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

Comparative proteome analyses of Staphylococcus aureus strains and their isogenic mutants in vitro and in host-pathogen interactions

Staphylococcus aureus can be a harmless colonizer of the human body, which colonizes about 20-30% of the population. If S. aureus overcomes the outer physical barrier of the body, comprised of the skin and mucous surfaces, it can also cause severe diseases such as endocarditis, pneumonia, or sepsis. S. aureus possesses a variety of secreted and surface bound virulence factors to mediate attachment and invasion into the host, to disseminate an infection and to modulate and evade the immune system. But not only the huge amount of virulence factors turn S. aureus into a dangerous human pathogen, also its resistances to a broad spectrum of commonly used antibiotics make infections hard to treat. During the last years it became apparent that S. aureus can be internalized by as well as replicate and persist in professional and non-professional phagocytic cells. It is suggested that the intracellular compartment protects S. aureus from antibiotic treatment and the immune system. To accomplish the adaptation to the intracellular compartment, S. aureus needs to regulate its gene expression by regulatory systems. One of these regulators is the alternative sigma factor SigB, which directly and indirectly regulates the expression of about 200 genes in vitro. However, the stimuli leading to the activation of SigB in S. aureus are barely known and also its role during an infection varies, depending on the S. aureus strain and infection model used. Therefore, the importance of SigB during the early adaption of S. aureus to the intracellular environment should be elucidated using a cell culture infection model. First, the existing cell culture infection workflow had to be modified to improve the data analysis and to increase the yield of identified proteins to comparatively monitor the adaption reaction of S. aureus HG001 and its isogenic ΔsigB mutant to the intracellular milieu of S9 human bronchial epithelial cells. The proteome analysis in conjunction with RT-qPCR analysis of the wild type and the ΔsigB mutant revealed a fast and transient activation of SigB directly after internalization. Quantitative analysis of the intracellular bacterial titer demonstrated a requirement of SigB for intracellular replication. Differences in the proteome composition of the ΔsigB mutant in comparison to the wild type after internalization reflected the different growth rates, resistance to antibiotics and toxic compounds, adaptation to oxidative stress, and protein quality control mechanisms. The accessory gene regulator (Agr) is like SigB also a global regulator of gene expression in S. aureus. To elucidate possible benefits in the intracellular survival of the co-occurrence of S. aureus wild type and Δagr mutant cells, like it can be found in sites of an infection, a co-infection assay was established. With the co-infection assay the simultaneous and competitive intracellular survival in comparison to the individual intracellular survival was followed for three days post-infection (p.i.). The single and the co-infection revealed that the wild type was able to replicate more efficiently during the first hours p.i. than the Δagr mutant, but the mutant was able to survive more efficiently. The extracellular proteome of S. aureus represents the key compartment for virulence factors. Virulence factors are secreted or bound to the surface of the S. aureus cell. With the infection workflow applied in this study, secreted proteins are lost during the enrichment of the intracellular bacteria for proteome analysis. Therefore, no information about the levels or the regulation of virulence factor expression can be acquired in the cell culture infection model using cell sorting approaches. Hence, the extracellular proteome of S. aureus was analyzed in vitro from shake flask experiments. To get a comprehensive overview of the regulatory impact of different global regulators onto the secretome, S. aureus LS1 mutants lacking the global regulators Agr, SarA and SigB were compared to the respective wild type. Additionally the protein level of the secretome of the well characterized and frequently used S. aureus strains 6850, CowanI, HG001, LS1, SH1000, and USA300 was comparatively analyzed. This project was performed in collaboration with the group of Prof. Löffler from the Institute of Medical Microbiology in Jena. The data of the extracellular proteome generated in this thesis were combined with phenotypic and toxicity data to explain strain differences in invasiveness, cytotoxicity, phagosomal escape, and intracellular persistence in infection experiments.Staphylococcus aureus ist ein harmloser Kommensale des Körpers, welcher 20-30% der Bevölkerung