VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

Here, we present a comparative proteome analysis of the highly pathogenicP. aeruginosa strain PA14 during colonization of C. elegans and growth onNGMII-agar (control), respectively. To this end, the nematodes werehomogenized after 24 h of „infection”, paramagnetic beads coupled to anti-Pseudomonas sp.-antibodies were used to enrich bacterial cells, and proteinswere extracted and trypsin-digested. The resulting mixture of bacterial andnematode-derived peptides was analyzed by reverse-phase liquidchromatography coupled to electrospray ionization tandem massspectrometry (MS). MS data were searched against a database containingprotein sequences of P. aeruginosa PA14 / PAO1 and C. elegans. A total of3940 C. elegans and 1500 P. aeruginosa proteins were identified from the„infected” nematode, while 2952 bacterial proteins were found in thecontrol. Numerous QS-regulated proteins like proteins involved inphenazine biosynthesis or iron sequestration were found to be highlyexpressed during the ILP. Overall these findings strikingly confirm thecentral role of QS-regulated protein expression for P. aeruginosapathogenicity.The obtained data are currently validated by testing P. aeruginosa mutantsdefective in selected proteins that were exclusively or highly expressedduring the ILP in the C. elegans pathogenicity model. In a next step, we willextend our metaproteome analyses to a chronic murine infection modelsystem to identify specific pathogenic traits underlying mammalian P.aeruginosa infections.MPV002A typical eukaryotic lipid in prokaryotic membranes:Synthesis and necessity of phosphatidylcholine inAgrobacterium tumefaciensJ. Gleichenhagen*, M. Wessel, M. Aktas, S. Klüsener, S. Hacker, C. Fritz,F. NarberhausDepartment of Biology of Microorganisms, Ruhr-University, Bochum,GermanyThe typical eukaryotic membrane lipid phosphatidylcholine (PC) is alsopresent in rather diverse bacteria. A number of pathogenic and symbioticPC-containing bacteria like Bradyrhizobium japonicum, Brucella abortusand Legionella pneumophila require PC for an efficient interaction with theirrespective hosts [1].A PC-deficient Agrobacterium tumefaciens mutant lacking pmtA and pcs isunable to elicit plant tumors [2]. This virulence defect is based on theimpaired expression of genes encoding the type IV secretion machinery.This machinery is responsible for transfer of the oncogenic T-DNA intoplant cells. PC-deficient mutants are hyper-sensitive towards the detergentSDS and show a growth defect at elevated temperature. Furthermore, thePC-deficient mutant is reduced in motility and enhanced in biofilmformation [2, 3].PC in A. tumefaciens is synthesised via two pathways: the methylationpathway and the phosphatidylcholine synthase (Pcs) pathway. Themethylation pathway involves a three-step methylation ofphosphatidylethanolamine catalysed by the phospholipid N-methyltransferase PmtA. S-adenosylmethionine (SAM) provides themethylgroup and is converted to S-adenosylhomocysteine (SAH) duringtransmethylation [3]. In a second pathway the membrane protein Pcscondenses CDP-diacylglycerol with choline to form PC.PmtA was recombinantly produced in E. coli and purified via affinitychromatography. PmtA is a monomer and methyltransferase activity isinhibited by PC and SAH. Moreover, SAM binding depends on lipid bindingand PmtA activity is stimulated by phosphatidylglycerol, one of the mainphospholipids in A. tumefaciens [4]. By combining in silico analysis andpoint mutagenesis the SAM-binding pocket was identified in the N-terminalpart of PmtA.[1] Aktas, M. et al (2010): Eur J Cell Biol 89, 888-894.