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GRK 1482 Jahrbuch 2011-2014

Publications [1] Farber, JM and PI Peterkin. Listeria monocytogenes, a food-borne pathogen. Microbiol. 1991, Rev. 55:476–511. [2] Greiffenberg L, Goebel W, Kwang SK, Weiglein I, Bubert A, Engelbrecht F, Stins M and Kuhn M. Interaction of Lis- teria monocytogenes with Human Brain Microvascular Endothelial Cells: InlB-Dependent Invasion, Long-Term Intracellular Growth, and Spread from Macrophages to EndothelialCells.InfectImmun.1998,66(11):5260–5267. [3] Dussurget O, Dehoux P, Lecuit M, Glaser P, Cossart P. Listeria monocytogenes bile salt hydrolase is a PrfA- regulated virulence factor involved in the intestinal and hepatic phases of listeriosis. Mol Microbiol. 2002, 45(4):1095-106. [4] Jydegaard-AxelsenAM,HøibyPE,HolmstrømK,RussellN, Knøchel S. CO2 and anaerobiosis-induced changes in physiology and gene expression of different L. monocy togenes strains. Appl Environ Microbiol. 2004, 70(7): 4111-7. PhD FELLOWS GRK Progress Report 2011-2014 | Page 43 Aim In this study we want to investigate the impact of the adaptati- on of L. monocytogenes to anaerobic niches on the colonizati- on of the intestine. To better understand the relation between adaptation to anaerobiosis and virulence we want to compare the adaptation to hypoxia of a pathogenic (L. monocytogenes) and a non-pathogenic (L. weihenstephanensis) Listeria spe- cies. Furthermore, we want to analyze specific adaptations of different L. monocytogenes strains to hypoxia and investigate if such strain specific differences could be linked to differences in colonization efficiencies. Methods and Results By comparing the adaptation of L. monocytogenes and the non-pathogenic L. weihenstephanensis to anaerobiosis we want to identify the key genes for the adaptation to anaerobic conditions in the intestine. Growth analysis for Listeria weihen- stephanensis was performed at 24 °C (optimal T0) and it was shown that L. weihenstephanensis is able to grow anaerobi- cally. A prerequisite for the transcriptional profiling of the adap- tation to anaerobiosis with Next Generation Sequencing (NGS) of cDNA is the genome information. For this purpose we started a genome project, using the Illumina MiSeq technology. After a first sequencing run and filtering of the reads by SolexaQA, FastQC and NGS QC software, we obtained a 3.3 Mb assembly distributed over 92 contigs using the ABySS program. Align- ment of the contigs and annotation of the sequence is carried out at the moment. To investigate specific adaptations of different L. monocyto genes strains to hypoxia, a strain set comprising different sero- types from different origins (outbreak strains, food strains, laboratory strains) was chosen. First, growth experiments were performed aerobically and anaerobically at 37°C in the presence and absence of fumarate. It was shown that fumarate supports the anaerobic growth of some, but not all strains. Further inves- tigations will be focused on the study of the role of the stimula- tory effect of fumarate under anaerobiosis on colonization. Knock-out mutants for genes implicated in the adaptation to hypoxia will be generated and used for in vitro and in vivo stu- dies. Before using the mouse model it would be useful to pre- screen several mutants in the easier to handle Caenorhabditis elegans model. This model, however, is useful for us just if its GI is microaerophilic/anaerobic. For this purpose we performed in vivo infections (Fig. 1) using the wildtype L. monocytogenes EGDe and a deletion mutant that is unable to proliferate anaero- bically. In the first experiment, the wildtype could be re-isolated from C. elegans after 6 days in contrast to the mutant. Results have to be confirmed with further experiments to prove that this alternative animal model is suitable for our approach. Outlook To investigate differences in the adaptation to anaerobiosis in a non-pathogenic (L. weihenstephanensis) and a pathogenic strain (L. monocytogenes), as well as specific adaptations of different L. monocytogenes strains, global transcriptional ana- lysis will be performed by NGS. Deletion mutants for genes dif- ferently expressed will be generated and used for in vivo and in vitro studies to analyze their role in the colonization process. Figure: (1) C. elegans are grown on a NGM OP50 plate (optimal agar con- taining E.Coli OP50 as food source) at 25°C. (2) The L4 larva worms are transferred in a prepared BHI plate, containing the Listeria strain to test. (3) The plates are incubated at 25°C for 4h and then (4) the C. elegans are transferred again in a fresh NGM OP50 plate and (5) grown overnight at 25°C. This procedure is repeated for 5 days to distinguish the parents from the progeny. (6) Then the worms are collected and homogenized with the FastPrep and (7) specific dilutions are plated on Palcam Agar (selective for Listeria). Plates are incubated at 37°C for 1-2 days. Supervisors Prof. Dr. Siegfried Scherer | TUM | Microbial Ecology Dr. Stefanie Müller-Herbst | TUM I Microbial Ecology Prof. Dr. Dirk Haller I TUM I Nutrition and Immunology Start of project: July 2012 Academic background: Studies of Molecular Biology at Padova University (Italy) and at Ludwig-Maximilians-Universität München