Small colony variants of Staphylococcus aureus reveal distinct protein profiles

Small-colony variants (SCVs) of Staphylococcus aureus represent a slow-growing subpopulation causing chronic and relapsing infections due to their physiological adaptation on an intracellular lifestyle. In this first proteomic study on physiological changes associated with a natural, clinically derived SCV, its proteomic profile was investigated in comparison to corresponding isogenic strains displaying normal (clinical wild-type strain, complemented hemB mutant and spontaneous revertant of the clinical SCV) and SCV phenotypes (hemB mutant and gentamicin-induced SCV). Applying an ultra-high resolution chromatography and high mass accuracy MS(E) -based label-free relative and absolute protein quantification approach, the whole cytoplasmic proteome of this strain sextet was investigated in a growth phase-controlled manner covering early-exponential, late-exponential and stationary phases. Of 1019 cytoplasmic proteins identified, 154 were found to be differently regulated between strains. All SCV phenotypes showed down-regulation of the tricarboxylic acid (TCA) cycle-related proteins and of a protein cluster involved in purine/pyrimidine and folate metabolism. In contrast to hemB mutant and gentamicin-induced SCVs, the clinically derived SCVs showed no prominent up-regulation of glycolytic proteins. The spontaneous switch into the normal phenotype resulted in up-regulation of TCA cycle-related parts, while oxidative stress-related proteins were down-regulated. However, the natural revertant from the clinical SCV retained also dominant protein features of the clinical SCV phenotype. In conclusion, physiological changes between normal and SCV S. aureus phenotypes are more complex than reflected by defined electron transport chain-interrupting mutants and their complemented counterparts.     

Gram‐positive bacteria produce membrane vesicles: Proteomics‐based characterization of Staphylococcus aureus‐derived membrane vesicles

Although archaea, Gram‐negative bacteria, and mammalian cells constitutively secrete membrane vesicles (MVs) as a mechanism for cell‐free intercellular communication, this cellular process has been overlooked in Gram‐positive bacteria. Here, we found for the first time that Gram‐positive bacteria naturally produce MVs into the extracellular milieu. Further characterizations showed that the density and size of Staphylococcus aureus‐derived MVs are both similar to those of Gram‐negative bacteria. With a proteomics approach, we identified with high confidence a total of 90 protein components of S. aureus‐derived MVs. In the group of identified proteins, the highly enriched extracellular proteins suggested that a specific sorting mechanism for vesicular proteins exists. We also identified proteins that facilitate the transfer of proteins to other bacteria, as well to eliminate competing organisms, antibiotic resistance, pathological functions in systemic infections, and MV biogenesis. Taken together, these observations suggest that the secretion of MVs is an evolutionally conserved, universal process that occurs from simple organisms to complex multicellular organisms. This information will help us not only to elucidate the biogenesis and functions of MVs, but also to develop therapeutic tools for vaccines, diagnosis, and antibiotics effective against pathogenic strains of Gram‐positive bacteria.     

Comparative Proteomic Profiling of Methicillin‐Susceptible and Resistant Staphylococcus aureus

Staphylococcus aureus is a highly successful human pathogen responsible for a wide range of infections. This study provides insights into the virulence, pathogenicity, and antimicrobial resistance determinants of methicillin‐susceptible and methicillin‐resistant S. aureus (MSSA; MRSA) recovered from non‐healthcare environments. Three environmental MSSA and three environmental MRSA are selected for proteomic profiling using isobaric tag for relative and absolute quantitation tandem mass spectrometry (iTRAQ MS/MS). Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway annotation are applied to interpret the functions of the proteins detected. 792 proteins are identified in MSSA and MRSA. Comparative analysis of MRSA and MSSA reveals that 8 of out 792 proteins are upregulated and 156 are downregulated. Proteins that have differences in abundance are predominantly involved in catalytic and binding activity. Among 164 differently abundant proteins, 29 are involved in pathogenesis, antimicrobial resistance, stress response, mismatch repair, and cell wall synthesis. Twenty‐two proteins associated with pathogenicity including SPA, SBI, CLFA, and DLT are upregulated in MRSA. Moreover, the upregulated pathogenic protein ENTC2 in MSSA is determined to be a super antigen, potentially capable of triggering toxic shock syndrome in the host. Enhanced pathogenicity, antimicrobial resistance, and stress response are observed in MRSA compared to MSSA.     

