A global Staphylococcus aureus proteome resource applied to the in vivo characterization of host-pathogen interactions.

Data-independent acquisition mass spectrometry promises higher performance in terms of quantification and reproducibility compared to data-dependent acquisition mass spectrometry methods. To enable high-accuracy quantification of Staphylococcus aureus proteins, we have developed a global ion library for data-independent acquisition approaches employing high-resolution time of flight or Orbitrap instruments for this human pathogen. We applied this ion library resource to investigate the time-resolved adaptation of S. aureus to the intracellular niche in human bronchial epithelial cells and in a murine pneumonia model. In epithelial cells, abundance changes for more than 400 S. aureus proteins were quantified, revealing, e.g., the precise temporal regulation of the SigB-dependent stress response and differential regulation of translation, fermentation, and amino acid biosynthesis. Using an in vivo murine pneumonia model, our data-independent acquisition quantification analysis revealed for the first time the in vivo proteome adaptation of S. aureus. From approximately 2.15 × 1

Charakterisierung des extrazellulären Proteoms von Staphylococcus aureus und Analyse der Wirt-Pathogen Interaktionen bei der Infektion humaner Epithelzellen mit Staphylococcus aureus

Staphylococcus aureus ist ein ubiquitär verbreitetes Bakterium. Häufig als Kommensale des Menschen vorkommend, zählt das Bakterium jedoch zu einem der wichtigsten Infektionserreger des 21. Jahrhunderts. Neben lokalen Infektionen (z. B. Furunkel) kann der Erreger nach einer Besiedlung auch systemische Erkrankungen in seinem Wirt (z. B. Sepsis, Endokarditis, Pneumonie) hervorrufen. Die pathogene Wirkung von S. aureus ist auf die Produktion und Sekretion von Pathogenitäts- bzw. Virulenzfaktoren, unter anderem Superantigene, hämolytische Toxine, Gewebe-zerstörende Enzyme und Oberflächenproteine, welche ihrerseits mit dem Immunsystem des Wirtes interferieren, zurückzuführen. Ziel dieser Arbeit war unter anderem die Analyse des extrazellulären Proteoms von S. aureus RN1HG in pMEM, ein an das bakterielle Wachstum adaptierte Zellkulturmedium. Bei den extrazellulären Proteomanalysen von S. aureus RN1HG konnten 39 Proteine identifiziert werden, welche dem Bakterium eine Interaktion mit dem Wirt (Clumping-Faktoren) ermöglichen, die Phagozytose (Protein A) verhindern oder die Ausbreitung im Gewebe (alpha-Hämolysin, gamma-Hämolysin, Lipase) erleichtern. Da die Zusammensetzung des extrazellulären Proteoms durch diverse Regulons (z. B. agr-System, sarA, sigB) bestimmt wird, stellte sich die Frage, inwiefern diese einen Einfluss auf die Virulenz des Stammes RN1HG-Stamm haben. Ein vielfach in der Literatur diskutierter Regulator ist SigB. Die vergleichende gelfreie LC-MS/MS-Analyse des extrazellulären Proteoms von S. aureus RN1HG mit einer sigB Deletion (RN1HG delta sigB) zeigte, dass sich im Vergleich zum Wildtyp die Zusammensetzung des extrazellulären Proteoms nicht grundsätzlich ändert. Jedoch konnte durch eine „labelfreie“ Quantifizierung eine verstärkte Akkumulation zahlreicher Virulenzfaktoren (z. B. Aureolysin, 1-Phosphatidylinositol- Phosphodiesterase, alpha-Hämolysin, gamma-Hämolysin, Lipase, Thermonuklease) in der delta sigB Mutante nachgewiesen werden. Die Serin-Proteasen A, C und E konnten nur für die delta sigB Mutante identifiziert werden. Adhäsine, darunter Clumping-Faktoren oder Elastin-Bindeprotein, wurden lediglich während der exponentiellen Wachstumsphase für die delta sigB Mutante nachgewiesen. Dies konnte für clf auch durch Transkriptomanalysen belegt werden. Die gelfreien Analysen wurden durch gelbasierte Verfahren (2D-Gelelektrophorese) ergänzt. Neben der Erstellung einer Referenzkarte des extrazellulären Proteoms von S. aureus RN1HG (Wildtyp und delta sigB Mutante) wurden quantitative gelbasierte Daten erhoben, die einerseits die Ergebnisse der gelfreien Analysen bestätigten, andererseits aber auch zeigten, dass SigB nur wenig Einfluss auf die Prozessierung und posttranslationale Modifikation extrazellulärer Proteine in S. aureus RN1HG hat. Die Zusammensetzung des extrazellulären Proteoms ist vor allem bei pathogenen Bakterien bedeutsam, da z. B. durch extrazelluläre Enzyme die Erschließung von Nährstoffquellen in extremen Habitaten begünstigt und durch Virulenzfaktoren sowohl die Kolonisierung als auch die Überlebensfähigkeit im Wirtsorganismus gesichert wird. Um die Erreger-Wirt Interaktion näher