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

126Finally, selected strains were characterized inin vitro screens using cellcultures. First results of these investigations are presented.MPP072Genetic and morphological analyses of the vancomycin anddaptomycin resistant Staphylococcus aureus strain VC40A. Berscheid* 1 , P. Sass 1,2 , A. Jansen 1 , M. Oedenkoven 1 , C. Szekat 1 ,P. François 3 , A. Strittmatter 4 , J. Schrenzel 3 , G. Gottschalk 4 , G. Bierbaum 11 University of Bonn, Institute of Medical Microbiology, Immunology andParasitology (IMMIP), Bonn, Germany2 Heinrich Heine University, Institute for Pharmaceutical Biology,Duesseldorf, Germany3 University of Geneva Hospitals, Genomic Research Laboratory, Divisionof Infectious Diseases, Geneva, Switzerland4 University of Göttingen, Institute of Microbiology and Genetics,Göttingen, GermanyGlycopeptide antibiotics (e.g. vancomycin) are the mainstay of therapy forserious infections caused by methicillin-resistant Staphylococcus aureus(MRSA). However, MRSA strains with reduced susceptibility toglycopeptides have emerged during the last decade. In times of increasedantibiotic treatment failure, there is an obvious need to understand howbacteria respond to the presence of antimicrobial compounds and developresistance. In order to study the impact of an elevated mutation frequencyon vancomycin resistance development, we had previously generated thehighly vancomycin resistant strain S. aureus VC40 (MIC: 64 g/ml) byserial passage of S. aureus RN4220mutS (MIC: 2 g/ml) in the presenceof increasing concentrations of vancomycin (1).In the current study, cross-resistance to daptomycin, a lipopeptideantibiotic recently introduced for the treatment of complicated skin andskin structure infections (cSSSI) caused by MRSA, was observed in strainVC40. To further elucidate the resistance phenotype of S. aureus VC40,the full genome sequences of this strain and its parent strainRN4220mutS were determined using 454 sequencing technology. A totalof 79 mutations in genes related to cell wall metabolism, transport andgene regulation were detected in strain VC40. Point mutations were alsofound in the histidine kinases of the two-component regulatory systemsVraSR and YycFG (WalKR), which significantly impact on thebiosynthesis and turnover rates of the bacterial cell wall (2,3), andaccompanying transcriptome analyses indeed showed an altered expressionof affected regulons in strain VC40. Further morphological analyses usingtransmission electron microscopy revealed that strain VC40 wascharacterized by an abnormal cell envelope morphology that may resultfrom deregulated VraSR or YycFG systems. Reintroduction of the VraSmutations into the parental background led to a significant increase inresistance against several cell wall-active antibiotics, includingvancomycin, daptomycin and the lantibiotic mersacidin. In conclusion,characterization of strain VC40 reveals a central role for VraS mutations inresistance development to cell envelope-active agents and may help to gaina better understanding of the mode of antibiotic resistance evolution in S.aureus.(1) Schaaff et al. 2002, AAC 46:3540-3548(2) Kuroda et al. 2003, Mol Microbiol 49(3):807-821(3) Dubrac et al. 2008, Mol Microbiol 70(6):1307-1322MPP073Antibiotic acyldepsipeptides inhibit bacterial cell division byinducing the ClpP peptidase-dependent degradation of the celldivision protein FtsZP. Sass* 1 , K. Famulla 1 , M. Josten 2 , H.-G. Sahl 2 , L. Hamoen 3 , H. Brötz-Oesterhelt 11 Heinrich Heine University of Duesseldorf, Institute for PharmaceuticalBiology, Duesseldorf, Germany2 University of Bonn, Institute of Medical Microbiology, Immunology andParasitology , Bonn, Germany3 Newcastle University, Centre for Bacterial Cell Biology, Institute for Celland Molecular Biosciences, Newcastle upon Tyne, United KingdomA novel class of antibacterial acyldepsipeptides (ADEPs) exerts prominentactivity against Gram-positive bacteria including multi-resistantStaphylococcus aureus in vitro and in vivo [1]. ADEPs act bydysregulating ClpP peptidase of the bacterial caseinolytic protease system.Usually, the activity of ClpP is tightly controlled by ATP-dependent Clp-ATPases and accessory proteins. ADEPs overcome these tight controlmechanisms, switching ClpP from a regulated to an uncontrolled proteasethat predominantly targets unfolded or flexible proteins as well as nascentpolypeptides in the absence of Clp-ATPases [1,2,3]. Although the activityof ADEPs can be explained on the molecular level of its target ClpP, thespecific events that finally lead to bacterial cell death remained unknown.In our study, we investigated the effect of ADEP treatment on differentGram-positive species using high-resolution microscopy. In the presenceof low inhibitory ADEP concentrations, the coccoid cells of S. aureus andStreptococcus pneumoniae swelled to more than 3-fold the volume of wildtype cells, and the rod-shaped cells of Bacillus