besiedelt. Falls S. aureus die physikalische Barriere des Körpers überwindet, kann es auch schwere Erkrankungen wie Endokarditis, Lungenentzündung oder Sepsis auslösen. S. aureus besitzt eine Vielzahl von sekretierten und Oberflächen-gebundenen Virulenzfaktoren um sich an den Wirt anzuheften und in ihn einzudringen, eine Infektion zu verursachen und um das Immunsystem zu umgehen. Jedoch sind es nicht nur die Virulenzfaktoren, die S. aureus zu einem gefährlichen human pathogenen Erreger machen, auch seine Resistenzen gegenüber fast allen häufig verwendeten Antibiotika erschweren die Behandlung von S. aureus ausgelösten Infektionen. Im Verlauf der letzten Jahre wurde es offensichtlich, dass S. aureus von profesionell und nicht-professionell phagozytierenden Zellen aufgenommen wird und sich in ihnen vermehren und persistieren kann. Es wird vermutet, dass der intrazelluläre Raum dem Bakterium Schutz vor Antibiotika und dem Immunsystem bietet. Um sich an die intrazelluläre Umgebung anzupassen, muss S. aureus seine Genexpression durch regulatorische Systeme regulieren. Einer dieser Regulatoren ist der alternative Sigmafaktor SigB, welcher direkt und indirekt die Expression von ca. 200 Genen in vitro kontrolliert. Jedoch sind die Reize, die zu einer Aktivierung von SigB in S. aureus führen bisher kaum bekannt und auch die Aufgabe von SigB wärend einer Infektion variiert mit dem verwendeten S. aureus Stamm und Infektionsmodel. Daher sollte die Bedeutung von SigB während der frühen Anpassungsphase von S. aureus an die intrazelluläre Umgebung in einem Zellkultur Infektionsmodel untersucht werden. Zunächst musste das bestehende Protokoll der Infektionsexperimente angepasst werden, um die Datenauswertung zu verbessern und die Ausbeute identifizierter Proteine zu erhöhen, um anschließend die Anpassung von S. aureus HG001 und seiner isogenen ΔsigB Mutante an das intrazelluläre Milieu von S9 humanen bronchialen Epithelzellen zu untersuchen. Die Proteomanalyse in Verbindung mit einer RT-qPCR Analyse des Wildtyps und der ΔsigB Mutante ergab, dass SigB direkt nach der Internalisierung schnell und transient aktiviert wurde. Die quantitative Bestimmung des intrazellulären bakteriellen Titers zeigte, dass SigB für eine erfolgreiche intrazelluläre Vermehrung vorhanden sein muss. Unterschiede der Proteinmengen in der ΔsigB Mutante im Vergleich zum Wildtyp nach der Internalisierung reflektierten das unterschiedliche Wachstumsverhalten, die Resistenz gegenüber Antibiotika und toxischer Verbindungen, die Anpassung an oxidativen Stress und Mechanismen für die Qualitätskontrolle von Proteinen. Der „accessory gene regulator“ (Agr) ist wie SigB ein globaler Regulator der Genexpression in S. aureus. Um mögliche Vorteile des gemeinsamen Vorkommens, wie es auch in Infektionen beobachtet werden kann, eines S. aureus Wildtypen und einer Δagr Mutante für das intrazelluläre Überleben zu beleuchten wurde ein Protokoll für ein Koinfektionsexperiment etabliert. Mit Koinfektionsexperimenten wurde das gleichzeitige intrazelluläre Überleben im Vergleich zum einzelnen intrazellulären Überleben drei Tage lang verfolgt. Die Einzel- und die Koinfektionen haben gezeigt, dass der Wildtyp in den ersten Stunden nach der Infektion in der lage ist sich effizienter zu vermehren als die Δagr Mutante. Jedoch konnte die Δagr Mutante drei Tage nach der Infektion in höheren Zellmengen überleben. Die meisten Virulenzfaktoren von S. aureus sind im extrazellulären Proteom zu finden, da sie entweder sekretiert werden oder an die Zelloberfläche gebunden sind. In dem in dieser Arbeit angewandten experimentellen Ablauf gehen sekretierte Proteine während der Anreicherung der intrazellulären Bakterien für anschließende Proteomanalysen verloren. Daher kann durch die Infektionsexperimente, in denen die Bakterien über Zellsortierungen angereichert werden, keine Informationen über die Mengen der Virulenzfaktoren oder ihre Regulation gesammelt werden. Deshalb wurde das extrazelluläre Proteom von S. aureus in in vitro Experimenten untersucht. Um einen umfassenden Überblick über das Sekretom zu erhalten, wurden S. aureus LS1 Mutanten, in denen die Gene für die globalen Regulatoren Agr, SarA und SigB deletiert wurden, im Vergleich zum Wildtyp analysiert. Zusätzlich wurden die Proteinmengen des Sekretoms der gut charakterisierten und häufig verwendeten S. aureus Stämme 6850, CowanI, HG001, LS1, SH1000 und USA300 vergleichend untersucht. Dieses Projekt wurde in Zusammenarbeit mit der Arbeitsgruppe von Prof. Löffler des Instituts für Medizinische Mikrobiologie in Jena durchgeführt. Die Daten des extrazellulären Proteoms konnten wichtige Erkenntnisse über die S. aureus Stämme beitragen, um Unterschiede in durchgeführten Infektionsexperimenten betreffend ihrer Invasivität, dem Auslösen von Zelltod, dem Ausbruch aus den Phagosomen und in ihrer intrazellulären Persistenz zu erklären

