The cholesterol-binding cytolysins, listeriolysin O (LLO) as well as pneumolysin (PLY), were expressed in Listeria innocua. Growth in the chemically defined minimal medium resulted in 2-fold increment in the expression of both cytolysins. Purification from supernatant fluids was achieved by ion exchange chromatography. The procedure resulted in about 75 % (LLO) or 70 % (PLY) yield of a hemolytically active, highly purified homogenous proteins.A new and previously uncharacterized target of the humoral immune response, the listerial ferritin protein (Frm), was discovered and characterized. Specific antibodies to Frm are detected in antisera of mice infected with the L. monocytogenes wild type strain but not in antisera of mice infected with a non-pathogenic L. innocua strain. Expression of Frm is both growth phase- and temperature- dependent. Using an isogenic delta frm mutant, ferritin was found to be essential for bacterial growth in minimal media but not in complex media such as BHI. Mutant bacteria also exhibited a defect in intracellular survival. The delta frm strain is hypersensitive to hydrogen peroxide. Mouse infection studies revealed that the listerial ferritin is required for efficacious bacterial growth early in the infectious process. Using the murine listeriosis model, it was investigated whether the induction and expansion of protective and inflammatory T cell responses may be modified by selective manipulation of virulence genes. This was accomplished in two ways; the first was through generation of isogenic Listeria monocytogenes mutant strains that harboured either a specific deletion within the actA gene and/or multiple deletions within the actA and plcB genes while the second way was through the complementation of the non-pathogenic Listeria innocua strain with the PrfA-dependent virulence gene cluster (vgc) of the wild type L. monocytogenes. In comparison to the wild type L. monocytogenes EGD-e strain, the mutant strains were extremely low in virulence and were rapidly eliminated by the host during the first days of infection. Nevertheless, a single immunization with mutant strains has efficiently induced and maintained effector memory CD8+ T cells and provided animals with a state of long-lasting protective immunity against wild type L. monocytogenes. Moreover, these mutants exhibited a significantly reduced ability to induce CD4+ T cell-mediated inflammation. Therefore, they can be used as live vaccines against the corresponding virulent pathogen and as carriers for heterologous antigens .
In an attempt to address the role of the pore forming listeriolysin O (LLO) in intracellular survival of L. monocytogenes as well as in mediating a protective T- cell response against the wild type L. monocytogenes strain, two approaches were conducted in this study. Firstly, the structural gene for the related cytolysin pneumolysin (PLY) was cloned on a plasmid vector downstream from the promoter and signal peptide sequences of hly, the gene encoding LLO, and expressed in the isogenic EGD-e hly mutant strain. The resultant recombinant strain secreted active PLY in culture supernatants and was able to escape phagosomes of phagocytic cells in vitro and spread from cell to cell to a limited time after which growth was aborted because of a cytotoxic effect on the host cell. This strain also showed a restricted in vivo survival in a mouse model of listeriosis but was able to protect mice against a lethal dose of the wild type L. monocytogenes and induced a specific T-cell responses against Listeria derived epitopes other than LLO such as the subdominant P60217-225 epitope.
The second approach was to study the role of the putative PEST-like sequence, found near the N-terminus of LLO, in virulence and intracellular compartmentalization of the wild type L. monocytogenes strain as well as in induction of a strong cell-mediated protective immunity to the wild type L. monocytogenes. Therefore, the 28 amino acids harbouring the PEST-like sequence at the N-terminus of LLO were deleted and the mutant LLO protein was expressed in a hly-negative isogenic mutant of L. monocytogenes. The mutant protein was secreted in normal amounts in the culture supernatant and was fully haemolytic. L. monocytogenes expressing this truncated LLO showed a reduced capacity to escape the phagosomes of J774 cell line macrophages and showed a 1000-fold decrease in virulence in the mouse model. Moreover, the mutant strain showed a low ability to induce both an early serum level of gamma interferon and gamma interferon-secreting T cells following infection of BALB/c mice and exhibited reduced levels of protection against virulent L. monocytogenes. These results suggest that the PEST-like sequence is crucial for conferring a long lasting immunity against the wild type Listeria monocytogenes through facilitating the bacterial escape into the cytosole of host cells for further processing and antigen presentation.
Verknüpfung zu Publikationen oder weiteren Datensätzen
