This proposal examines infection with the facultative intracellular pathogen Listeria monocytogenes (LM), focusing on the early stage when LM is multiplying in tissues at a high exponential rate. During this period, precisely by the 48th hour, there is extensive lymphocyte death by apoptosis in lymphoid organs. The death is caused by the LM virulent pore forming protein Listeriolysin O (LLO). This proposal is based on three observations, the finding of extensive lymphocyte apoptosis in infective foci, our finding ex vivo and in vivo, that LLO is apoptogenic to lymphocytes;and observations indicating that the lymphocyte death stage may be linked to a period of lower resistance. The lower resistance became evident when SCID mice were found to handle the exponential growth phase better than normal mice. Pointedly, SCID mice injected with lymphocytes at the same time of infection, reverted to the pattern seen in normal mice, with higher LM burden. The plans include examining the molecular basis of LLO action on cells, to in vivo analysis of how LLO may be acting, plus an evaluation of the changes that take place in lymphoid organs at the time of the apoptotic phase. Accordingly, how LLO induces apoptotic death of lymphocytes is examined in detail. Preliminary evidence indicates that LLO initiates an intracellular proteolytic pathway that results in the death program. Experiments are planned to determine the nature of this pathway. Having a better understanding and pinpointing critical molecules in the death pathway may allow the infection to be modulated by targeting such molecules in a number of ways. Another important issue to examine is whether lymphocytes vary in their susceptibility to LLO apoptosis depending on their set and/or activation states: this issue is tackled by both in vivo and ex vivo experiments with purified cells. A LM that expresses LLO as a membrane protein is being engineered, in order to explore whether it is extracellular LLO that causes the lymphocyte death. The proposal includes a number of different approaches in order to determine whether lymphocyte apoptosis modulates in a negative way the infectious process. In summary, listeriosis is a powerful system to examine the relevance of lymphocyte death to the outcome of an infectious disease.
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