Impacts of the Type I Toxin-Antitoxin System, SprG1/SprF1, on Staphylococcus aureus Gene Expression.

Type I toxin-antitoxin (TA) systems are widespread genetic modules in bacterial genomes. They express toxic peptides whose overexpression leads to growth arrest or cell death, whereas antitoxins regulate the expression of toxins, acting as labile antisense RNAs. The Staphylococcus aureus (S. aureus) genome contains and expresses several functional type I TA systems, but their biological functions remain unclear. Here, we addressed and challenged experimentally, by proteomics, if the type I TA system, the SprG1/SprF1 pair, influences the overall gene expression in S. aureus. Deleted and complemented S. aureus strains were analyzed for their proteomes, both intracellular and extracellular, during growth. Comparison of intracellular proteomes among the strains points to the SprF1 antitoxin as moderately downregulating protein expression. In the strain naturally expressing the SprG1 toxin, cytoplasmic proteins are excreted into the medium, but this is not due to unspecific cell leakages. Such a toxin-driven release of the cytoplasmic proteins may modulate the host inflammatory response that, in turn, could amplify the S. aureus infection spread.

Human skin microbiota-friendly lysostaphin.

Growing antibiotic resistance of bacteria is a burning problem of human and veterinary medicine. Expansion and introduction of novel microbicidal therapeutics is highly desirable. However, antibiotic treatment disturbs the balance of physiological microbiota by changing its qualitative and/or quantitative composition, resulting in a number of adverse effects that include secondary infections. Although such dysbiosis may be reversed by the treatment with probiotics, a more attractive alternative is the use of antibiotics that target only pathogens, while sparing the commensals. Here, we describe lysostaphin LSp222, an enzyme produced naturally by Staphylococcus pseudintermedius 222. LSp222 is highly effective against S. aureus, including its multi-drug resistant strains. Importantly, the inhibitory concentration for S. epidermidis, the predominant commensal in healthy human skin, is at least two orders of magnitude higher compared to S. aureus. Such significant therapeutic window makes LSp222 a microbiota-friendly antibacterial agent with a potential application in the treatment of S. aureus-driven skin infections.

Application of Two-Dimensional Difference Gel Electrophoresis in Identification of Factors Responsible for Virulence of Staphylococcus aureus.

Staphylococcus aureus is a dangerous opportunistic pathogen of humans and animals. Highly virulent and multi-antibiotic-resistant strains are of particular concern due to high invasiveness and limited array of useful treatment options. Proteomics allows identification and investigation of staphylococcal virulence factors to better understand and treat the related disease. Two-dimensional difference gel electrophoresis (2D DIGE) is a powerful method for identification of differences in staphylococcal proteomes, both intracellular and secretory. Not only the presence of particular proteins and their quantities may be determined, but also each modification changing the molecular mass and/or isoelectric point of a protein is trackable. Especially, 2D DIGE allows for detection of posttranslational modifications, including processing and degradation by proteases. For differential analysis, protein samples are labeled with spectrally distinguishable fluorescent dyes, mixed and separated according to their isoelectric point (first dimension), and then electrophoresed in the presence of sodium dodecyl sulfate according to their molecular mass (second dimension). Exceptional resolution of 2D DIGE allows to obtain focused and sharp protein spots, and identify a large number of differentiating proteins. Here we provide protocols for TRI Reagent-based preparation of high-quality samples for 2D DIGE, sample separation, and ways of handling differentiating protein spots which lead to samples ready for protein identification using MS.

Cold-modulated small proteins abundance in winter triticale (x Triticosecale, Wittm.) seedlings tolerant to the pink snow mould (Microdochium nivale, Samuels and Hallett) infection.

Two winter triticale (x Triticosecale Wittmack) model cultivars: Hewo (tolerant to pink snow mould) and Magnat (sensitive) were used to test the effect of cold-hardening (4 weeks at 4°C) on soluble ≤50 kDa protein profiles of the seedling leaves. The presence and abundance of individual proteins were analysed via two-dimensional gel electrophoresis (2-DE) and Surface-Enhanced Laser Desorption/Ionization Time-of-Flight (SELDI-TOF). Up to now, no proteomics analysis of triticale response to hardening has been performed. Thus, the present paper is the first in the series describing the obtained results. In our experiments, the exposure to the low temperature-induced only quantitative changes in the leaves of both cultivars, causing either an increase or decrease of 4-50 kDa protein abundance. Among proteins which were cold-accumulated in cv. Hewo's leaves, we identified two thioredoxin peroxidases (chloroplastic thiol-specific antioxidant proteins) as well as mitochondrial- ß-ATP synthase subunit and ADP-binding resistance protein. On the contrary, in hardened seedlings of this genotype, we observed the decreased level of chloroplastic RuBisCO small subunit PW9 and epidermal peroxidase 10. Simultaneous SELDI-TOF analysis revealed several low mass proteins better represented in cold-hardened plants of tolerant genotype in comparison to the sensitive one and the impact of both genotype and temperature on their level. Based on those results, we suggest that indicated proteins might be potential candidates for molecular markers of cold-induced snow mould resistance of winter triticale and their role is worth to be investigated in the further inoculation experiments.