Proteolysis during long‐term glucose starvation in Staphylococcus aureus COL

A combination of pulse‐chase experiments and 2‐D PAGE revealed that protein degradation appears to play a crucial role for the cell physiology of Staphylococcus aureus COL during extended periods of glucose starvation. The synthesis rate of virtually all cytosolic and radioactively labeled proteins from growing cells seemed dramatically reduced in the first 3.5 h of glucose starvation. The stability of proteins synthesized in growing cells was monitored by a pulse‐chase approach on a proteome wide scale. Especially, enzymes involved in nucleic acid and amino acid biosyntheses, energy metabolism and biosynthesis of cofactors were found rather rapidly degraded within the onset of the stationary phase, whereas the majority of glycolytic and tricarboxylic acid cycle enzymes remained more stable. Furthermore, single enzymes of biosynthetic pathways were differentially degraded. A metabolite analysis revealed that glucose completely depleted from the medium in the transient phase, and amino acids such as alanine and glycine were taken up by the cells in the stationary phase. We suggest that vegetative proteins no longer required in non‐growing cells and thus no longer protected by integration into functional complexes were degraded. Proteolysis of putative non‐substrate‐bound or “unemployed” proteins appears to be a characteristic feature of S. aureus in order to access nutrients as an important survival strategy under starvation conditions.     

Altered immune proteome of Staphylococcus aureus under iron‐restricted growth conditions

Staphylococcus aureus is one of the major causative agents of severe infections, and is responsible for a high burden of morbidity and mortality. Strains of increased virulence have emerged (e.g. USA300) that can infect healthy individuals in the community and are difficult to treat. To add to the knowledge about the pathophysiology of S. aureus, the adaption to iron restriction, an important in vivo stressor, was studied and the corresponding immune response of the human host characterized. Using a combination of 1D and 2D immune proteomics, the human antibody response to the exoproteomes of S. aureus USA300Δspa grown under iron restriction or with excess iron was compared. Human antibody binding to the altered exoproteome under iron restriction showed a 2.7‐ to 6.2‐fold increase in overall signal intensity, and new antibody specificities appeared. Quantification of the secreted bacterial proteins by gel‐free proteomics showed the expected strong increase in level of proteins involved in iron acquisition during iron‐restricted growth compared to iron access. This was accompanied by decreased levels of superantigens and hemolysins. The latter was corroborated by functional peripheral blood mononuclear cell proliferation assays. The present data provide a comprehensive view of S. aureus exoproteome adaptation to iron restriction. Adults have high concentrations of serum antibodies specific for some of the newly induced proteins. We conclude that iron restriction is a common feature of the microenvironment, where S. aureus interacts with the immune system of its human host.     

Time‐resolved quantitative proteome profiling of host–pathogen interactions: The response of Staphylococcus aureus RN1HG to internalisation by human airway epithelial cells

Staphylococcus aureus is a versatile Gram‐positive pathogen that gains increasing importance due to the rapid spreading of resistances. Functional genomics technologies can provide new insights into the adaptational network of this bacterium and its response to environmental challenges. While functional genomics technologies, including proteomics, have been extensively used to study these phenomena in shake flask cultures, studies of bacteria from in vivo settings lack behind. Particularly for proteomics studies, the major bottleneck is the lack of sufficient proteomic coverage for low numbers of cells. In this study, we introduce a workflow that combines a pulse‐chase stable isotope labelling by amino acids in cell culture approach with high capacity cell sorting, on‐membrane digestion, and high‐sensitivity MS to detect and quantitatively monitor several hundred S. aureus proteins from a few million internalised bacteria. This workflow has been used in a proof‐of‐principle experiment to reveal changes in levels of proteins with a function in protection against oxidative damage and adaptation of cell wall synthesis in strain RN1HG upon internalisation by S9 human bronchial epithelial cells.     

