Interaction of Staphylococcus aureus and Host Cells upon Infection of Bronchial Epithelium during Different Stages of Regeneration

The primary barrier that protects our lungs against infection by pathogens is a tightly sealed layer of epithelial cells. When the integrity of this barrier is disrupted as a consequence of chronic pulmonary diseases or viral insults, bacterial pathogens will gain access to underlying tissues. A major pathogen that can take advantage of such conditions is Staphylococcus aureus, thereby causing severe pneumonia. In this study, we investigated how S. aureus responds to different conditions of the human epithelium, especially nonpolarization and fibrogenesis during regeneration using an in vitro infection model. The infective process was monitored by quantification of the epithelial cell and bacterial populations, fluorescence microscopy, and mass spectrometry. The results uncover differences in bacterial internalization and population dynamics that correlate with the outcome of infection. Protein profiling reveals that, irrespective of the polarization state of the epithelial cells, the invading bacteria mount similar responses to adapt to the intracellular milieu. Remarkably, a bacterial adaptation that was associated with the regeneration state of the epithelial cells concerned the early upregulation of proteins controlled by the redox-responsive regulator Rex when bacteria were confronted with a polarized cell layer. This is indicative of the modulation of the bacterial cytoplasmic redox state to maintain homeostasis early during infection even before internalization. Our present observations provide a deeper insight into how S. aureus can take advantage of a breached epithelial barrier and show that infected epithelial cells have limited ability to respond adequately to staphylococcal insults

Bordetella pertussis outer membrane vesicles as virulence factor vehicles that influence bacterial interaction with macrophages

Gram-negative pathogenic bacteria constitutively shed outer membrane vesicles(OMVs) which play a significant role in the host-pathogen interaction, eventually determiningthe outcome of the infection. We previously found that Bordetella pertussis, the etiologicalagent of whooping cough, survives the innate interaction with human macrophages remainingalive inside these immune cells. Adenylate cyclase (CyaA), one of the main toxins of thispathogen, was found involved in the modulation of the macrophage defense response,eventually promoting bacterial survival within the cells. We here investigated whether B.pertussis OMVs, loaded with most of the bacterial toxins and CyaA among them, modulatethe macrophage response to the bacterial infection. We observed that the pre-incubation ofmacrophages with OMVs led to a decreased macrophage defense response to the encounterwith the bacteria, in a CyaA dependent way. Our results suggest that CyaA delivered by B.pertussis OMVs dampens macrophages protective function by decreasing phagocytosis andthe bactericidal capability of these host cells. By increasing the chances of bacterial survivalto the innate encounter with the macrophages, B. pertussis OMVs might play a relevant rolein the course of infection, promoting bacterial persistence within the host and eventually,shaping the whole infection process.Fil: Blancá, Bruno Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Alvarez Hayes, Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Surmann, Kristin. University Medicine Greifswald; AlemaniaFil: Valdez, Hugo Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Hentschker, Christian. University Medicine Greifswald; AlemaniaFil: Lamberti, Yanina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Völker, Uwe. University Medicine Greifswald; AlemaniaFil: Rodriguez, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; Argentin

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

Proteomic characterization of host-pathogen interactions using human cell lines infected with Staphylococcus aureus HG001 as a model

