Reduced interleukin-18 secretion by human monocytic cells in response to infections with hyper-virulent Streptococcus pyogenes.

BACKGROUND: Streptococcus pyogenes (group A streptococcus, GAS) causes a variety of diseases ranging from mild superficial infections of the throat and skin to severe invasive infections, such as necrotizing soft tissue infections (NSTIs). Tissue passage of GAS often results in mutations within the genes encoding for control of virulence (Cov)R/S two component system leading to a hyper-virulent phenotype. Dendritic cells (DCs) are innate immune sentinels specialized in antigen uptake and subsequent T cell priming. This study aimed to analyze cytokine release by DCs and other cells of monocytic origin in response to wild-type and natural covR/S mutant infections. METHODS: Human primary monocyte-derived (mo)DCs were used. DC maturation and release of pro-inflammatory cytokines in response to infections with wild-type and covR/S mutants were assessed via flow cytometry. Global proteome changes were assessed via mass spectrometry. As a proof-of-principle, cytokine release by human primary monocytes and macrophages was determined. RESULTS: In vitro infections of moDCs and other monocytic cells with natural GAS covR/S mutants resulted in reduced secretion of IL-8 and IL-18 as compared to wild-type infections. In contrast, moDC maturation remained unaffected. Inhibition of caspase-8 restored secretion of both molecules. Knock-out of streptolysin O in GAS strain with unaffected CovR/S even further elevated the IL-18 secretion by moDCs. Of 67 fully sequenced NSTI GAS isolates, 28 harbored mutations resulting in dysfunctional CovR/S. However, analyses of plasma IL-8 and IL-18 levels did not correlate with presence or absence of such mutations. CONCLUSIONS: Our data demonstrate that strains, which harbor covR/S mutations, interfere with IL-18 and IL-8 responses in monocytic cells by utilizing the caspase-8 axis. Future experiments aim to identify the underlying mechanism and consequences for NSTI patients.

ACTN2 Mutant Causes Proteopathy in Human iPSC-Derived Cardiomyocytes.

Genetic variants in α-actinin-2 (ACTN2) are associated with several forms of (cardio)myopathy. We previously reported a heterozygous missense (c.740C>T) ACTN2 gene variant, associated with hypertrophic cardiomyopathy, and characterized by an electro-mechanical phenotype in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Here, we created with CRISPR/Cas9 genetic tools two heterozygous functional knock-out hiPSC lines with a second wild-type (ACTN2wt) and missense ACTN2 (ACTN2mut) allele, respectively. We evaluated their impact on cardiomyocyte structure and function, using a combination of different technologies, including immunofluorescence and live cell imaging, RNA-seq, and mass spectrometry. This study showed that ACTN2mut presents a higher percentage of multinucleation, protein aggregation, hypertrophy, myofibrillar disarray, and activation of both the ubiquitin-proteasome system and the autophagy-lysosomal pathway as compared to ACTN2wt in 2D-cultured hiPSC-CMs. Furthermore, the expression of ACTN2mut was associated with a marked reduction of sarcomere-associated protein levels in 2D-cultured hiPSC-CMs and force impairment in engineered heart tissues. In conclusion, our study highlights the activation of proteolytic systems in ACTN2mut hiPSC-CMs likely to cope with ACTN2 aggregation and therefore directs towards proteopathy as an additional cellular pathology caused by this ACTN2 variant, which may contribute to human ACTN2-associated cardiomyopathies.

Alternative fluorescent labeling strategies for characterizing gram-positive pathogenic bacteria: Flow cytometry supported counting, sorting, and proteome analysis of Staphylococcus aureus retrieved from infected host cells.

Staphylococcus aureus is a Gram-positive opportunistic pathogen that is able to cause a broad range of infectious diseases in humans. Furthermore, S. aureus is able to survive inside nonprofessional phagocytic host cell which serve as a niche for the pathogen to hide from the immune system and antibiotics therapies. Modern OMICs technologies provide valuable tools to investigate host-pathogen interactions upon internalization. However, these experiments are often hampered by limited capabilities to retrieve bacteria from such an experimental setting. Thus, the aim of this study was to develop a labeling strategy allowing fast detection and quantitation of S. aureus in cell lysates or infected cell lines by flow cytometry for subsequent proteome analyses. Therefore, S. aureus cells were labeled with the DNA stain SYTO® 9, or Vancomycin BODIPY® FL (VMB), a glycopeptide antibiotic binding to most Gram-positive bacteria which was conjugated to a fluorescent dye. Staining of S. aureus HG001 with SYTO 9 allowed counting of bacteria from pure cultures but not in cell lysates from infection experiments. In contrast, with VMB it was feasible to stain bacteria from pure cultures as well as from samples of infection experiments. VMB can also be applied for histocytochemistry analysis of formaldehyde fixed cell layers grown on coverslips. Proteome analyses of S. aureus labeled with VMB revealed that the labeling procedure provoked only minor changes on proteome level and allowed cell sorting and analysis of S. aureus from infection settings with sensitivity similar to continuous gfp expression. Furthermore, VMB labeling allowed precise counting of internalized bacteria and can be employed for downstream analyses, e.g., proteomics, of strains not easily amendable to genetic manipulation such as clinical isolates. © 2016 International Society for Advancement of Cytometry.

