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The Pathogen and the Host
Keynote Speaker: Ulrich Schaible, Forschungszentrum Borstel, Borstel, Germany
Analysing life forms of pulmonary bacterial pathogens
Forschungszentrum Borstel, Germany
The lung is target organ of a number of bacterial pathogens including the notorious tubercle bacillus (Mycobacterium tuberculosis, Mtb) as well as various agents causing pneumonia including the emerging Gram-negative opportunist Stenotrophomonas maltophilia (Sm). Mtb is able to live and proliferate inside host macrophages and other phagocytes by manipulating its host cell's intracellular vesicle transport system.
In order to dissect the virulence properties of intracellular tubercle bacilli, we used different separation methods to purify intracellular mycobacteria for proteomics, transcriptomics and lipidomics. To mimick the mycobacteria-host interphase, we employ magnetic beads coupled to Mtb structures such as glycolipids to study host-pathogen interaction in a simplified model. Obligate extracellular bacterial pathogens of the lung such as Sm often can generate biofilms, which may interfere with phagocytosis by innate host defense cells and enhance drug resistance. In order to reveal the differential living conditions in Sm biofilms, we compared biofilm-forming vs. planctonic GFP expressing Sm by proteomics demonstrating a biofilm-specific proteome.
The characterization of distinct life forms, which arise during a life cycle of pathogenic bacteria, such as intra- vs. extracellular or biofilm vs. planctonic, is facilitated by specific separation methods.
In vivo proteome analysis of internalized Staphylococcus aureus by human bronchial epithelial cells
1Department of Functional Genomics, Ernst-Moritz-Arndt-University of Greifswald, Germany; 2ZIK-FunGen Junior Group Applied Proteomics, Ernst-Moritz-Arndt-University of Greifswald, Germany; 3Medical Microbiology and Hygiene, Eberhard Karls University of Tübingen, Germany
Staphylococcus aureus , the cause of a wide spectrum of severe community-acquired and nosocomial infections is traditionally not considered to be an intracellular pathogen. However, there is evidence that S. aureus can be internalized and persist in non-professional phagocytic cells in vitro . During the internalization process, S. aureus has to adapt to the new intracellular environment to survive or even persist within the host. Due to low numbers of bacteria available from an internalization experiment, the investigation of the bacterial adaptation to the host environment is still a challenge and therefore little is known about these adaptive changes on the proteome level.
We were recently able to analyze internalized S. aureus RN1HG wild type cells with our newly developed workflow, which combines a classical infection assay with pulse-chase labeling, high capacity cell sorting, and gel-free proteomics.
Before internalization, S. aureus RN1HG cells were fully labeled with heavy amino acids using the SILAC method and then co-cultivated with S9 human bronchial epithelial cells allowing internalization. Due to the continuous GFP expression of S. aureus , bacteria could be enriched after lysis of the S9 cells by FACS on a filter membrane. The Staphylococci on the membrane were subjected to tryptict digestion and generated peptides were measured by nanoLC-ESI-MS/MS. Using a sorting time of 45 min, 300 to 700 S. aureus proteins from only 2-5 ∙ 10^6 cells could be identified and quantified over a period of 6.5 hours post-internalization.
In a further experiment we compared the adaptive changes of RN1HG and its isogenic ∆agr mutant. The most promising approach to compare these adaptive and competitive changes would be simultaneous co-infection of the wild type and the mutant. The striking advantage of a co-infection assay is that both strains internalize and adapt to the host under exactly the same conditions.
For distinct enrichment of RN1HG and the ∆agr mutant, the mutant had to be labeled with a fluorescent marker allowing the separation from GFP of the wild type. After validation, the fluorescent marker gpCerulean showed a good intensity and a clear distinction to GFP in FACSAria. Therefore, we are now able to sort co-internalized wild type and ∆agr mutant parallel in distinct wells of a 96-well plate.
In a first proteomics experiment, we have enriched 10^3 - 10^4 co-internalized Staphylococci via FACSAria and identified about 200 - 300 proteins. The pros and cons of the approach and observed adaptations in the protein profiles will be discussed in the presentation.
