The peptidoglycan layer of gram-positive bacteria contains various components that are crucial for interactions with the environment and for host invasion. In this study, the gram-positive pathogen Listeria monocytogenes was used to study the importance of three cell wall constituents for viability and virulence: the cell-wall degrading murein hydrolases (autolysins), teichoic acids and lipoproteins.The first section of this study addresses cell wall hydrolases and their role in deter-mining the morphology, stability and activity of the bacterial cell. Numerous reports have previously documented the spontaneous generation of rough strains from otherwise smooth colonies isolated from clinical samples or the environment. Here I identify the molecular basis of this transition by examining two murein hydrolases, MurA (murA) and p60 (iap), whose activity are critical for this transition. Whereas single mutation of the respective genes results in a smooth phenotype with chains of bacterial cells, double mutation generates rough colony morphology. Combinatory deletions of each autolysin with other known hydrolases in L. monocytogenes (Ami, Auto and p45) were assessed for the smooth-rough transition. None of these mutants exhibit a rough phenotype or altered cell morphology. It was observed that strains lacking murA, iap or ami display altered abilities in autolysis, ethanol tolerance, flagellar motility and biofilm formation. Bioinformatic analysis of the listerial genome was used to identify proteins with putative murein hydrolase activity. Characterization of respective deletion mutants revealed that none exhibit changes in morphology or in lytic activity. However, all newly generated bacterial strains are more vulnerable to ethanol stress when compared to their parental strain. For some mutants, impaired flagellar motility and decreased host invasion could also be observed.The second part of my thesis deals with the modulation of teichoic acids via incorporation of D-alanine to resist attack by cationic antimicrobial peptides (CAMPs). The VirR/S regulon of L. monocytogenes has been shown to control various genes, among those resistance genes for CAMPs, dltA-D and mprF. Deletion mutants lacking corresponding genes were generated to determine their sensitivity towards CAMPs and their function in host invasion. Using quantitative RT-PCR, the dltB and mprF genes were found to be up-regulated in L. monocytogenes EGDe in the presence of CAMPs. The virR and virS deletion strains have decreased tolerance to various cationic peptides. They also display very low invasion rates in Caco2 cells but wild-type-like rates in Hela cells. This discrepancy may be due to the different abilities of Caco2 and Hela cells to produce cationic peptides. These data suggest that the VirR/S two-component system is involved in the control of the dlt and mprF resistance genes when L. monocytogenes is in the presence of cationic peptides.The final section of my thesis examines the role of lipoproteins for listerial virulence and host immune activation. Recently, a deletion strain of L. monocytogenes lacking the prolipoprotein diacylglyceryl transferase (lgt) gene was generated that is unable to produce lipoproteins due to the loss of diacylglycerol modification activity. The absence of lipoproteins on the bacterial surface renders the strain insensitive to TLR2 recognition and host cells exposed to mutant lgt bacteria show highly delayed proinflammatory cytokine production. Infection studies of wild-type and TLR2-deficient mice demonstrate attenuation of the lgt deletion mutant, implying multiple roles of lipoproteins during infection. Further characterization of the lgt mutant revealed that it is impaired in both invasion and intracellular survival, and that it exhibits increased susceptibility to cationic peptides and ethanol. Lipoproteins are identified as the immunologically active ligands of TLR2 and as relevant contributors to the pathogenicity at various stages of infection.
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