Proteome data of whole saliva which are associated with development of oral mucositis in head and neck cancer patients undergoing radiotherapy.

Saliva as major human body fluid may act as an indicator of oral disease status. Oral mucositis is a common and often treatment-limiting side effect of radiotherapy for head and neck cancer patients. In this dataset, we provide the complete proteome dataset (raw and search files) of the patients at baseline of radiotherapy treatment in patients undergoing radiotherapy analyzed by nano liquid chromatography coupled to mass spectrometry (LC-MS/MS). In the data set, 5323 tryptic peptides were identified which can be assigned to 487 distinct proteins (≥2 peptides). The MS data have been deposited to the ProteomeXchange ("ProteomeXchange provides globally coordinated proteomics data submission and dissemination" [1]) via the PRIDE partner repository with the dataset identifier PRIDE: PXD003230. The data are associated with the previously published work, "Differences in the whole saliva baseline proteome profile associated with development of oral mucositis in head and neck cancer patients undergoing radiotherapy" [2].

Differences in the whole saliva baseline proteome profile associated with development of oral mucositis in head and neck cancer patients undergoing radiotherapy.

Oral mucositis (OM) is a common, painful and often treatment-limiting side effect of radiotherapy (RT) for head and neck cancer (HNC) patients. Unstimulated saliva was collected before the first radiotherapy application in 50 HNC patients. 41 out of 50 patients developed OM (grade III) during radiotherapy, of which 14 patients even displayed an early OM (grade III) at a low radiation dose of 30Gy. Nine patients did not develop OM (grade III). Using an LC-MS/MS approach 5323 tryptic peptides were assigned to 487 distinct proteins (≥2 peptides) in the data set. The levels of 48 proteins differed significantly (p<0.05) between patients developing OM or not. 17 proteins displayed increased levels (≥1.3-fold) and 31 proteins decreased in level in OM, respectively. Furthermore, using partial least square analysis protein patterns could be used to distinguish subjects which did not develop grade III OM even after 70Gy total dose (n=9) and those displaying early OM (grade III at <30Gy total dose, n=14). Using leave one out cross validation 37 of 41 patients (90%) developing OM could be correctly assigned indicating that prognostic proteome signatures may help identify patients that should be specifically monitored to increase overall effectiveness of RT treatment.

Cohort profile: Greifswald approach to individualized medicine (GANI_MED).

BACKGROUND: Individualized Medicine aims at providing optimal treatment for an individual patient at a given time based on his specific genetic and molecular characteristics. This requires excellent clinical stratification of patients as well as the availability of genomic data and biomarkers as prerequisites for the development of novel diagnostic tools and therapeutic strategies. The University Medicine Greifswald, Germany, has launched the "Greifswald Approach to Individualized Medicine" (GANI_MED) project to address major challenges of Individualized Medicine. Herein, we describe the implementation of the scientific and clinical infrastructure that allows future translation of findings relevant to Individualized Medicine into clinical practice. METHODS/DESIGN: Clinical patient cohorts (N > 5,000) with an emphasis on metabolic and cardiovascular diseases are being established following a standardized protocol for the assessment of medical history, laboratory biomarkers, and the collection of various biosamples for bio-banking purposes. A multi-omics based biomarker assessment including genome-wide genotyping, transcriptome, metabolome, and proteome analyses complements the multi-level approach of GANI_MED. Comparisons with the general background population as characterized by our Study of Health in Pomerania (SHIP) are performed. A central data management structure has been implemented to capture and integrate all relevant clinical data for research purposes. Ethical research projects on informed consent procedures, reporting of incidental findings, and economic evaluations were launched in parallel.

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.

Bone marrow-derived macrophages from BALB/c and C57BL/6 mice fundamentally differ in their respiratory chain complex proteins, lysosomal enzymes and components of antioxidant stress systems.

