conditions with 157 protein synthesis profiles specifically induced in response to a single stimulus. The respective proteins might serve as marker proteins for the corresponding stimulus. By contrast, proteins whose synthesis was increased or repressed in response to more than four stimuli are rather exceptional. The only protein that was induced by six stimuli is the universal stress protein SACOL1759. Most strikingly, cluster analyses of synthesis profiles of proteins differentially synthesized under at least one condition revealed only in rare cases a grouping that correlated with known regulon structures. The most prominent examples are the GapR, Rex, and CtsR regulon. In contrast, protein synthesis profiles of proteins belonging to the CodY and σB regulon are widely distributed. In summary, Aureolib is by far the most comprehensive protein expression database for S. aureus and provides an essential tool to decipher more complex adaptation processes in S. aureus during host pathogen interaction.
Bacillus subtilis is exposed to a variety of antimicrobial compounds in the soil. In this paper, we report on the response of B. subtilis to the fungal-related antimicrobials 6-brom-2-vinyl-chroman-4-on (chromanon) and 2-methylhydroquinone (2-MHQ) using proteome and transcriptome analyses. Chromanon, a derivative of aposphaerins from Aposphaeria species caused predominant protein damage in B. subtilis as indicated by the induction of the HrcA, CtsR, and Spx regulons. The expression profile of the ganomycin-related substance 2-MHQ was similar to that of catechol as reflected by the common induction of the thiol-specific oxidative stress response. Several putative ring-cleavage dioxygenases and oxidoreductases were differentially up-regulated by 2-MHQ, catechol, and chromanon including yfiDE, ydfNOP, yodED, ycnDE, yodC, and ykcA. The nitroreductase encoding yodC gene is induced in response to catechol, 2-MHQ, and chromanon, which depend on the MarR-type repressor YodB. The yfiDE (catDE) operon encodes a catechol-2,3-dioxygenase which is most strongly induced by catechol. The yodED (mhqED), ydfNOP (mhqNOP) operons, and ykcA (mhqA) respond most strongly to 2-MHQ and encode putative hydroquinone-specific extradiol dioxygenases. The ycnDE operon was most strongly induced by chromanon. Mutational analyses revealed that the putative hydroquinone-specific dioxygenases MhqO and MhqA confer resistance to 2-MHQ in B. subtilis.
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