subtilis grew into very longfilaments, which reached 60- to 100-fold the length of untreated cells,clearly indicating stalled bacterial cell division. To gain further insightsinto the underlying molecular mechanism, we followed the events that ledto the inhibition of cell division. We observed that ADEP treatmentresulted in the inhibition of septum formation in S. aureus and B. subtilis,while chromosome segregation was rather unaffected. Localization studieswith GFP-labeled cell division proteins revealed that the ADEP-ClpPcomplex interferes with key components of early cell division andtherefore perturbs normal divisome formation. By analyzing cell extractsof ADEP-treated bacteria, immunoblotting revealed that treated cellsshowed a significantly decreased abundance of the essential FtsZ protein,which consequently ends in bacterial cell death [4]. Specific degradation ofFtsZ by ADEP-activated ClpP was confirmed by in vitro studies usingpurified ClpP protein. ADEPs demonstrate that beside their interestingantibacterial potency they are excellent tools to examine centralmechanism of bacterial physiology, like cell division and regulatedproteolysis.[1] Brötz-Oesterhelt et al. 2005, Nat. Med. 11: 1082-87[2] Kirstein et al. 2009, EMBO Mol. Med. 1: 37-49[3] Lee et al. 2010, Nat. Struct. Mol. Biol. 1787: 1-8[4] Sass et al. 2011, Proc Natl Acad Sci U S A. 108(42):17474-9MPP074Molecular mode of action of acyldepsipeptide antibiotics inmycobacteriaK. Famulla* 1 , P. Sass 1 , T. Akopian 2 , O. Kandror 2 , R. Kalscheuer 3 ,A. Goldberg 2 , H. Broetz-Oesterhelt 11 Heinrich-Heine University Duesseldorf, Institute of Pharmaceutical Biologyand Biotechnology, Duesseldorf, Germany2 Harvard Medical School, Department of Cell Biology, Boston, MA, United States3 Heinrich-Heine University Duesseldorf, Institute for Medical Microbiology,Duesseldorf, GermanyAcyldepsipeptides (designated ADEPs) are a novel class of antibiotics,which act through an unprecedented mechanism by dysregulating thebacterial caseinolytic protease ClpP [1], which is otherwise controlled byClp-ATPases and adapter proteins. Binding of ADEPs to ClpP prevents theinteraction of the peptidase with corresponding Clp-ATPases and leads tothe inhibition of all natural functions of ClpP [2]. Additionally, ADEPsinduce opening of the entrance pore to the proteolytic chamber, whichconfers independent proteolytic activity to the peptidase. ADEP-activatedClpP degrades nascent polypeptides at the ribosome and flexible proteinsin the absence of Clp-ATPases [3]. Recently, it has been shown that FtsZ,an essential cell division protein, is a particularly sensitive target forADEP-activated ClpP [4].Although ADEPs demonstrated promising antibacterial activity againststaphylococci, streptococci and enterococci in vitro and in infectionmodels, their development was hampered by the fact that ClpP is notstrictly essential in these genera and prone to mutation. To this end, one ofour aims is to identify pathogens, which are less susceptible for mutationsand therefor have the potential for a slower development of ADEPresistance. A special feature of mycobacteria is that they encode twochromosomal copies of ClpP [5] and recent observations have shown thatboth genes are essential in these organisms [6, 7]. In this study, we arefocusing on the efficacy of ADEPs against mycobacteria. We demonstratethat ADEPs are active against Mycobacterium bovis BCG, which is closelyrelated to M. tuberculosis, a pathogen of global importance and thecausative agent of tuberculosis. We further observed that ADEPs activatethe ClpP complex of M. tuberculosis in vitro to degrade several modelsubstrates including the flexible protein casein. Thus, ADEPs are idealtools to study the function of these unique ClpP proteins in mycobacteria.Furthermore, ClpP represents a promising new drug target due to itsessentiality in these organisms, and ADEPs are interesting lead structuresfor the development of new anti-tuberculosis drugs.[1] Brötz-Oesterhelt et al. 2005, Nat. Med. 11: 1082-87[2] Kirstein et al. 2009, EMBO Mol. Med. 1: 37-49[3] Lee et al. 2010, Nat. Struct. Mol. Biol. 1787: 1-8[4] Sass et al. 2011, Proc Natl Acad Sci U S A. 108(42):17474-9[5] Chandu et al. 2004, Res. Microbiol. 155: 710-719[6] Sasetti et al. 2001, PNAS, 98: 12712-12717[7] Ollinger et al. 2011, J. Bacteriol., epub. ahead of print.MPP075Will not be presented!MPP076Interference of quinoles and aminocoumarines regardingRecA mediated response in Staphylococcus aureusW. Schröder*, C. Goerke, C. WolzAG Wolz, Medizinische Mikrobiologie, Tübingen, GermanyDifferent gyrase inhibitors bind to different moieties of the gyrase bothresulting in arrest of DNA replication. The chinolones are known to inducethe bacterial SOS response through the generation of double strand breakesresulting in RecA activation. RecA dependent cleavage of the LexArepressor results in error prone repair, which favours mutations andBIOspektrum | Tagungsband 2012