Macrophages are essential components of the innate immune system and crucial for pathogen elimination in early stages of infection. We previously observed that bone marrow-derived macrophages (BMMs) from C57BL/6 mice exhibited increased killing activity against Burkholderia pseudomallei compared to BMMs from BALB/c mice. This effect was particularly pronounced when cells were treated with IFN-γ. To unravel mechanisms that could explain these distinct bactericidal effects, a comparative combined proteome and transcriptome analysis of untreated and IFN-γ treated BALB/c and C57BL/6 BMMs under standardized serum-free conditions was carried out. We found differences in gene expression/protein abundance belonging to cellular oxidative and antioxidative stress systems. Genes/proteins involved in the generation of oxidant molecules and the function of phagosomes (respiratory chain ATPase, lysosomal enzymes, cathepsins) were predominantly higher expressed/more abundant in C57BL/6 BMMs. Components involved in alleviation of oxidative stress (peroxiredoxin, mitochondrial superoxide dismutase) were more abundant in C57BL/6 BMMs as well. Thus, C57BL/6 BMMs seemed to be better equipped with cellular systems that may be advantageous in combating engulfed pathogens. Simultaneously, C57BL/6 BMMs were well protected from oxidative burst. We assume that these variations co-determine differences in resistance between BALB/c and C57BL/6 mice observed in many infection models. BIOLOGICAL SIGNIFICANCE: In this study we performed combined transcriptome and proteome analyses on BMMs derived from two inbred mouse strains that are frequently used for studies in the field of host-pathogen interaction research. Strain differences between BALB/c and C57BL/6 BMMs were found to originate mainly from different protein abundance levels rather than from different gene expression. Differences in abundance of respiratory chain complexes and lysosomal proteins as well as differential regulation of components belonging to various antioxidant stress systems help to explain long-known differences between the mouse strains concerning their different susceptibility in several infection models.

Proteomic analyses of age related changes in A.BY/SnJ mouse hearts.

BACKGROUND: A.BY/SnJ mice are used to study pathological alterations in the heart due to enteroviral infections. Since age is a well-known factor influencing the susceptibility of mice to infection, response to stress and manifestation of cardiovascular diseases, the myocardial proteome of A.BY/SnJ mice aged 1 and 4 months was comparatively studied using two dimensional-differential in-gel electrophoresis (2D-DIGE) and liquid chromatography tandem mass spectrometry (LC-MS/MS). RESULTS: Complementary analyses by 2D-DIGE and gel-free LC-MS/MS revealed 96 distinct proteins displaying age associated alterations in their levels. Proteins related to protein transport, and transport chain, lipid metabolism and fatty acid transport showed significant changes in 4 months old mouse hearts compared to juvenile hearts. Proteins involved in lipid metabolism and transport were identified at significantly higher levels in older mice and dysregulation of proteins of the respiratory transport chain were observed. CONCLUSION: The current proteomics study discloses age dependent changes occurring in the hearts already in young mice of the strain A.BY/SnJ. Besides alterations in protein transport, we provide evidence that a decrease of ATP synthase in murine hearts starts already in the first months of life, leading to well-known low expression levels manifested in old mice thereby raising the possibility of reduced energy supply. In the first few months of murine life this seems to be compensated by an increased lipid metabolism. The functional alterations described should be considered during experimental setups in disease related studies.

Identification of periodontitis associated changes in the proteome of whole human saliva by mass spectrometric analysis.

AIM: Interest in human saliva proteomics for disease-specific biomarker screening increased in the last decade. We used whole saliva samples from periodontally healthy and diseased subjects with chronic periodontitis to screen for disease-associated differences in the protein pattern. MATERIAL AND METHODS: We selected 20 periodontally healthy and 20 periodontally diseased subjects from the population-based cross-sectional Study of Health in Pomerania (SHIP-2 and SHIP-Trend). Saliva collection was performed with commercially available Salivette(®) (Sarstedt, Nümbrecht, Germany). Whole saliva proteins were analysed after trichloroacetic acid (TCA) precipitation and proteolytic digestion with trypsin by LC-MS/MS. MS-data were analysed and quantified using the Rosetta Elucidator software package. RESULTS: In whole saliva we identified 344 human protein groups across all samples. For label free quantitation we only considered 152 proteins identified with more than one unique peptide. In total, 20 proteins showed 1.5-fold difference in abundance between controls and patients (p < 0.05); the majority of these proteins showed higher abundance in the periodontally diseased subjects. Functional annotation of proteins linked the periodontally diseased status with acute phase response and inflammatory processes. CONCLUSION: Label free proteomic analysis of whole saliva is a powerful tool to characterize the periodontal disease status and differentiate between healthy and periodontally diseased subjects.