A peptide resource for the analysis of Staphylococcus aureus in host–pathogen interaction studies

Staphylococcus aureus is an opportunistic human pathogen, which can cause life‐threatening disease. Proteome analyses of the bacterium can provide new insights into its pathophysiology and important facets of metabolic adaptation and, thus, aid the recognition of targets for intervention. However, the value of such proteome studies increases with their comprehensiveness. We present an MS–driven, proteome‐wide characterization of the strain S. aureus HG001. Combining 144 high precision proteomic data sets, we identified 19 109 peptides from 2088 distinct S. aureus HG001 proteins, which account for 72% of the predicted ORFs. Peptides were further characterized concerning pI, GRAVY, and detectability scores in order to understand the low peptide coverage of 8.7% (19 109 out of 220 245 theoretical peptides). The high quality peptide‐centric spectra have been organized into a comprehensive peptide fragmentation library (SpectraST) and used for identification of S. aureus‐typic peptides in highly complex host–pathogen interaction experiments, which significantly improved the number of identified S. aureus proteins compared to a MASCOT search. This effort now allows the elucidation of crucial pathophysiological questions in S. aureus‐specific host–pathogen interaction studies through comprehensive proteome analysis. The S. aureus‐specific spectra resource developed here also represents an important spectral repository for SRM or for data‐independent acquisition MS approaches. All MS data have been deposited in the ProteomeXchange with identifier PXD000702 ( http://proteomecentral.proteomexchange.org/dataset/PXD000702 ).     

Improved accuracy of cell surface shaving proteomics in Staphylococcus aureus using a false‐positive control

Proteolytic treatment of intact bacterial cells is an ideal means for identifying surface‐exposed peptide epitopes and has potential for the discovery of novel vaccine targets. Cell stability during such treatment, however, may become compromised and result in the release of intracellular proteins that complicate the final analysis. Staphylococcus aureus is a major human pathogen, causing community and hospital‐acquired infections, and is a serious healthcare concern due to the increasing prevalence of multiple antibiotic resistances amongst clinical isolates. We employed a cell surface “shaving” technique with either trypsin or proteinase‐K combined with LC‐MS/MS. Trypsin‐derived data were controlled using a “false‐positive” strategy where cells were incubated without protease, removed by centrifugation and the resulting supernatants digested. Peptides identified in this fraction most likely result from cell lysis and were removed from the trypsin‐shaved data set. We identified 42 predicted S. aureus COL surface proteins from 260 surface‐exposed peptides. Trypsin and proteinase‐K digests were highly complementary with ten proteins identified by both, 16 specific to proteinase‐K treatment, 13 specific to trypsin and three identified in the control. The use of a subtracted false‐positive strategy improved enrichment of surface‐exposed peptides in the trypsin data set to approximately 80% (124/155 peptides). Predominant surface proteins were those associated with methicillin resistance–surface protein SACOL0050 (pls) and penicillin‐binding protein 2′ (mecA), as well as bifunctional autolysin and the extracellular matrix‐binding protein Ebh. The cell shaving strategy is a rapid method for identifying surface‐exposed peptide epitopes that may be useful in the design of novel vaccines against S. aureus.     

Influence of Sae‐regulated and Agr‐regulated factors on the escape of Staphylococcus aureus from human macrophages

Although Staphylococcus aureus is not a classical intracellular pathogen, it can survive within phagocytes and many other cell types. However, the pathogen is also able to escape from cells by mechanisms that are only partially understood. We analysed a series of isogenic S. aureus mutants of the USA300 derivative JE2 for their capacity to destroy human macrophages from within. Intracellular S. aureus JE2 caused severe cell damage in human macrophages and could efficiently escape from within the cells. To obtain this full escape phenotype including an intermittent residency in the cytoplasm, the combined action of the regulatory systems Sae and Agr is required. Mutants in Sae or mutants deficient in the Sae target genes lukAB and pvl remained in high numbers within the macrophages causing reduced cell damage. Mutants in the regulatory system Agr or in the Agr target gene psmα were largely similar to wild‐type bacteria concerning cell damage and escape efficiency. However, these strains were rarely detectable in the cytoplasm, emphasizing the role of phenol‐soluble modulins (PSMs) for phagosomal escape. Thus, Sae‐regulated toxins largely determine damage and escape from within macrophages, whereas PSMs are mainly responsible for the escape from the phagosome into the cytoplasm. Damage of macrophages induced by intracellular bacteria was linked neither to activation of apoptosis‐related caspase 3, 7 or 8 nor to NLRP3‐dependent inflammasome activation.     

