Staphylococcus aureus lipoproteins - TLR2-mediated activation of innate and adaptive immunity

Staphylococcus (S.) aureus is a very successful pathogen due to its immune evasion strategies. Besides toxins and adhesins, it expresses membrane lipoproteins (Lpp), which are bound by the pattern recognition receptor Toll-like receptor (TLR) 2 in the host. Recognition of Lpp activates the MyD88-signaling pathway, which allows mounting a strong inflammatory response. Interestingly, evolution did not select for S. aureus mutants deficient in lipid modification of proteins suggesting that Lpp, besides their signaling potential to the host, offer an advantage for S. aureus. The aim of this thesis was to identify the benefit of Lpp maturation for S. aureus and the contribution of Lpp-TLR2-signaling to the pathogenesis of staphylococcal disease in mouse infection models. In the first part, we demonstrate the strong cytokine-activating potential of Lpp in murine macrophages, which was associated with the presence of TLR2 in the host. In a systemic infection model, Lpp-TLR2 activation was less contributing to inflammation and S. aureus killing than MyD88-signaling. This indicates that other receptors signaling to MyD88 participate in the antimicrobial response. We further showed in systemic infection that maturation of Lpp facilitates survival of S. aureus in organs due to improved iron acquisition. Studies on growth, uptake, and intracellular storage of iron in vitro confirmed the iron dependence of S. aureus. It is long known, that the immunocompetent host restricts iron in an infection. We show that Lpp enhanced S. aureus growth in the iron-overloaded immunocompetent host, while they were not required in the iron rich environment in the MyD88-deficient host. Interestingly, iron-restricted S. aureus could not profit from Lpp for growth as long as the infected mice were fully immunocompetent. Only in mice deficient in MyD88-dependent inflammation iron-restricted S. aureus used Lpp for growth. In summary, the results in part 1 strongly suggest that Lpp confer a growth and survival advantage although allowing innate immune responses mediated through TLR2-MyD88-signaling. In the second part, data are presented showing that Lpp released during growth activate TLR2-signaling but engulfment of S. aureus enhances cytokine production. Lpp enhanced phagocytosis by macrophages and intracellular survival of S. aureus. Moreover, Lpp-TLR2-signaling induced cathepsin B-mediated cytotoxicity in macrophages. An effect of Lpp on various interactions of S. aureus with PMN was not found in vitro and in vivo, whereas Lpp enhanced invasion of S. aureus in endothelial cells in vitro. These results point to an additional survival advantage by maturation of Lpp in S. aureus due to improved evasion from extracellular killing, better intracellular survival, and escape from the phagosome. In the third part, we demonstrate that Lpp-TLR2-MyD88-signaling is important for activation of DCs to induce differentiation of na•ve CD4+ into IFN-g- and IL-17-producing T cells in vitro. Induction of Lpp-TLR2-signaling was also required to promote IFN-g release by na•ve CD8+ T cells. In systemic infection, restimulated spleen T cells produced MyD88-dependent IFN-g and TLR2-MyD88-dependent IL-17. Surprisingly, the presence of B and T cells diminished eradication of S. aureus from organs during early sepsis. These data show that detection of invading S. aureus by DCs leads to the development of adaptive immune responses, which are not always beneficial for eradication of S. aureus. In the fourth part, the role of other pattern recognition receptors (PRRs) in staphylococcal infection was examined. TLR9 and NOD2, in contrast to IL-1R, had a positive effect on cytokine induction in macrophages and in systemic infection, whereas killing was not affected. In inflammation and bacterial killing during S. aureus infection, TLR2 and TLR9, which both require the MyD88-adaptor, were found to cooperate. These data suggest that concurrent activation of different PRRs elicit a strong antimicrobial defense in response to various molecules of S. aureus

Process Analysis for Material Flow Systems

This paper describes a generic approach for analysis of internal behavior of logistic systems based on event logs. The approach is demonstrated by an example of event data from the simulation model of an automated material handling system (MHS) in a manufacturing company. The purpose of the analysis is the identification of design and operation problems and their causes, prospectively. As a result, the simulation model developer obtains the condensed and ranked information on events. These events describe the internal system behavior with anomalies pointing at either possible problems or capacity reserves