Flow Cytometry as Efficient Tool in Filarial Research
1Universitiy Hosital Bonn, Germany; 2Muséum National d’Histoire Naturelle, France
Lymphatic filariasis (LF) and onchocerciasis are parasitic nematode infections that are responsible for a major disease burden in the African continent. Disease symptoms are induced by the immune reactions of the host, with lymphoedema and hydrocoele in LF, and dermatitis and ocular inflammation in onchocerciasis. The infection is acquired during a blood meal of an insect vector thereby transmitting infective filarial stages onto the human host, where they develop into adults and produce millions of new larvae, the microfilariae (Mf). Current disease elimination strategies of the WHO are based on mass drug administration (MDA) of microfilaricides.
However, success of MDA is compromised by logistical aspects such as long-time treatment, as well as by adverse events to therapy and suboptimal responses to the drugs. Alternative approaches are urgently needed and a vaccine targeting the blood-circulating Mf stage would be a pivotal step in disease control in order to stop transmission. Here we show a successful immunization protocol against the MF stage in the murine model for filariasis.
Flowcytometry has become one of the most powerful tools in immunological research and consequently has also been used to study LF, e.g. for staining immune cells from the site of infection in the murine model of LF. Here we show that flowcytometry can be used to count and analyze the multi-cellular 80-100 µm tall filarial Mf stage. Flowcytometric analysis of the Mf stage may become a powerful tool to investigate the mechanisms that form the basis for the Mf-specific responses of the successful immunization.
In addition we show a flowcytometry-based characterization of immune cells from the site of infection in the murine model of LF. Given that the cellular content from the site of infection is composed of several different cell types, we finally want to illustrate some of the “pitfalls” that have to be considered in the interpretation of that kind of data.
Interaction of platelets with bacteria visualized by correlated light and electron microscopy and tomography
1Medical University of Vienna, Center of Anatomy and Cell Biology, Dept. for Cell Biology and Ultrastructure Research, Austria; 2Blood donation Center of the Austrian Red Cross for Vienna, Lower Austria and Burgenland, Austria
Platelets (PLT) obtained by thrombapheresis were spiked with bacteria to see how they interact with each other in respect to PLT activation, sequestration or phagocytosis. Since bacteria can be covered by the surface connected open canalicular system (OCS) of PLT we used ruthenium red (RR) as a tracer for the OCS which enabled discriminating completely engulfed bacteria from those that are only partially covered by the OCS. In addition, the reorganization of the cytoskeleton including the circumferential microtubular coil and the actin microfilaments was investigated.
PLT were spiked with commercially available preparations of Staphylococcus aureus and Escherichia coli (pHrodoTM, Invitrogen) in a ratio of approximately 1:10. After 30 minutes of coincubation, PLT were allowed to adhere to glass coverslips and spiked with bacteria or incubated in PBS; both treatments lasted for 30 min. Alternatively, spiking with bacteria was carried out in suspension or when PLT in suspension interacted with bacteria immobilized on a glass surface. The samples were fixed 15 min. with 1% formaldehyde for light microscopy or with 2.5% glutaraldehyde (60 min.) and 1% OsO4 (2 h.) for electron microscopy. OCS tracing was carried out with 2% OsO4 and 3% RR in 0.1M cacodylate buffer, for 90 min. Electron tomography (ET) was carried on 300 nm semithin sections by acquisition of tilting series of ±65° with an increment of 1° and reconstruction using the weighted back projection method. F-actin and α-tubulin staining was performed with phalloidin-TRITC and an Alexa Fluor 488 anti-tubulin-α MoAb respectively.
PLT, adherent to glass surfaces, formed a granulomer and a hyalomer. In TEM, slightly adherent PLT showed the circumferential microtubular coil with adjacent lacunae of the dense tubular system (DTS). In firmly adherent PLT, this ring was centralized, lining the granulomer containing organelles. This centralization of the microtubular coil could be also shown by fluorescence microscopy..
Both species of bacteria formed cell contacts with PLT. Activated PLT extended filopodia forming aggregates by free cell contacts sequestering bacteria. Conspicuous PLT aggregates occurred also due to interaction with immobilized bacteria. RR decorated only particular OCS compartments while other remained separated from the PLT surface which could be convincingly underlined by ET. Bacteria in the PLT suspension induced a significant PLT activation demonstrated by centralization of the microtubular coil and by accumulation of F-actin around the granulomer. The delivery of α granules could also be verified at the EM level.
Further investigations should clarify, whether PLT are able to kill bacteria using their lysosomal enzymes or are only able to transport them to sites of degradation or to deposit them to particular sites of the body, where bacteria-induced inflammation could be induced. Possible consequences for transfusion medicine and human pathology will be discussed.