Proteomic characterization of early lung response to breast cancer metastasis in mice.

INTRODUCTION: The tumor-promoting rearrangement of the lungs facilitates the process of cancer cell survival in a foreign microenvironment and enables their protection against immune defense. The study aimed to define the fingerprint of the early rearrangement of the lungs via the proteomic profiling of the lung tissue in the experimental model of tumor metastasis in a murine 4T1 mammary adenocarcinoma. MATERIALS AND METHODS: The studies were performed on 7-8-week-old BALB/c female mice. Viable 4T1 cancer cells were orthotopically inoculated into the right mammary fat pad. The experiment was performed in the early phase of the tumor metastasis one and two weeks after cancer cell inoculation. The comparative analysis of protein profiles was carried out with the aid of the two-dimensional difference in gel electrophoresis (2D-DIGE). Proteins, of which expression differed significantly, were identified using nano-liquid chromatography coupled to a high-resolution mass spectrometry (nanoLC/hybrid ion trap- Orbitrap XL Discovery). RESULTS: Palpable primary tumors were noted in the 2nd week after cancer cell inoculation. The investigated period preceded the formation of numerous macrometastases in the lungs, however the metastasis-promoting changes were visible very early. Primary tumor-induced inflammation developed in the lungs as early as after the 1st week and progressed during the 2nd week, accompanied by increased concentration of 2-OH-E+, an oxidative stress marker, and imbalance in nitric oxide metabolites, pointing to endothelium dysfunction. The early proteomic changes in the lungs in the 1st week after 4T1 cell inoculation resulted in the reorganization of lung tissue structure [actin, cytoplasmic 1 (Actb), tubulin beta chain (Tubb5), lamin-B1 (Lmnb1), serine protease inhibitor A3K (Serpina3k)] and activation of defense mechanisms [selenium-binding protein 1 (Selenbp1), endoplasmin (Hsp90b1), stress 70 protein, mitochondrial (Hspa9), heat shock protein HSP 90-beta (Hsp90ab1)], but also modifications in metabolic pathways [glucose-6-phosphate 1-dehydrogenase X (G6pdx), ATP synthase subunit beta, mitochondrial (Atp5b), L-lactate dehydrogenase B chain (Ldhb)]. Further development of the solid tumor after the 2nd week following cancer cell inoculation, secretion of prolific tumor-derived factors as well as the presence of the increasing number of circulating cancer cells and extravasation processes further impose reorganization of the lung tissue [Actb, vimentin (Vim), clathrin light chain A (Clta)], altering additional metabolic pathways [annexin A5 (Anxa5), Rho GDP-dissociation inhibitor 2 (Arhgdib), complement 1 Q subcomponent-binding protein, mitochondrial (C1qbp), 14-3-3 protein zeta/delta (Ywhaz), peroxiredoxin-6 (Prdx6), chitinase-like protein 4 (Chi3l4), reticulocalbin-1 (Rcn1), EF-hand domain-containing protein D2 (Efhd2), calumenin (Calu)]. Interestingly, many of differentially expressed proteins were involved in calcium homeostasis (Rcn1, Efhd2, Calu, Actb, Vim, Lmnb1, Clta, Tubb5, Serpina3k, Hsp90b1, Hsp90ab1, Hspa9. G6pdx, Atp5b, Anxa5, Arhgdib, Ywhaz). CONCLUSION: The analysis enabled revealing the importance of calcium signaling during the early phase of metastasis development, early cytoskeleton and extracellular matrix reorganization, activation of defense mechanisms and metabolic adaptations. It seems that the tissue response is an interplay between pro- and anti-metastatic mechanisms accompanied by inflammation, oxidative stress and dysfunction of the barrier endothelial cells.

Joint Genomic and Proteomic Analysis Identifies Meta-Trait Characteristics of Virulent and Non-virulent Staphylococcus aureus Strains.