Melanin in the human pathogenic fungus Aspergillus fumigatur: discovery of a novel melanin in the fungus and the use of conidial melanin as a target for camelid heavy-chain antibodies

Aspergillus fumigatus is the causative agent of the invasive aspergillosis which is often fatal for immunocompromised patients. This work aimed at: (i) studying the role of melanisation as pathogenicity mechanism and (ii) detecting the fungus with the help of a melanin specific antibody. A camelid antibody from a recombinant VHH-domain library was successfully selected against melanised conidia applying phage-display and a newly developed filtration approach. Its recombinant production in Escherichia coli by high cell density fermentation provided a pure antibody which bound with high affinity to melanised conidia of different Aspergillus species. A less stringent binding to synthetic DOPA-melanin and Sepia-melanin was observed in ELISA studies. Furthermore, the biotinylated antibody demonstrated melanisation of conidia and hyphae by an immunofluorescent staining approach. Furthermore, melanisation of A. fumigatus was studied by means of deletion mutants of genes coding for key enzymes of the DHN-melanin and tyrosine degradation pathway. The loss of a functional Arp2 enzyme, a reductase involved in the DHN-melanin biosynthesis, did not result in a distinct phenotype from wild type despite the colour change of the conidia from grey-green to pinkish. Hence, the modified DHN-melanin is as protective as the grey-green wild-type pigment. By the deletion of the genes encoding homogentisate dioxygenase (HmgA) and 4-hydroxyphenylpyruvate dioxygenase (HppD) it was shown that homogentisic acid is the major intermediate in the L-tyrosine degradation pathway which yields in the formation of pyomelanin. Despite activation of the genes by tyrosine and during infiltrational growth in the mouse lung and despite the increased sensitivity of the germlings of the hppD strain to ROI, the mutant was not attenuated in its virulence in the mouse infection model. Hence, this work provides the proof for the presence of pyomelanin in A. fumigatus

The CRESST II Dark Matter Search

Direct Dark Matter detection with cryodetectors is briefly discussed, with particular mention of the possibility of the identification of the recoil nucleus. Preliminary results from the CREEST II Dark Matter search, with 730 kg-days of data, are presented. Major backgrounds and methods of identifying and dealing with them are indicated.Comment: Talk at DSU workshop, ITP Beijing, Oct. 2011. 9 figures, 2 table

Results from 730 kg days of the CRESST-II Dark Matter Search

The CRESST-II cryogenic Dark Matter search, aiming at detection of WIMPs via elastic scattering off nuclei in CaWO$_4$ crystals, completed 730 kg days of data taking in 2011. We present the data collected with eight detector modules, each with a two-channel readout; one for a phonon signal and the other for coincidently produced scintillation light. The former provides a precise measure of the energy deposited by an interaction, and the ratio of scintillation light to deposited energy can be used to discriminate different types of interacting particles and thus to distinguish possible signal events from the dominant backgrounds. Sixty-seven events are found in the acceptance region where a WIMP signal in the form of low energy nuclear recoils would be expected. We estimate background contributions to this observation from four sources: 1) "leakage" from the e/\gamma-band 2) "leakage" from the \alpha-particle band 3) neutrons and 4) Pb-206 recoils from Po-210 decay. Using a maximum likelihood analysis, we find, at a high statistical significance, that these sources alone are not sufficient to explain the data. The addition of a signal due to scattering of relatively light WIMPs could account for this discrepancy, and we determine the associated WIMP parameters.Comment: 17 pages, 13 figure

Status of the CRESST Dark Matter Search

The CRESST experiment aims for a detection of dark matter in the form of WIMPs. These particles are expected to scatter elastically off the nuclei of a target material, thereby depositing energy on the recoiling nucleus. CRESST uses scintillating CaWO4 crystals as such a target. The energy deposited by an interacting particle is primarily converted to phonons which are detected by transition edge sensors. In addition, a small fraction of the interaction energy is emitted from the crystals in the form of scintillation light which is measured in coincidence with the phonon signal by a separate cryogenic light detector for each target crystal. The ratio of light to phonon energy permits the discrimination between the nuclear recoils expected from WIMPs and events from radioactive backgrounds which primarily lead to electron recoils. CRESST has shown the success of this method in a commissioning run in 2007 and, since then, further investigated possibilities for an even better suppression of backgrounds. Here, we report on a new class of background events observed in the course of this work. The consequences of this observation are discussed and we present the current status of the experiment.Comment: Proceedings of the 13th International Workshop on Low Temperature Detectors, 4 pages, 3 figure

