VAAM-Jahrestagung 2012 18.–21. März in Tübingen

122MPP054BopC is a type III secretion effector protein of BurkholderiapseudomalleiS. Muangman* 1 , S. Korbsrisate 1 , V. Muangsombut 1 , V. Srinon 1 , N.L. Adler 2 ,G.N. Schroeder 3 , G. Frankel 3 , E.E. Galyov 2,31 Mahidol University, Immunology, Faculty of Medicine Siriraj Hospital,Bangkok, Thailand2 University of Leicester, Infection, Immunity and Inflammation, Leicester,United Kingdom3 Imperial College London, Centre for Molecular Microbiology and Infection,Division of Cell and Molecular Biology, London, United KingdomQuestion: Burkholderia pseudomallei is the causative agent ofmelioidosis, the disease endemic in southeast Asia and northern Australia.The promising component causing pathogenesis is the Bsa type IIIsecretion system (T3SS). Only two Bsa-secreted effectors have beenconclusively identified to date. Here we explored the T3SS-dependentsecretion and the virulence mechanism of a B. pseudomallei putativeeffector protein BopC (BPSS1516), which is encoded from the bpss1516gene adjacent to its putative chaperone bpss1517.Methods: B. pseudomallei bopC gene was cloned as Glutathione S-transferase (GST)-tagged constructs and expressed in Escherichia coli.Pull down and co-purification assays were conducted to address theinteraction between BopC and its putative chaperone BPSS1517.Translocation assay was performed to investigate the importance of the N-terminal amino acids of BopC. B. pseudomallei bopC mutant wasconstructed by insertion mutagenesis. A549 lung epithelial cells wereemployed for invasion assay.Results: Immunoblotting demonstrated that BopC was secreted intoculture supernatant by the wild-type B. pseudomallei strain, but itssecretion was abolished in the bsaZ T3SS mutant, suggesting that BopC issecreted via T3SS. Pull down and co-purification assays confirmed thatBopC interacts with its putative chaperone, BPSS1517, in vitro. The first20 N-terminal amino acids of BopC were found to be sufficient to mediatethe T3SS-dependent translocation of a reporter protein from a heterologousenteropathogenic E. coli host into mammalian cells. Finally, B.pseudomallei bopC mutant was found to be less invasive than the wildtypestrain in the epithelial cells.Conclusions: B. pseudomallei BopC is a newly identified type III effectorprotein. The secretion of BopC is dependent on Bsa T3SS. FurthermoreBopC is implicated in the B. pseudomallei invasion into epithelial cells.Muangman S, Korbsrisate S, Muangsombut V, Srinon V, Adler N L, Schroeder G N, et al., BopC isa type III secreted effector protein of Burkholderia pseudomallei. FEMS microbiology letters,2011;323 (1): 75-82.MPP055Proteomic characterization of the different Legionellapneumophila life stageT. Gerlach* 1 , P. Aurass 1 , B. Voigt 2 , D. Becher 2 , M. Hecker 2 , L. Jänsch 3 ,T. Goldmann 4 , M. Steinert 5 , A. Flieger 11 Robert Koch-Institut, Division of Bacterial Infections (FG11),Wernigerode, Germany2 Ernst-Moritz-Arndt-Universität, Division of Microbial Physiology andMolecular Biology, Greifswald, Germany3 Helmholtz Centre for Infection Research, Cellular Proteom Research,Braunschweig, Germany4 Leibnitz-Zentrum Borstel, Clinical and Experimental Pathology, Borstel,Germany5 Technische Universität Braunschweig, Department of Life SciencesInstitute of Microbiology, Braunschweig, GermanyThe Gram-negative bacterium Legionella pneumophila is the causativeagent of a severe and often fatal human pneumonia, Legionnaires’ disease.In the natural environment, L. pneumophila inhabits freshwater andbiofilms and parasitizes protozoan hosts. The intracellular life cycle of L.pneumophila is divided into two distinct stages: the replicative phase (RP),where the bacteria multiply until the nutrients cease, and the transmissivephase (TP), where the bacteria render virulent and invasive. Uponprolonged periods of stress (such as nutrient deprivation, temperaturechange, etc.), L. pneumophila may enter into the viable but not culturable(VBNC) state where the bacteria only show a very low level of metabolicactivity and do not grow on standard media. Remarkably, VBNC state L.pneumophila may resuscitate and thereby regain culturability as well asvirulence after passage through a eucaryotic host. In consequence, VBNCstate L. pneumophila have to be considered as a public health hazard. Tocharacterize the distinct stages of life for L. pneumophila, in this study weperformed a systematic proteomic comparison of broth-grown RP and TPand stress-induced VBNC states. To induce VBNC cell formation in L.pneumophila bacteria, different stress conditions like cold and heat stress,nutrient limitation, and several chemical agents were tested. During heatstress (42°C), the number of CFU decreased to zero within 68 dayswhereas the microcosms remained stable with respect to culturability at4°C and 21°C for at least 140 days. Despite the drastic decrease in CFUcounts, 40% of the bacteria remained viable according to microscopiclive/dead analysis. For