Proteome data from a host-pathogen interaction study with Staphylococcus aureus and human lung epithelial cells

To simultaneously obtain proteome data of host and pathogen from an internalization experiment, human alveolar epithelial A549 cells were infected with Staphylococcus aureus HG001 which carried a plasmid (pMV158GFP) encoding a continuously expressed green fluorescent protein (GFP). Samples were taken hourly between 1.5 h and 6.5 h post infection. By fluorescence activated cell sorting GFP-expressing bacteria could be enriched from host cell debris, but also infected host cells could be separated from those which did not carry bacteria after contact (exposed). Additionally, proteome data of A549 cells which were not exposed to S. aureus but underwent the same sample processing steps are provided as a control. Time-resolved changes in bacterial protein abundance were quantified in a label-free approach. Proteome adaptations of host cells were monitored by comparative analysis to a stable isotope labeled cell culture (SILAC) standard. Proteins were extracted from the cells, digested proteolytically, measured by nanoLC–MS/MS, and subsequently identified by database search and then quantified. The data presented here are related to a previously published research article describing the interplay of S. aureus HG001 and human epithelial cells (Surmann et al., 2015 [1]). They have been deposited to the ProteomeXchange platform with the identifiers PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD002384 for the S. aureus HG001 proteome dataset and PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD002388 for the A549 proteome dataset. Keywords: Epithelial cells, Flow cytometry, Internalization, Host-pathogen interaction, Proteomics, SILAC, Staphylococcus aureu

Metabolic cross-talk between human bronchial epithelial cells and internalized Staphylococcus aureus as a driver for infection

Staphylococcus aureus is infamous for causing recurrent infections of the human respiratory tract. This is a consequence of its ability to adapt to different niches, including the intracellular milieu of lung epithelial cells. To understand the dynamic interplay between epithelial cells and the intracellular pathogen, we dissected their interactions over four days by mass spectrometry. Additionally, we investigated the dynamics of infection through live cell imaging, immunofluorescence and electron microscopy. The results highlight a major role of often overlooked temporal changes in the bacterial and host metabolism, triggered by fierce competition over limited resources. Remarkably, replicating bacteria reside predominantly within membrane-enclosed compartments and induce apoptosis of the host within ~24 hours post infection. Surviving infected host cells carry a subpopulation of non-replicating bacteria in the cytoplasm that persists. Altogether, we conclude that, besides the production of virulence factors by bacteria, it is the way in which intracellular resources are used, and how host and intracellular bacteria subsequently adapt to each other that determines the ultimate outcome of the infectious process

Sigma Factor SigB Is Crucial to Mediate <i>Staphylococcus aureus</i> Adaptation during Chronic Infections