Staphylococcus aureus is an opportunistic pathogen of humans and warm-blooded animals and presents a growing threat in terms of multi-drug resistance. Despite numerous studies, the basis of staphylococcal virulence and switching between commensal and pathogenic phenotypes is not fully understood. Using genomics, we show here that S. aureus strains exhibiting virulent (VIR) and non-virulent (NVIR) phenotypes in a chicken embryo infection model genetically fall into two separate groups, with the VIR group being much more cohesive than the NVIR group. Significantly, the genes encoding known staphylococcal virulence factors, such as clumping factors, are either found in different allelic variants in the genomes of NVIR strains (compared to VIR strains) or are inactive pseudogenes. Moreover, the pyruvate carboxylase and gamma-aminobutyrate permease genes, which were previously linked with virulence, are pseudogenized in NVIR strain ch22. Further, we use comprehensive proteomics tools to characterize strains that show opposing phenotypes in a chicken embryo virulence model. VIR strain CH21 had an elevated level of diapolycopene oxygenase involved in staphyloxanthin production (protection against free radicals) and expressed a higher level of immunoglobulin-binding protein Sbi on its surface compared to NVIR strain ch22. Furthermore, joint genomic and proteomic approaches linked the elevated production of superoxide dismutase and DNA-binding protein by NVIR strain ch22 with gene duplications.

Identification of Secreted Exoproteome Fingerprints of Highly-Virulent and Non-Virulent Staphylococcus aureus Strains.

Staphylococcus aureus is a commensal inhabitant of skin and mucous membranes in nose vestibule but also an important opportunistic pathogen of humans and livestock. The extracellular proteome as a whole constitutes its major virulence determinant; however, the involvement of particular proteins is still relatively poorly understood. In this study, we compared the extracellular proteomes of poultry-derived S. aureus strains exhibiting a virulent (VIR) and non-virulent (NVIR) phenotype in a chicken embryo experimental infection model with the aim to identify proteomic signatures associated with the particular phenotypes. Despite significant heterogeneity within the analyzed proteomes, we identified alpha-haemolysin and bifunctional autolysin as indicators of virulence, whereas glutamylendopeptidase production was characteristic for non-virulent strains. Staphopain C (StpC) was identified in both the VIR and NVIR proteomes and the latter fact contradicted previous findings suggesting its involvement in virulence. By supplementing NVIR, StpC-negative strains with StpC, and comparing the virulence of parental and supplemented strains, we demonstrated that staphopain C alone does not affect staphylococcal virulence in a chicken embryo model.

A peptide factor secreted by Staphylococcus pseudintermedius exhibits properties of both bacteriocins and virulence factors.

Staphylococcus pseudintermedius is a common commensal bacterium colonizing the skin and mucosal surfaces of household animals. However, it has recently emerged as a dangerous opportunistic pathogen, comparable to S. aureus for humans. The epidemiological situation is further complicated by the increasing number of methicillin-resistant S. pseudintermedius infections and evidence of gene transmission driving antibiotic resistance between staphylococci colonizing human and zoonotic hosts. In the present study, we describe a unique peptide, BacSp222, that possesses features characteristic of both bacteriocins and virulence factors. BacSp222 is secreted in high quantities by S. pseudintermedius strain 222 isolated from dog skin lesions. This linear, fifty-amino-acid highly cationic peptide is plasmid-encoded and does not exhibit significant sequence similarities to any other known peptides or proteins. BacSp222 kills gram-positive bacteria (at doses ranging from 0.1 to several micromol/l) but also demonstrates significant cytotoxic activities towards eukaryotic cells at slightly higher concentrations. Moreover, at nanomolar concentrations, the peptide also possesses modulatory properties, efficiently enhancing interferon gamma-induced nitric oxide release in murine macrophage-like cell lines. BacSp222 appears to be one of the first examples of multifunctional peptides that breaks the convention of splitting bacteriocins and virulence factors into two unrelated groups.

Proteomics in studies of Staphylococcus aureus virulence.