Staphylococcus aureus is a commensal that colonizes the skin and mucosa of 20-30% of the human population without leading to symptoms of diseases. However, it is also the most important cause of nosocomial infections. Those range from minor skin infections to life-threatening diseases such as pneumonia, endocarditis or septicaemia. Development of strains with resistance against many antibiotics complicates the situation further. The variety of strains with their various properties is one reason why no successful vaccine has been introduced to the market, yet. Therefore, efficient strategies for prevention and therapy of these dangerous infections are urgently needed. To accomplish these goals, the understanding of molecular interactions between host and pathogen is indispensable. Within this dissertation, several internalization experiments were performed aiming to investigate the interaction of S. aureus HG001 and human cell lines upon infection on the protein level. In order to obtain sufficient amounts of proteins for comprehensive physiological interpretations, it is necessary to enrich bacteria, secreted bacterial proteins or infected host cells upon internalization. In the framework of this thesis, bacteria which continuously produce green fluorescent protein (GFP) were employed. With that it was possible to sort bacteria from lysed host cells by flow cytometry or to separate host cells carrying bacteria after contact from those which did not. Subsequently, the proteins were proteolytically digested and peptides were analyzed by mass spectrometry in a gel-free proteomics approach. To allow such analyses also for staphylococci which do not produce GFP, such as clinical isolates, an additional protocol was developed. Prior to the infection, bacteria were labeled with fluorescent or para-magnetic nanoparticles. Afterwards bacteria could be separated from host cell debris by fluorescence-based cell sorting or with the help of a strong magnet. In order to cover also important secreted virulence factors of S. aureus HG001, phagosomes and engulfed bacteria and secreted proteins were isolated from infected host cells. Further steps of protocol optimization included improved bacterial cell counting by fluorescence-based flow cytometry, enhanced data analysis by combination of different search algorithms, and comprehensive functional annotation of proteins of the applied strain by sequence comparison with other strains and organisms. First, the proteome adaptation of internalized S. aureus HG001 and the infected A549 host cells was investigated during the first hours of infection. It became clear, that the bacteria replicate inside the host during the first 6.5 h. After internalization the levels of bacterial enzymes involved in protein biosynthesis decreased. Furthermore, bacteria adapted their proteome to the harsh intracellular conditions such as oxygen limitation, cell wall stress, host defense in terms of oxidative stress, and nutrient limitation. After contact to S. aureus HG001, A549 cells produced increased amounts of cytokines (e.g. IL-8, IFN-γ) in comparison to non-treated A549 cells. In addition, activation of the immunoproteasome and hints of early apoptosis activity were observed. Afterwards, the response of S. aureus HG001 to internalization by A549, S9 or HEK 293 cells was compared on the proteome level. It was obvious, that the adaptation to stress and the reduced protein synthesis are conserved mechanisms. Host dependent differences were detected especially in the energy metabolism and the synthesis of some amino acids. Additionally, bacteria showed different intracellular replication patterns depending on the host cell line. A higher percentage of extracellular bacterial proteins was found in isolated phagosomes compared to the sorted samples. Selected low abundant virulence factors could be quantified at two points in time after infection with the help of the sensitive single reaction monitoring (SRM) method. Further, a heterogeneous mixture of several phagosomal maturation steps was present during the first 6.5 h after infection. Finally, the gel-free proteome analyses could be applied to investigate Bordetella pertussis, the cause of whooping cough, during iron limitation and after internalization, and the results were compared to the S. aureus HG001 data.Staphylococcus aureus ist ein Kommensale, der Haut und Schleimhäute von 20-30% der Menschen besiedelt ohne Krankheitssymptome auszulösen, aber es ist auch der bedeutendste humanpathogene Erreger nosokomialer Infektionen. Das Krankheitsspektrum umfasst leichtere Hautinfektionen sowie lebensbedrohliche Infektionen wie Lungenentzündung, Endokarditis oder Sepsis. Erschwerend kommt hinzu, dass sich hochpathogene Stämme entwickelt haben, die gegen den Großteil derzeit verfügbarer Antibiotika resistent sind. Die Vielzahl der Stämme mit unterschiedlichen Eigenschaften und Anpassungsmöglichkeiten ist ein Grund dafür, dass noch kein erfolgreich einsetzbarer Impfstoff für Menschen auf dem Markt ist. Daher ist es dringend notwendig neue effektive Strategien für Vorbeugung und Therapie zu entwickeln. Dafür ist das Verständnis des Zusammenspiels von Erreger und Wirt auf molekularer Ebene unabdingbar. In dieser Dissertation wurden Internalisierungsexperimente durchgeführt, die es erlaubten das Wechselspiel zwischen S. aureus HG001 und humanen Zelllinien infolge einer Infektion auf Proteinebene zu untersuchen. Um ausreichende Proteinmengen für physiologische Rückschlüsse zu erhalten, ist es nötig Bakterien, sezernierte bakterielle Proteine, beziehungsweise infizierte Wirtszellen nach einer Internalisierung anzureichern. In dieser Arbeit wurden Bakterien verwendet, die kontinuierlich das grün fluoreszierende Protein (GFP) produzieren. Damit war es möglich mittels Durchflusszytometrie Bakterien aus lysierten Wirtszellen zu sortieren. Zudem konnten Wirtszellen, die Bakterien aufgenommen hatten, von denen getrennt werden, die keine Bakterien enthielten. Anschließend wurden die Proteine proteolytisch verdaut und die Peptide mittels gel-freier Proteomanalysen massenspektrometrisch untersucht. Um mit Staphylokokken arbeiten zu können, die kein GFP produzieren, wie zum Beispiel klinische Isolate, wurde ein weiteres Protokoll entwickelt. Dabei wurden die Bakterien vor einer Infektion mit fluoreszierenden oder para-magnetischen Nanopartikeln markiert. Anschließend konnten die Bakterien mittels Durchflusszytometrie oder mit einem starken Magneten von Wirtszelltrümmern getrennt werden. Um wichtige sezernierte Virulenzfaktoren von S. aureus HG001 erfassen zu können, wurden Phagosomen und die darin enthaltenen Bakterien und sezernierten Proteine aus infizierten Zellen angereichert. Weitere Schritte der Protokolloptimierung in dieser Doktorarbeit umfassten unter anderem eine Optimierung der Zellzählung mittels fluoreszenz-gestützter Durchflusszytometrie, verbesserte Datenanalyse durch Kombination verschiedener Suchalgorithmen sowie eine umfangreichere Annotierung der Proteinfunktionen des verwendeten Stammes durch Sequenzvergleiche mit anderen Stämmen bzw. Organismen. Mithilfe der optimierten oder entwickelten Protokolle konnten umfassende Proteomanalysen zu den Wechselwirkungen zwischen S. aureus HG001 sowie den humanen Zelllinien A549, S9 und HEK 293 durchgeführt werden. Zuerst wurden die Proteomanpassungen internalisierter S. aureus HG001 Zellen sowie die der infizierten A549 Wirtszellen in den ersten Stunden der Infektion untersucht. Dabei wurde deutlich, dass sich die Bakterien in den ersten 6,5 h im Wirt vermehren. Nach Internalisierung verringerte sich die Menge der bakteriellen Proteinsyntheseenzyme im Vergleich zu Kontrollbakterien. Außerdem passten die Bakterien ihr Proteom an die harschen intrazellulären Bedingungen wie Sauerstoffmangel, Zellwandstress, Wirtsabwehr in Form von oxidativem Stress sowie Nährstoffmangel an. Nach Kontakt mit S. aureus HG001 produzierten A549 Zellen verstärkt Zytokine (v.a. IL-8, IFN-γ) im Vergleich zu unbehandelten A549 Zellen. Weiterhin wurden eine Aktivierung des Immunoproteasoms sowie Hinweise auf frühe Apoptoseaktivität detektiert. Anschließend wurde die Anpassung von S. aureus HG001 auf Proteomebene nach Internalisierung durch A549, S9 oder HEK 293 Zellen verglichen. Hier wurde deutlich, dass die Anpassung an Stress und eine geringere Proteinsynthese konservierte Antwortmechanismen an eine Internalisierung darstellen. Unterschiede wurden vor allem im Energiestoffwechsel und bei der Synthese von Aminosäuren beobachtet. Zudem zeigten die Bakterien unterschiedliche intrazelluläre Replikationsmuster in Abhängigkeit vom Wirt. In isolierten Phagosomen wurde ein erhöhter Anteil extrazellulärer Bakterienproteine im Vergleich zu den sortierten Proben detektiert. Ausgewählte niedrigabundante Virulenzfaktoren konnten mithilfe der sensitiven SRM Methode zu zwei Zeitpunkten nach der Infektion quantifiziert werden. Zudem wurde festgestellt, dass eine heterogene Mischung verschiedener Phagosomenreifungsstufen in den ersten 6,5 h nach Infektion vorlag. Schließlich konnten die gel-freien Proteomanalysen angewendet werden um den Keuchhustenerreger Bordetella pertussis unter Eisenmangel und nach Internalisierung zu untersuchen und die Ergebnisse mit den Daten für S. aureus HG001 zu vergleichen