proteome analysis, it is essential to separate theVBNC-Legionella from dead bacteria. To this purpose, we usedfluorescence-activated cell sorting (FACS). Our work will contribute to adeeper understanding of the modification processes within bacteria inresponse to different conditions, including adaptation to long-term stress.MPP056Carolacton cause inhibition of Streptococcus mutans biofilmsthrough the serine/threonine protein kinase PknBM. Reck* 1 , B. Kunze 1 , J. Tomasch 1 , S. Schulz 2 , I. Wagner-Döbler 11 Helmholtz Centre for Infection Research, Microbial Communication,Braunschweig, Germany2 Technical University Braunschweig, Institute for Organic Chemistry,Braunschweig, GermanyBiofilm forming bacteria are often significantly more resistant to drugtreatments than their planktonic counterparts and are associated to variouspathological conditions in humans as e.g. cystic fibrosis, colonisation ofindwelling medical devices and dental plaque formation. Therefore newsubstances and therapies aiming to erase biofilms are urgently needed. Onepossible strategy to cope with this demand is to disturb signal-transductionin biofilms.Carolacton, a secondary metabolite isolated from the myxobacteriumSorangium cellulosum was proven to disturb biofilm viability at nanomolarconcentrations. Treated biofilms showed a leakage of cytoplasmic content(proteins and DNA) in growing cells at low pH. Using a ß-galactosidasereporter strain and quantitative PCR the efflux-dynamics of intracellularproteins and DNA were quantified. The strong acidification occurringduring biofilm growth was shown to be responsible for the biofilm-specificactivity of carolacton.A chemical conversion of the of the ketocarbonic function of the moleculeto a methlyester did not impact its activity, indicating that carolacton is notfunctionally activated at low pH by a change of its net charge. Besidemultiple genes involved in cell wall metabolism the VicKRX and ComDEtwo-component signal transduction systems were found to play anessential role in the cellular response to carolacton treatment as identifiedby time-resolved microarray analysis. The influence of carolacton on denovo cell wall metabolism and cell division was further investigated byfluorescence microscopy using a fluorescent vancomycin derivative.A sensitivity testing of mutants with deletions of all 13 viable histidinekinases and the serine/threonine protein kinase PknB identified only thepknB mutant to be insensitive to carolacton treatment. Furthermore astrong overlap between the PknB-regulon in S. mutans and the genesaffected by carolacton treatment was found. In conclusion the data suggestthat carolacton interferes with PknB-mediated signalling in growing cells.The altered cell wall metabolism and architecture cause membrane damageand cell death at low pH.MPP057Systems biology analysis of metabolic adaptation of Staphylococcusaureus and analysis of the impact of protein complexesM. Burian* 1 , C. Liang 2 , T. Dandekar 2 , U. Völker 11 Interfaculty Institute for Genetics and Functional Genomics, Departmentof Functional Genomics, Greifswald, Germany2 Biocenter, University of Würzburg, Department of Bioinformatics,Würzburg, GermanySystems biology approaches, combining modern OMICs techniques withbioinformatics and mathematical modeling allow us to explore howadaptation of cell physiology and metabolic processes affect pathogenicityin a more “panoramic view” and thus may accomplish a new level ofunderstanding. The emergence of methicillin-resistant Staphylococcusaureus (MRSA) strains causing serious infections even in healthyindividuals (CA-MRSA) represents a major threat and underscores theneed for a comprehensive understanding of virulence mechanisms. Since itis known that the basic cell physiology determines not only growth butpathogenicity as well, we want to identify the concentrations and complexformation of proteins involved in central carbon metabolism. Twophysiological adaptation scenarios, which are very likely encountered by S.aureus during infection settings are addressed in vitro: i) changes in thesupply of carbon sources and ii) the aerobic/anaerobic shift. In addition,protein concentrations of internalized S. aureus cells by non-professionalphagocytes will be determined.For absolute quantification of proteins we use the QconCAT technologywhich allows quantification of up to 15 proteins within a syntheticstandard protein. The design as well as the construction (heterologousexpression, labeling with stable isotopes, purification and quality control)of 4 QconCAT proteins in total (covering all enzymes of the glycolysis,TCA-cycle, gluconeogenesis, pentosephosphate pathway and proteinsinvolved in the aerobic/anaerobic shift) is completed. For experimentalscreening of protein complexes we use in vivo cross-linking and taggingexperiments. Enzymes of the central carbon metabolism are used as a baitprotein,tagged with a Strep-tag and inserted into the plasmid pMADBIOspektrum | Tagungsband 2012