New changes in the plasma‐membrane‐associated proteome of rice roots under salt stress

To gain a better understanding of salt stress responses in plants, we used a proteomic approach to investigate changes in rice (Oryza sativa) root plasma‐membrane‐associated proteins following treatment with 150 mmol/L NaCl. With or without a 48 h salt treatment, plasma membrane fractions from root tip cells of a salt‐sensitive rice cultivar, Wuyunjing 8, were purified by PEG aqueous two‐phase partitioning, and plasma‐membrane‐associated proteins were separated by IEF/SDS‐PAGE using an optimized rehydration buffer. Comparative analysis of three independent biological replicates revealed that the expressions of 18 proteins changed by more than 1.5‐fold in response to salt stress. Of these proteins, nine were up‐regulated and nine were down‐regulated. MS analysis indicated that most of these membrane‐associated proteins are involved in important physiological processes such as membrane stabilization, ion homeostasis, and signal transduction. In addition, a new leucine‐rich‐repeat type receptor‐like protein kinase, OsRPK1, was identified as a salt‐responding protein. Immuno‐blots indicated that OsRPK1 is also induced by cold, drought, and abscisic acid. Using immuno‐histochemical techniques, we determined that the expression of OsRPK1 was localized in the plasma membrane of cortex cells in roots. The results suggest that different rice cultivars might have different salt stress response mechanisms.     

Structure and function of the bacillithiol‐S‐transferase BstA from Staphylococcus aureus

Bacillithiol is a low‐molecular weight thiol produced by many gram‐positive organisms, including Staphylococcus aureus and Bacillus anthracis. It is the major thiol responsible for maintaining redox homeostasis and cellular detoxification, including inactivation of the antibiotic fosfomycin. The metal‐dependent bacillithiol transferase BstA is likely involved in these sorts of detoxification processes, but the exact substrates and enzyme mechanism have not been identified. Here we report the 1.34 Å resolution X‐ray crystallographic structure of BstA from S. aureus. Our structure confirms that BstA belongs to the YfiT‐like metal‐dependent hydrolase superfamily. Like YfiT, our structure contains nickel within its active site, but our functional data suggest that BstA utilizes zinc for activity. Although BstA and YfiT both contain a core four helix bundle and coordinate their metal ions in the same fashion, significant differences between the protein structures are described here.     

Penicillin-induced changes in the cell wall composition of Staphylococcus aureus before the onset of bacteriolysis

To analyze if chemical cell wall alterations contribute to penicillin-induced bacteriolysis, changes in the amount, stability, and chemical composition of staphylococcal cell walls were investigated. All analyses were performed before onset of bacteriolysis i.e. during the first 60 min following addition of different penicillin G doses. Only a slight reduction of the amount of cell wall material incorporated after penicillin addition at the optimal lytic concentration was observed as compared to control cells. However, the presence of higher penicillin G concentrations reduced the incorporation of wall material progressively without bacteriolysis. Losses of wall material during isolation of dodecylsulfate insoluble cell walls were monitored to assess the stability of the wall material following penicillin addition. Wall material grown at the lytic penicillin concentration was least stable but about 30% of the newly incorporated wall material withstood even the harsh conditions of mechanical breakage and dodecylsulfate treatment. Dodecylsulfate insoluble cell walls were used for chemical analyses. While peptidoglycan chain length was unaffected in the presence of penicillin, other wall parameters were considerably altered: peptide cross-linking was reduced in the wall material synthesized after addition of penicillin; reductions from approx. 85% in controls to about 60% were similar for lytic and also for very high penicillin concentrations leading to nonlytic death. O-acetylation was also reduced after treatment with penicillin; this effect paralleled the occurence of subsequent bacteriolysis at different drug concentrations. The results are not consistent with hypotheses explaining penicillin-induced lysis as a result of an overall weakened cell wall structure or an overall activation of autolytic wall enzymes but not conflicting with the model that ascribes penicillin-induced bacteriolysis as the result of a very restricted, local perforation of the peripheral cell wall (murosome-induced bacteriolysis).Abbreviations CL Cross-linking - DNFB 2,4-dinitro-1-fluorobenzole - MIC Minimal inhibitory concentration - OD Optical density at 578 nm - PEN Penicillin G     

Anti‐infectious activity of synbiotics in a novel mouse model of methicillin‐resistant Staphylococcus aureus infection