Staphylococcus aureus is a widespread, opportunistic pathogen that causes community and hospital acquired infections. Its high pathogenicity is driven by multifactorial and complex mechanisms determined by the ability of the bacterium to express a wide variety of virulence factors. The proteome secreted into extracellular milieu is a rich reservoir of such factors which include mainly nonenzymatic toxins and enzymes. Simultaneously, membrane proteins, membrane-cell wall interface proteins and cell wall-associated proteins also strongly influence staphylococcal virulence. Proteomics shows a great potential in exploring the role of the extracellular proteome in cell physiology, including the pathogenic potential of particular strains of staphylococci. In turn, understanding the bacterial physiology including the interconnections of particular factors within the extracellular proteomes is a key to the development of the ever needed, novel antibacterial strategies. Here, we briefly overview the latest applications of gel-based and gel-free proteomic techniques in the identification of the virulence factors within S. aureus secretome and surfacome. Such studies are of utmost importance in understanding the host-pathogen interactions, analysis of the role of staphylococcal regulatory systems and also the detection of posttranslational modifications emerging as important modifiers of the infection process.

A regulatory role for Staphylococcus aureus toxin-antitoxin system PemIKSa.

Toxin-antitoxin systems were shown to be involved in plasmid maintenance when they were initially discovered, but other roles have been demonstrated since. Here we identify and characterize a novel toxin-antitoxin system (pemIKSa) located on Staphylococcus aureus plasmid pCH91. The toxin (PemKSa) is a sequence-specific endoribonuclease recognizing the tetrad sequence U↓AUU, and the antitoxin (PemISa) inhibits toxin activity by physical interaction. Although the toxin-antitoxin system is responsible for stable plasmid maintenance our data suggest the participation of pemIKSa in global regulation of staphylococcal virulence by alteration of the translation of large pools of genes. We propose a common mechanism of reversible activation of toxin-antitoxin systems based on antitoxin transcript resistance to toxin cleavage. Elucidation of this mechanism is particularly interesting because reversible activation is a prerequisite for the proposed general regulatory role of toxin-antitoxin systems.

A novel member of the thermolysin family, cloning and biochemical characterization of metalloprotease from Staphylococcus pseudintermedius.

Thermolysins constitute a family of secreted bacterial metalloproteases expressed, among others, by several pathogens. Strains of Staphylococcus pseudintermedius isolated from diseased dogs and judged as protease-positive, by skim milk agar plate culture, were investigated for protease content. No proteolytic activity was detected when the bacteria were grown in regular liquid media. Unexpectedly, supplementation of the medium with calcium ions resulted in expression of a metalloprotease and profound changes in the profile of extracellular proteins. On the basis of homology to other staphylococcal metalloproteases, the nucleotide sequence of the gene encoding this protease (Pst) and its flanking regions was determined. The full-length pst codes for a protein with an open reading frame of 505 amino acids. The internal region contains the HEXXH catalytic domain that is conserved in members of the thermolysin family. Regardless of the presence of calcium in the medium, the expression of the protease gene was of the same intensity. This suggests that regulation of the metalloprotease production by calcium ions is at a post-transcriptional level. Isolates of S. pseudintermedius exhibit a proteolytic phenotype due to the metalloprotease expression, however only in presence of calcium ions, which most probably stabilize the structure of the protease.

Rat brain proteome in morphine dependence.

The aim of this study was to reveal potential markers associated with drug dependence, using the proteomic approach. Gels containing samples derived from morphine-treated and control animals were compared and analyzed. Inspection of protein profiles, following TCA/acetone precipitation and the use of nano-scale liquid chromatography coupled to tandem mass spectrometry, allowed for identification of eleven potential dependence markers, mainly cytoplasmic and mitochondrial enzymes, e.g. proteins that belong to GTPase and GST superfamilies, ATPase, asparaginase or proteasome subunit p27 families.

Identification of major cellular proteins synthesized in response to interleukin-1 and interleukin-6 in human hepatoma HepG2 cells.

Interleukin-1 (IL-1) and interleukin-6 (IL-6) are principal proinflammatory cytokines inducing the acute phase response of various tissues, including liver. Cultured human hepatoma HepG2 cells were stimulated with IL-1 (10 ng/ml) and IL-6 (10 ng/ml). After 24 h the cells were collected and disrupted by sonication in a lysis buffer containing 8M urea. The extracted cellular proteins were separated by 2D polyacrylamide gel electrophoresis. The gels were stained with Coomassie Brilliant Blue R-250 and the protein spots showing different intensities in comparison to control (unstimulated) cells were excised and subjected to analysis by LC-MS/MS. Alternatively, proteins were stained with SYPRO Ruby. These differentially expressed proteins include seven up-regulated and two down-regulated intracellular proteins of various functions. The identification of three cytokine-responsive proteins was confirmed by biosynthetic labeling with [35S]methionine after incubation of HepG2 cells, and by western blot with specific antisera.