Composite CaWO4 Detectors for the CRESST-II Experiment

CRESST-II, standing for Cryogenic Rare Events Search with Superconducting Thermometers phase II, is an experiment searching for Dark Matter. In the LNGS facility in Gran Sasso, Italy, a cryogenic detector setup is operated in order to detect WIMPs by elastic scattering off nuclei, generating phononic lattice excitations and scintillation light. The thermometers used in the experiment consist of a tungsten thin-film structure evaporated onto the CaWO4 absorber crystal. The process of evaporation causes a decrease in the scintillation light output. This, together with the need of a big-scale detector production for the upcoming EURECA experiment lead to investigations for producing thermometers on smaller crystals which are glued onto the absorber crystal. In our Run 31 we tested composite detectors for the first time in the Gran Sasso setup. They seem to produce higher light yields as hoped and could provide an additional time based discrimination mechanism for low light yield clamp events.Comment: Proceedings of the Thirteenth International Workshop on Low Temperature Detectors 4 pages, 9 figure

Effects of deletion of the Streptococcus pneumoniae lipoprotein diacylglyceryl transferase gene lgt on ABC transporter function and on growth in vivo

Lipoproteins are an important class of surface associated proteins that have diverse roles and frequently are involved in the virulence of bacterial pathogens. As prolipoproteins are attached to the cell membrane by a single enzyme, prolipoprotein diacylglyceryl transferase (Lgt), deletion of the corresponding gene potentially allows the characterisation of the overall importance of lipoproteins for specific bacterial functions. We have used a Δlgt mutant strain of Streptococcus pneumoniae to investigate the effects of loss of lipoprotein attachment on cation acquisition, growth in media containing specific carbon sources, and virulence in different infection models. Immunoblots of triton X-114 extracts, flow cytometry and immuno-fluorescence microscopy confirmed the Δlgt mutant had markedly reduced lipoprotein expression on the cell surface. The Δlgt mutant had reduced growth in cation depleted medium, increased sensitivity to oxidative stress, reduced zinc uptake, and reduced intracellular levels of several cations. Doubling time of the Δlgt mutant was also increased slightly when grown in medium with glucose, raffinose and maltotriose as sole carbon sources. These multiple defects in cation and sugar ABC transporter function for the Δlgt mutant were associated with only slightly delayed growth in complete medium. However the Δlgt mutant had significantly reduced growth in blood or bronchoalveolar lavage fluid and a marked impairment in virulence in mouse models of nasopharyngeal colonisation, sepsis and pneumonia. These data suggest that for S. pneumoniae loss of surface localisation of lipoproteins has widespread effects on ABC transporter functions that collectively prevent the Δlgt mutant from establishing invasive infection

Disruption of Coronin 1 Signaling in T Cells Promotes Allograft Tolerance while Maintaining Anti-Pathogen Immunity

The ability of the immune system to discriminate self from non-self is essential for eradicating microbial pathogens but is also responsible for allograft rejection. Whether it is possible to selectively suppress alloresponses while maintaining anti-pathogen immunity remains unknown. We found that mice deficient in coronin 1, a regulator of naive T cell homeostasis, fully retained allografts while maintaining T cell-specific responses against microbial pathogens. Mechanistically, coronin 1-deficiency increased cyclic adenosine monophosphate (cAMP) concentrations to suppress allo-specific T cell responses. Costimulation induced on microbe-infected antigen presenting cells was able to overcome cAMP-mediated immunosuppression to maintain anti-pathogen immunity. In vivo pharmacological modulation of this pathway or a prior transfer of coronin 1-deficient T cells actively suppressed allograft rejection. These results define a coronin 1-dependent regulatory axis in T cells important for allograft rejection and suggest that modulation of this pathway may be a promising approach to achieve long-term acceptance of mismatched allografts