The anti‐infectious activity of synbiotics against methicillin‐resistant Staphylococcus aureus (MRSA) infection was evaluated using a novel lethal mouse model. Groups of 12 mice treated with multiple antibiotics were infected orally with a clinical isolate of MRSA at an inoculum of 10⁸ CFU on day 7 after starting the antibiotics. A dose of 400 mg/kg 5‐fluorouracil (5‐FU) was injected intraperitoneally on day 7 after the infection. A dose of 10⁸ CFU Bifidobacterium breve strain Yakult and 10 mg of galactooligosaccharides (GOS) were given orally to mice daily with the antibiotic treatment until day 28. The intestinal population levels of MRSA in the mice on multiple antibiotics were maintained stably at 10⁸ CFU/g of intestinal contents after oral MRSA infection and the subsequent 5‐FU treatment killed all the mice in the group within 14 days. B. breve administration saved most of the mice, but the synbiotic treatment saved all of the mice from lethal MRSA infection. The synbiotic treatment was effective for the treatment of intestinal infection caused by four MRSA strains with different toxin productions. There was a large difference among the six Bifidobacteria strains that were naturally resistant to the antibacterial drugs used. B. breve in combination with GOS is demonstrated to have valuable preventive and curative effects against even fatal MRSA infections.     

Growth inhibition of Staphylococcus aureus induced by low‐frequency electric and electromagnetic fields

Magnetic field therapy is an established technique in the treatment of pseudarthrosis. In cases of osteomylitis, palliation is also observed. This study focuses on the impact of different electric and electromagnetic fields on the growth of Staphylococcus aureus by in vitro technologies. Cultures of Staphylococcus aureus in fluid and gel‐like medium were exposed to a low‐frequency electromagnetic field, an electromagnetic field combined with an additional electric field, a sinusoidal electric field and a static electric field. In gel‐like medium no significant difference between colony‐forming units of exposed samples and non‐exposed references was detected. In contrast, Staphylococcus aureus concentrations in fluid medium could clearly be reduced under the influence of the four different applied fields within 24 h of experiment. The strongest effects were observed for the direct current electric field which could decrease CFU/ml of 37%, and the low‐frequency electromagnetic field with additional induced electric alternating field with a decrease of Staphylococci concentration by 36%. The effects of the electromagnetic treatment on Staphylococci within fluid medium are significantly higher than in gel‐like medium. The application of low‐frequency electromagnetic fields corroborates clinical situations of bone infections during magnetic field therapy. Bioelectromagnetics 30:270–279, 2009. © 2009 Wiley‐Liss, Inc.     

Involvement of endotoxin in the mortality of mice with gut‐derived sepsis due to methicillin‐resistant Staphylococcus aureus

MRSA causes a wide diversity of diseases, ranging from benign skin infections to life‐threatening diseases, such as sepsis. However, there have been few reports of the pathophysiology and mechanisms of sepsis resulting from the gut‐derived origin of MRSA. Therefore, we established a murine model of gut‐derived sepsis with MRSA and factors of MRSA sepsis that cause deterioration. We separated mice into four groups according to antibiotic treatment as follows: (i) ABPC 40 mg/kg; (ii) CAZ 80 mg/kg; (iii) CAZ 80 mg/kg + endotoxin 10 μg/mouse; and (iv) saline‐treated control groups. Gut‐derived sepsis was induced by i.p. injection of cyclophosphamide after colonization of MRSA strain 334 in the intestine. After the induction of sepsis, significantly more CAZ‐treated mice survived compared with ABPC‐treated and control groups. MRSA were detected in the blood and liver among all groups. Endotoxin levels were significantly lower in the CAZ‐treated group compared to other groups. Inflammatory cytokine levels in the serum were lower in the CAZ‐treated group compared to other groups. Fecal culture showed a lower level of colonization of E. coli in the CAZ‐treated group compared to other groups. In conclusion, we found that CAZ‐treatment ameliorates infection and suppresses endotoxin level by the elimination of E. coli from the intestinal tract of mice. However, giving endotoxin in the CAZ‐treated group increased mortality to almost the same level as in the ABPC‐treated group. These results suggest endotoxin released from resident E. coli in the intestine is involved in clinical deterioration resulting from gut‐derived MRSA sepsis.