Dual non-coding RNA profiles of host and bacteria and their role in the regulation of innate immune response during Listeria monocytogenes infection
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During the course of infection, the pathogens follow several strategies to evade from the host defense system and to adapt to the host environment for efficient survival. Meanwhile the host imposes several cellular processes, innate and adaptive immune systems to fight back against the pathogens. At molecular level, the pathogens deploy different strategies, mainly by modulating their gene expression profiles as per the intercellular and intracellular compartments of the host. Similarly, the host also alters its gene expression profile specific to each pathogen to protect itself. For a longtime, proteins were considered to be predominant molecules to regulate gene expression. With the discovery of regulatory non-coding RNAs (ncRNAs) in both prokaryotes and eukaryotes this assumption had to be reconsidered. These regulatory ncRNAs not only opened a new branch in the understanding of gene regulation but also furthermore represented possible biomarkers for diagnostic purposes or leverage points for drug targets. In this context, we took effort to study the functional roles of these eukaryotic non-coding RNAs (miRNAs) from the perspective of host-pathogen interactions and host immune responses during infections. In this thesis, efforts are made to reveal host miRNA response to model pathogen L. monocytogenes in different infection models (Caco-2 cells and G. mellonella) along with bacterial ncRNA (asRNAs) profilling during their intracellular survival in P388D1 macrophages. The first part of the study revealed the altered miRNA response in Caco-2 cells that were infected with L. monocytogenes. Using different mutant strains (deltahly and deltainlAB), which are unable to invade epithelial cells and escape from phagocytic vacuoles, we demonstrated that miRNA response is dependent on the subcellular localization of L. monocytogenes and its virulence determinants. Even the purified endotoxin LLO from L. monocytogenes is able to regulate significant miRNAs in Caco-2 cells. The correlation of selected miRNAs and their predicted target gene expression levels uncovered roles of miRNAs in fine-tuning of immune related gene expression during L. monocytogenes infection. In continuation of the first study, the second part involved elucidation of miRNA response to L. monocytogenes infection in invertebrate infection model G. mellonella and its role in host-microbial interactions. Insect specific miRNA microarray analysis demonstrated the deregulation of miRNA response with upregulation of 39 and downregulation of 58 miRNAs upon infection of L. monocytogenes in G. mellonella. Some of the miRNAs regulation patterns are conserved between vertebrates and invertebrates, as we observed the downregulation of conserved miRNAs miR-133 and miR-998 (homologous to miR-29) both in higher mammals and G. mellonella with bacterial infections. We observed pathogen/non-pathogen specific miRNA regulation in this insect model, when we compared miRNAs expression patterns with pathogenic L. monocytogenes and non-pathogenic L. innocua infections. Later, we established a public database, which would be very useful to study insect-microbial interactions that can correlate even with higher animals. qRT-PCR analysis of predicted target genes such as spätzle, MAP kinase and optineurin demonstrated the role of miRNAs in Toll pathway, MAP-kinase pathway and autophagy process with pathogenic L. monocytogenes infection.In the third part of the thesis, bacterial ncRNAs especially asRNAs that are involved in metabolic adaptations and virulence of L. monocytogenes are unveiled using different sequencing technologies such as SOLiD and Ion torrent technologies. By using SOLiD platform, several antisense RNAs were identified and some of them were associated with regulation of housekeeping genes like purA, fumC and pgi, thereby involved in metabolic adaptations of L. monocytogenes. In another study, Ion torrent based sequencing of intracellularly grown L. monocytogenes RNA revealed the existence of long noncoding RNAs that overlap internalins, methylases and potassium uptake system (kdpABCD operon), highlighting their probable role in regulation of various group of genes.In conclusion, the thesis provides crucial insights over the ncRNAs mediated regulation of host immune response, role in listerial adaptations to intracellular environment as well as the interplay between host and pathogens.Verknüpfung zu Publikationen oder weiteren Datensätzen
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Giessen : VVB Laufersweiler Verlag