Comparative transcriptomics of intracellular survival of Listeria : A bioinformatics approach to determine the minimal genome required for intracellular survival of Listeria monocytogenes

摘要

The rapid technical development in recent years within the scope of sequence detection ranging from microarray via tilingarray to direct RNA sequencing enables new insights into gene expression as well as gene regulation of hitherto unparalleled accuracy and quality.This thesis chronologically describes the use of currently available technologies to analyse the intracellular transcriptome of pathogenic gram-positive germs, especially Listeria mon-ocytogenes EGD-e. Necessary adaptations and recent developments of the bioinformatics workflows facilitated, among other things, comparative analysis of extra- and intracellular transcription profiles to identify specific adaptations for intracellular survival of bacteria.For this purpose, a sequence of operations composed of existing and new software has been developed to ensure a standardized procedure for microarray analysis. Concerning this, the MARS Suite was extended, with the result that MARS II, in combination with MARSlab, is capable of uptake and processing of raw data, statistics, analysis, archiving and publication. Several studies were published using this framework.In search for specific adaptations to the survival under intracellular conditions, transcription of intergenic regions was investigated by whole genome tiling arrays. A further devel-opment of the existing microarray workflow permitted insights into the regulation of inter-genic regions of L. monocytogenes. For the first time, small non-coding RNAs, large anti-sense transcripts as well as long untranslated regions were detected under intracellular conditions based on this technology.Due to the technically limited resolution and the uncertainty of results, the first in-tracellular transcriptome of the pathogen L. monocytogenes has been sequenced with 454 Life Sciences technology. A completely new workflow had to be developed for the analy-sis of this new technology. The resulting software, sncRAS (small non-coding RNA analy-sis suite) is able to process information from several sequencing technologies, performs quality controls and enhances sequence quality through e.g. sequence trimming. Further-more sncRAS implements a variety of algorithms for mapping reads against reference ge-nomes as well as for the execution of different analyses and statistics. These supports the preparation of lists of sRNA candidates assisted by further data such as promoters, termi-nators, sRNA predictions, and experimentally verified candidates. Additionally, sncRAS allows the generation of several listings and graphics to assist the interpretation of the data, as well as the export of experimental data to public databases such as Array Express to facilitate the publication of the data. This workflow was successfully deployed in the pub-lication of intracellular small RNAs.Using genomic as well as transcriptomic analyses of four intracellular Listeria strains rep-resenting all three lineages by employing the MARS II pipeline, the major differences be-tween extra- and intracellular growth could be obtained. Initially, complete sequencing and annotation of strain L. monocytogenes 4a L99 were conducted. Bioinformatic analysis found that a reduction or loss of both, virulence genes as well as surface proteins, has an attenuating effect on the intracellular survival. In addition, the loss of several repeats with-in the actA gene leads to a deterioration of mobility which in turn results in a reduced in-tracellular survival rate. In contrast, duplication of PTS transporters and presence of differ-ent prophages had a positive impact on intracellular growth. Furthermore, a switch of the metabolic flow from regular glycolysis towards the pentose phosphate metabolism may serve multiple purposes including the production of NADPH countering oxidative stress.Another challenge was presented by the investigation of gene regulation of bacteria, which were extracted from IFN-γ activated macrophages. Due to the experimental setup, resulting signals were recognized near the detection limit. The analysis revealed a significant shift on the transcriptional level to counter oxidative as well as nitrosative stress in combination with an increased demand for tryptophan during replication.In the following course of the dissertation, the intracellular intergenic transcriptome of L. monocytogenes was examined in detail for the first time using modern RNA-Seq technolo-gy. As a result, 71 previously unknown sRNAs, of which 29 were expressed specifically intracellular, could be added to the collection. Based on phenotypic studies of 12 deletion mutants a strong influence of sRNAs upon intracellular survival could be demonstrated.In conclusion, bioinformatic workflows developed in this work can be used as a gen-eral tool for the analysis of genomes and transcriptomes as well as for the special applica-tion in search of new structures such as small non-coding RNAs.