zu charakterisieren, wurde die Reaktion von humanen bronchialen Epithelzellen (S9-Zellen) auf eine Infektion mit S. aureus RN1GH pMV158 untersucht. Die Durchführung der Infektionsstudien mit einem GFP-markierten RN1HG-Stamm ermöglichte die Sortierung der infizierten S9-Zellen durch die Durchflusszytometrie. Da im Epithelverband nicht jede Zelle mit S. aureus infiziert ist, lag der Vorteil der Sortierung darin, dass Proteomanalysen spezifisch für die S9-Zellen mit internalisierten Staphylokokken durchgeführt werden konnten. Infolge einer Internalisierung von S. aureus durch die S9-Epithelzellen kam es zunächst zu einer Integrin-vermittelten Adhäsion. Eine zunehmende Inkubation mit S. aureus führte zu inflammatorischen Prozessen. Die Invasion pathogener Bakterien in Wirtzellen führt somit zum Remodelling biologischer Prozesse, die dem Wirt die Auseinandersetzung mit dem Pathogen ermöglichen.Staphylococcus aureus is an ubiquitous microorganism. Apart from being a permanently colonizer of the human skin, the bacteria is one of the most important pathogen in infection disease of the 21th century. In addition to local infections (e.g. furuncles) the pathogen causes systemic diseases (e.g. septicemia, endocarditis, pneumonia). The pathogenicity of S. aureus is based on the production and secretion of virulence factors, including superantigens, hemolysines, tissue-disruptive enzymes and surface proteins, inferring with the immune system of the host. One aim of this project was the analysis of the extracellular proteome of S. aureus RN1HG in pMEM, an eukaryotic cell culture media adapted to bacterial growth. The analysis of the extracellular proteome analysis of S. aureus RN1HG revealed 39 proteins, which are involved in the interaction with the host (clumping-factors), avoiding the phagocytosis (protein A) or facilitating the bacterial spread (alpha-hemolysin, gamma-hemolysin, lipase). Since the composition of the extracellular proteome depends on different regulons (e.g. agr-system, sarA, sigB) their influence on the impact of virulence in the strain RN1HG has to be examined. One of the most important regulon is SigB. A comparative gelfree LC-MS/MS analysis of the extracellular proteome of S. aureus RN1HG and its sigB deletion mutant (RN1HG delta sigB) revealed that the composition of the extracellular proteome is not significantly different. However, “labelfree” quantification of the proteins displayed an increased accumulation of virulence factors (e.g. aureolysin, 1-phosphatidylinositol-phosphodiesterase, alpha-hemolysin, gamma-hemolysin, lipase, thermonuclease) in the delta sigB mutant. Serine proteases A, C and E were only identified in the delta sigB mutant. Adhesion proteins, among them clumping-factors or elastin binding protein, were detected during the exponential growth phase in the delta sigB mutant. For clf the data were confirmed on transcriptome level. Additionally, classical gelbased approaches (2D-gelelectrophoreses) were established. Besides a reference map of the extracellular proteome of S. aureus RN1HG (wildtype and delta sigB mutant) quantitative analyses were performed, confirming the results from gelfree analysis and showing only little influence of SigB on the processing and posttranslational modification of extracellular proteins in S. aureus RN1HG as well. The composition of the extracellular proteome is mainly important for pathogenic bacteria in facilitating the exploitation of nutrients in extreme habitats through extracellular enzymes or saving the colonization as well as the survival in host organisms. For a detailed characterization of the host-pathogen interaction, the reaction of human bronchial epithelial cells (S9-cell line) after infection with S. aureus RN1HG pMV158 was examined. The use of a GFP-labeled RN1HG strain in the infection experiments allowed the sorting of S9 infected cells via flow cytometry. Since not every cell in a cell layer was infected with S. aureus, the sorting enabled a particular proteomic analysis of S9-cells with internalized staphylococci. As a result of bacterial infection, in the S9-cells an integrin-mediated adhesion was observed in the beginning whereas a prolonged incubation with S. aureus lead to inflammatory processes. In conclusion the data show, that the invasion of pathogenic bacteria leads to a remodeling of biological processes in the host cells displaying a protective response of the host against the pathogen

Study of the neutering impact in canine control population per administrative district in São Paulo