[2] Wessel, M. et al (2006): Mol Microbiol 62, 906-915[3] Klüsener, S. et al (2009): J Bacteriol 191, 365-374[4] Aktas, M. and F. Narberhaus, F. (2009): J Bacteriol 191, 2033-2041.MPV003Virulence properties of Legionella pneumophila GDSLlipolytic enzymes: Proteolytic activation of PlaCacyltransferase activityC. Lang*, E. Rastew, B. Hermes, E. Siegbrecht, S. Banerji, A. FliegerBacterial Infections (FG11), Robert Koch Institute, Wernigerode, GermanyLegionella pneumophila infects both mammalian cells and environmentalhosts, such as amoeba, and expresses a multitude of lipolytic enzymesbelonging to three different lipase families. One of these families, the GDSLhydrolases, comprises enzymes of prokaryotic and eukaryotic origin withphospholipase, acyltransferase, and hemolytic activities. Enzymatic activitydepends on a conserved nucleophilic serine embedded into the GDSL motifas well as on the residues aspartate and histidin together building up thecatalytic triad. The L. pneumophila genome codes for three GDSL-hydrolasegenes: plaA, plaC and plaD. The three enzymes show lysophospholipase A(LPLA) and phospholipase A (PLA) activity with PlaA being the majorsecreted LPLA. The sequences of PlaA and PlaC harbour N-terminal signalpeptides for Sec and subsequent type II-dependent protein export, whereasthe secretion mode of PlaD is still unclear. PLA/ LPLA activity of GDSLenzymestherefore contributes to the hydrolysis of eukaryotic membranephospholipids and leads to the release of toxic lysophosphatidylcholine,which can function as signal transducer and stimulator of inflammatoryresponse. We here aimed further characterization of L. pneumophila PlaCwhich in addition to PLA and LPLA activities, transfers free fatty acids fromphospholipids to cholesterol and ergosterol. This GCAT activity is posttranscriptionallyregulated by ProA, a secreted zinc metalloprotease and wehere studied the specific cleavage site of ProA within PlaC. Sincecholesterol is an important compound of mammalian cell membranes andegosterol of amoeba membranes, GCAT activity might be a tool for host cellremodelling during Legionella infection. As phospholipases are importantvirulence factors that have been shown to promote bacterial survival, spreadand host cell modification or damage, we here also aimed to investigate thecontribution of GDSL enzymes to L. pneumophila virulence and furtheraddressed the impact of GDSL-enzyme combinations on host infection.MPV004A yjbH-homologue in S. aureus: a new role of athioredoxin-like protein in ß-lactam resistanceN. Göhring*, I. Fedtke, D. Mader, S. Heinrich, D. Kühner, U. Bertsche,A. PeschelInstitute for Microbiology and Infection Medicine, Cellular and MolecularMicrobiology, Eberhard-Karls-University, Tübingen, GermanyBacteria have to adapt to a variety of environmental stresses and therebytrying to keep their cellular reduction/oxidation (redox) status in balance.Using low-molecular-weight thiols, like thioredoxins, help aerobic bacteriato maintain a reducing cytoplasm in which protein cysteines are kept in theirthiol (-SH) state. The breakage of unwanted disulphide bonds is oftenmediated via a conserved active site loop motif (CXXC) within thesethioredoxins by interprotein exchange reactions [1]. As Staphylococcusaureus lacks alternative thiol redox systems, the thioredoxin system istherefore essential for growth [1].YjbH as a thioredoxin-like protein has been describe in Bacillus subtilis as anovel effector within the disulphide stress regulation. Cells lacking YjbHshow pleiotropic defects in growth, sporulation and display a reducedsensitivity to the thiol oxidant diamide [2].Hence, the current research project deals with the characterisation of theYjbH-homologue in Staphylococcus aureus, its active site motif (CXC) andthe role in responding to thiol oxidants. In addition, the defined knock-outmutant (∆yjbH) showed an influence on beta-lactam resistance andpeptidoglycan cross-linking. Taken together, these results indicate anadditional functionality of the YjbH protein in Staphyloccus aureus.[1] Messens, J. et al (2004): J. Mol. Biol. 339.[2] Larsson, J.T.et al (2007): Mol. Micro. 66.spektrum | Tagungsband 2011