The role of the two-component systems Cpx and Arc in protein alterations upon gentamicin treatment in Escherichia coli

Abstract Background The aminoglycoside antibiotic gentamicin was supposed to induce a crosstalk between the Cpx- and the Arc-two-component systems (TCS). Here, we investigated the physical interaction of the respective TCS components and compared the results with their respective gene expression and protein abundance. The findings were interpreted in relation to the global proteome profile upon gentamicin treatment. Results We observed specific interaction between CpxA and ArcA upon treatment with the aminoglycoside gentamicin using Membrane-Strep-tagged protein interaction experiments (mSPINE). This interaction was neither accompanied by detectable phosphorylation of ArcA nor by activation of the Arc system via CpxA. Furthermore, no changes in absolute amounts of the Cpx- and Arc-TCS could be determined with the sensitive single reaction monitoring (SRM) in presence of gentamicin. Nevertheless, upon applying shotgun mass spectrometry analysis after treatment with gentamicin, we observed a reduction of ArcA ~ P-dependent protein synthesis and a significant Cpx-dependent alteration in the global proteome profile of E. coli. Conclusions This study points to the importance of the Cpx-TCS within the complex regulatory network in the E. coli response to aminoglycoside-caused stress

A <i>Bordetella pertussis</i> MgtC homolog plays a role in the intracellular survival