Procurou-se avaliar o impacto das ações de esterilização animal na dinâmica populacional canina no município de São Paulo, bem como sugerir um método de controle a fim de se manter todos os distritos administrativos com uma população homogeneamente castrada. Os resultados foram comparados com as ações realizadas atualmente pelo órgão municipal de controle animal. Foram avaliadas taxas de esterilização de 10, 20, 30, 40, 50 e 60% ano-1 para a população canina total ao longo de 5, 10, 15 e 20 anos aplicando-se modelo matemático de dinâmica populacional para dois sexos, sem diferenciação etária. Demonstrou-se que com uma taxa de 60% ano-1 ocorre diminuição de 56,05% da população canina após 20 anos de programa permanente de esterilização animal. Para se obter 20% da população canina homogeneamente castrada por distrito administrativo no município após 10 anos foi avaliada a necessidade de se utilizar uma taxa mínima de esterilização de 5% ano-1. Verificou-se que a taxa de esterilização de 2,7% realizada em 2011 e 2012 pelo município é baixa para se diminuir a população canina total e que há necessidade de se melhor alocar os locais onde se realizam cirurgias no município, sendo o local de maior urgência a região sul do município, A distribuição homogênea de clínicas conveniadas à prefeitura em cada um dos distritos administrativos apresentaria melhores resultados em relação aos mutirões.This work aimed to evaluate the impact in the canine population dynamics through the neutering activities done by São Paulo city, as well as suggest a control method in order to maintain a homogenous neutered population in each administrative districts. The results were compared with the activities currently done by the animal control organization. 10, 20, 30, 40, 50 and 60% annual neutering rates were evaluated applying a mathematical model for two sexes, without age differentiation. It was demonstrated that applying a 60% annual rate produces 56,05% diminishment in the canine population after 20 years of neutering permanent activities. In order to obtain 20% of the canine population homogeneously neutered per administrative district a minimum 5% neuter rate would be necessary. It was verified that the 2,7% neuter rate applied by the municipality in 2011 and 2012 was low in order to diminish the canine population and that it is necessary to relocate the places where most the surgeries are done, more urgently the south region of São Paulo. The homogenous distribution of hired clinics by the municipality in each administrative district would present better results than the neutering campaigns currently done by the animal control organization

Two monoclonal antibodies against glycoprotein Gn protect mice from Rift Valley Fever challenge by cooperative effects.

Rift Valley fever virus (RVFV) is a zoonotic arbovirus that causes severe disease in humans and ruminants. The infection is characterized by abortions in pregnant animals, high mortality in neonates as well as febrile illness in humans that develop in 1% of cases encephalitis or hemorrhagic fever. There is presently no specific antiviral treatment for RVFV infection available. In this study, two monoclonal antibodies (mAbs), raised against glycoprotein Gn, were applied in a therapeutic study. Treatment of RVFV infected mice with neutralizing mAb Gn3 alone at two different time points (30 minutes before or 30 minutes after virus challenge) showed only moderate efficacy of about 58.3% survival in both applications. However, a combination therapy together with non-neutralizing mAb Gn32 demonstrated complete protection (100% survival) when applied 30 minutes after the lethal challenge dose. The increase of mAb efficacy is probably based on cooperative neutralization effects. These data suggest that a combination therapy with mAbs Gn3 and Gn32 could be an effective treatment option against RVFV infection

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

Rift Valley Fever Virus Non-Structural Protein S Is Associated with Nuclear Translocation of Active Caspase-3 and Inclusion Body Formation

Rift Valley fever phlebovirus (RVFV) causes Rift Valley fever (RVF), an emerging zoonotic disease that causes abortion storms and high mortality rates in young ruminants as well as severe or even lethal complications in a subset of human patients. This study investigates the pathomechanism of intranuclear inclusion body formation in severe RVF in a mouse model. Liver samples from immunocompetent mice infected with virulent RVFV 35/74, and immunodeficient knockout mice that lack interferon type I receptor expression and were infected with attenuated RVFV MP12 were compared to livers from uninfected controls using histopathology and immunohistochemistry for RVFV nucleoprotein, non-structural protein S (NSs) and pro-apoptotic active caspase-3. Histopathology of the livers showed virus-induced, severe hepatic necrosis in both mouse strains. However, immunohistochemistry and immunofluorescence revealed eosinophilic, comma-shaped, intranuclear inclusions and an intranuclear (co-)localization of RVFV NSs and active caspase-3 only in 35/74-infected immunocompetent mice, but not in MP12-infected immunodeficient mice. These results suggest that intranuclear accumulation of RVFV 35/74 NSs is involved in nuclear translocation of active caspase-3, and that nuclear NSs and active caspase-3 are involved in the formation of the light microscopically visible inclusion bodies

Vaccine efficacy of self-assembled multimeric protein scaffold particles displaying the glycoprotein Gn head domain of rift valley fever virus

Compared to free antigens, antigens immobilized on scaffolds, such as nanoparticles, generally show improved immunogenicity. Conventionally, antigens are conjugated to scaffolds through genetic fusion or chemical conjugation, which may result in impaired assembly or hetero-geneous binding and orientation of the antigens. By combining two emerging technologies—i.e., self-assembling multimeric protein scaffold particles (MPSPs) and bacterial superglue—these short-comings can be overcome and antigens can be bound on particles in their native conformation. In the present work, we assessed whether this technology could improve the immunogenicity of a candidate subunit vaccine against the zoonotic Rift Valley fever virus (RVFV). For this, the head domain of glycoprotein Gn, a known target of neutralizing antibodies, was coupled on various MPSPs to further assess immunogenicity and efficacy in vivo. The results showed that the Gn head domain, when bound to the lumazine synthase-based MPSP, reduced mortality in a lethal mouse model and protected lambs, the most susceptible RVFV target animals, from viremia and clinical signs after immunization. Furthermore, the same subunit coupled to two other MPSPs (Geobacillus stearothermophilus E2 or a modified KDPG Aldolase) provided full protection in lambs as well.</p