<div><p><i>Staphylococcus aureus</i> is a major human pathogen that causes a range of infections from acute invasive to chronic and difficult-to-treat. Infection strategies associated with persisting <i>S</i>. <i>aureus</i> infections are bacterial host cell invasion and the bacterial ability to dynamically change phenotypes from the aggressive wild-type to small colony variants (SCVs), which are adapted for intracellular long-term persistence. The underlying mechanisms of the bacterial switching and adaptation mechanisms appear to be very dynamic, but are largely unknown. Here, we analyzed the role and the crosstalk of the global <i>S</i>. <i>aureus</i> regulators <i>agr</i>, <i>sarA</i> and SigB by generating single, double and triple mutants, and testing them with proteome analysis and in different <i>in vitro</i> and <i>in vivo</i> infection models. We were able to demonstrate that SigB is the crucial factor for adaptation in chronic infections. During acute infection, the bacteria require the simultaneous action of the <i>agr</i> and <i>sarA</i> loci to defend against invading immune cells by causing inflammation and cytotoxicity and to escape from phagosomes in their host cells that enable them to settle an infection at high bacterial density. To persist intracellularly the bacteria subsequently need to silence <i>agr</i> and <i>sarA</i>. Indeed <i>agr</i> and <i>sarA</i> deletion mutants expressed a much lower number of virulence factors and could persist at high numbers intracellularly. SigB plays a crucial function to promote bacterial intracellular persistence. In fact, Δ<i>sigB</i>-mutants did not generate SCVs and were completely cleared by the host cells within a few days. In this study we identified SigB as an essential factor that enables the bacteria to switch from the highly aggressive phenotype that settles an acute infection to a silent SCV-phenotype that allows for long-term intracellular persistence. Consequently, the SigB-operon represents a possible target to develop preventive and therapeutic strategies against chronic and therapy-refractory infections.</p></div

Differential expression of the regulators <i>agr</i>, <i>sarA</i> and <i>sigB</i> during the course of host cell infection in wild-type LS1 and the corresponding mutants.

<p>Endothelial cells were infected with <i>S</i>. <i>aureus</i> strain LS1 or the corresponding mutants as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004870#ppat.1004870.g003" target="_blank">Fig 3A</a> and infected cells were analysed for up to 7 days. Host cells infected with wild-type strain LS1 were lysed after 2 (acute phase) and 7 days (chronic phase) and the whole RNA was extracted and was used to determine changes in bacterial gene expression for <i>agrA/hla</i> (α-hemolysin) (A), <i>sarA/aur</i> (aureolysin) (B) and <i>sigB/asp23</i> (C) during the course of infection by real-time PCR. The values of all experiments represent the mean ± SD of 5 independent experiments measured in triplicate. * P≤0.05 ANOVA comparing levels of gene expression in the wild-type strains and the corresponding mutants at each time point. The fold change is the result of normalized expression to the housekeeping genes <i>gyr</i>, <i>aroE</i> and <i>gmk</i>. Uninfected cells were used as controls (Control = 1).</p

The interplay of <i>agr</i>, <i>sarA</i> and <i>sigB</i> is required to settle and maintain an infection in a local rat chronic osteomyelitis model.

<p>A local chronic osteomyelitis model was used to study the effects of wild-type SH1000 and corresponding mutants. (A) After 4 days of infection slices of bone tissue were performed for histology and stained by hematoxylin-eosin to detect the influx of immune cells to bone tissue. For each strain tested representative photomicrographs are shown in low and high magnification and typical histological features are described. (B, C) Bacterial persistence in host tissue was analyzed 4 days p.i. (acute) and 14 weeks p.i. (chronic) by plating homogenized bone tissue on agar plates and counting the CFU the following day. (D, E) The osteomyelitis index was measured 4 days p.i. (acute) and 14 weeks p.i. (chronic) from each infected tibiae in comparison with the non-infected tibiae from the same animal. The experiments were performed with 12 animals per group and the results shown are means ± SD. * P≤0.05 ANOVA test comparing persisting CFU and osteomyelitis index caused by wild-type SH1000 and the corresponding mutants. (F) Photographs of infected (wild-type SH1000) and non-infected tibiae recovered after 14 weeks.</p