Comparative evaluation of saliva collection methods for proteome analysis.

BACKGROUND: Saliva collection devices are widely used for large-scale screening approaches. This study was designed to compare the suitability of three different whole-saliva collection approaches for subsequent proteome analyses. METHODS: From 9 young healthy volunteers (4 women and 5 men) saliva samples were collected either unstimulated by passive drooling or stimulated using a paraffin gum or Salivette® (cotton swab). Saliva volume, protein concentration and salivary protein patterns were analyzed comparatively. RESULTS: Samples collected using paraffin gum showed the highest saliva volume (4.1±1.5 ml) followed by Salivette® collection (1.8±0.4 ml) and drooling (1.0±0.4 ml). Saliva protein concentrations (average 1145 µg/ml) showed no significant differences between the three sampling schemes. Each collection approach facilitated the identification of about 160 proteins (≥2 distinct peptides) per subject, but collection-method dependent variations in protein composition were observed. CONCLUSION: Passive drooling, paraffin gum and Salivette® each allows similar coverage of the whole saliva proteome, but the specific proteins observed depended on the collection approach. Thus, only one type of collection device should be used for quantitative proteome analysis in one experiment, especially when performing large-scale cross-sectional or multi-centric studies.

Viral myocarditis induced by Coxsackievirus B3 in A.BY/SnJ mice: analysis of changes in the myocardial proteome.

Enteroviral myocarditis displays highly diverse clinical phenotypes ranging from mild dyspnoea or chest pain to cardiogenic shock and death. Despite detailed studies of the virus life cycle in vitro and in vivo, the molecular interplay between host and virus in disease progression is largely unresolved. Murine models of Coxsackievirus B3 (CVB3)-induced myocarditis well mimic the human disease patterns and can thus be explored to study mechanisms leading from acute to chronic myocarditis. Here, we present a 2-D gel-based proteomic survey of the changes in the murine cardiac proteome that occurs following infection with CVB3. In total, 136 distinct proteins were affected. Proteins, which are involved in immunity and defense and protein metabolism/modification displayed pronounced changes in intensity not only during acute but also at later stages of CVB3 myocarditis. Proteins involved in maintenance of cell structure and associated proteins were particularly influenced in the acute phase of myocarditis, whereas reduction of levels of metabolic enzymes was observed in chronic myocarditis. Studies about changes in protein intensities were complemented by an analysis of protein phosphorylation that revealed infection-associated changes in the phosphorylation of myosin binding protein C, atrial and ventricular isoforms of myosin regulatory light chain 2, desmin, and Rab GDP dissociation inhibitor beta-2.

Proteomic analysis of doxorubicin-induced changes in the proteome of HepG2cells combining 2-D DIGE and LC-MS/MS approaches.

HepG-2 cells are widely used as a cell model to investigate hepatocellular carcinomas and the effect of anticancer drugs such as doxorubicin, an effective antineoplastic agent, which has broad antitumoral activity against many solid and hematological malignancies. To investigate the effect of doxorubicin on the protein pattern, we used complementary proteomic workflows including 2-D gel-based and gel-free methods. The analysis of crude HepG2 cell extracts by 2-D DIGE provided data on 1835 protein spots which was then complemented by MS-centered analysis of stable isotope labeling by amino acids in cell culture-labeled cells. The monitoring of more than 1300 distinct proteins, including proteins of the membrane fraction provides the most comprehensive overview on the proteome of the widely used model cell line HepG2. Of the proteins monitored in total, 155 displayed doxorubicin-induced changes in abundance. Functional analysis revealed major influences of doxorubicin on proteins involved in protein synthesis, DNA damage control, electron transport/mitochondrial function, and tumor growth. The strongest decrease in level was found for proteins involved in DNA replication and protein synthesis, whereas proteins with a function in DNA damage control and oxidative stress management displayed increased levels following treatment with doxorubicin compared with control cells. Furthermore, the doxorubicin-associated increase in levels of multiple forms of keratins 8, 18, and 19 and other structural proteins revealed an influence on the cytoskeleton network.

Synthesis of new N-analogous corollosporine derivatives with antibacterial activity by laccase-catalyzed amination.

Corollosporine isolated from the marine fungus Corollospora maritima and N-analogous corollosporines are antimicrobial substances. Owing to the basic structure of the N-analogous corollosporines, they have become an attractive target for laccase-catalyzed derivatisation. In this regard we report on the straightforward laccase-catalyzed amination of dihydroxylated arenes with N-analogous corollosporines. In biological assays the obtained amination products are more active than the parent compounds.