Bordetella pertussis, the causative agent of whooping cough, has the capability to survive inside the host cells. This process requires efficient adaptation of the pathogen to the intracellular environment and the associated stress. Among the proteins produced by the intracellular B. pertussis we identified a protein (BP0414) that shares homology with MgtC, a protein which was previously shown to be involved in the intracellular survival of other pathogens. To explore if BP0414 plays a role in B. pertussis intracellular survival a mutant strain defective in the production of this protein was constructed. Using standard in vitro growth conditions we found that BP0414 is required for B. pertussis growth under low magnesium availability or low pH, two environmental conditions that this pathogen might face within the host cell. Intracellular survival studies showed that MgtC is indeed involved in B. pertussis viability inside the macrophages. The use of bafilomycin A1, which inhibits phagosome acidification, abolished the survival defect of the mgtC deficient mutant strain suggesting that in intracellular B. pertussis the role of MgtC protein is mainly related to the bacterial adaptation to the acidic conditions found inside the of phagosomes. Overall, this work provides an insight into the importance of MgtC in B. pertussis pathogenesis and its contribution to bacterial survival within immune cells.Facultad de Ciencias ExactasCentro de Investigación y Desarrollo en Fermentaciones Industriale

A Bordetella pertussis MgtC homolog plays a role in the intracellular survival.

Bordetella pertussis, the causative agent of whooping cough, has the capability to survive inside the host cells. This process requires efficient adaptation of the pathogen to the intracellular environment and the associated stress. Among the proteins produced by the intracellular B. pertussis we identified a protein (BP0414) that shares homology with MgtC, a protein which was previously shown to be involved in the intracellular survival of other pathogens. To explore if BP0414 plays a role in B. pertussis intracellular survival a mutant strain defective in the production of this protein was constructed. Using standard in vitro growth conditions we found that BP0414 is required for B. pertussis growth under low magnesium availability or low pH, two environmental conditions that this pathogen might face within the host cell. Intracellular survival studies showed that MgtC is indeed involved in B. pertussis viability inside the macrophages. The use of bafilomycin A1, which inhibits phagosome acidification, abolished the survival defect of the mgtC deficient mutant strain suggesting that in intracellular B. pertussis the role of MgtC protein is mainly related to the bacterial adaptation to the acidic conditions found inside the of phagosomes. Overall, this work provides an insight into the importance of MgtC in B. pertussis pathogenesis and its contribution to bacterial survival within immune cells

Improving Proteome Coverage for Small Sample Amounts: An Advanced Method for Proteomics Approaches with Low Bacterial Cell Numbers

Abstract Proteome analyses are often hampered by the low amount of available starting material like a low bacterial cell number obtained from in vivo settings. Here, the single pot solid‐phase enhanced sample preparation (SP3) protocol is adapted and combined with effective cell disruption using detergents for the proteome analysis of bacteria available in limited numbers only. Using this optimized protocol, identification of peptides and proteins for different Gram‐positive and Gram‐negative species can be dramatically increased and, reliable quantification can also be ensured. This adapted method is compared to already established strain‐specific sample processing protocols for Staphylococcus aureus, Streptococcus suis, and Legionella pneumophila. The highest species‐specific increase in identifications is observed using the adapted method with L. pneumophila samples by increasing protein and peptide identifications up to 300% and 620%, respectively. This increase is accompanied by an improvement in reproducibility of protein quantification and data completeness between replicates. Thus, this protocol is of interest for performing comprehensive proteomics analyses of low bacterial cell numbers from different settings ranging from infection assays to environmental samples

Improving Proteome Coverage for Small Sample Amounts: An Advanced Method for Proteomics Approaches with Low Bacterial Cell Numbers

Abstract Proteome analyses are often hampered by the low amount of available starting material like a low bacterial cell number obtained from in vivo settings. Here, the single pot solid‐phase enhanced sample preparation (SP3) protocol is adapted and combined with effective cell disruption using detergents for the proteome analysis of bacteria available in limited numbers only. Using this optimized protocol, identification of peptides and proteins for different Gram‐positive and Gram‐negative species can be dramatically increased and, reliable quantification can also be ensured. This adapted method is compared to already established strain‐specific sample processing protocols for Staphylococcus aureus, Streptococcus suis, and Legionella pneumophila. The highest species‐specific increase in identifications is observed using the adapted method with L. pneumophila samples by increasing protein and peptide identifications up to 300% and 620%, respectively. This increase is accompanied by an improvement in reproducibility of protein quantification and data completeness between replicates. Thus, this protocol is of interest for performing comprehensive proteomics analyses of low bacterial cell numbers from different settings ranging from infection assays to environmental samples

Exploring virulence factors and alternative therapies against Staphylococcus aureus pneumonia

Pneumonia is an acute pulmonary infection associated with high mortality and an immense financial burden on healthcare systems. Staphylococcus aureus is an opportunistic pathogen capable of inducing S. aureus pneumonia (SAP), with some lineages also showing multidrug resistance. Given the high level of antibiotic resistance, much research has been focused on targeting S. aureus virulence factors, including toxins and biofilm-associated proteins, in an attempt to develop effective SAP therapeutics. Despite several promising leads, many hurdles still remain for S. aureus vaccine research. Here, we review the state-of-the-art SAP therapeutics, highlight their pitfalls, and discuss alternative approaches of potential significance and future perspectives