Final Report Summary - IMMUNITY TO LISTERIA (Mechanisms of innate immune activation of the intracellular bacterial pathogen L. monocytogenes.)
In the research program “immunity to Listeria” funded by the Marie Curie FP7 program, we aimed to study the interactions between the human intracellular bacterial pathogen Listeria monocytogenes and the innate immune system. We specifically proposed to perform forward genetic screens using L. monocytogenes mutant libraries to identify bacterial molecular determinants that contribute to activation of host innate immune pathways. We also suggested studying further the previously identified multidrug transporters (MDRs) as modulators of Type I interferon response in macrophage cells and their physiological role in vivo. Finally, we aimed on identifying novel immuno-stimulatory ligands that secreted by the MDR transporters. During the 4 years of the program, we have made major progress in all the stated aims.
The genetic screens were done in the first year and yielded several interesting bacterial mutants that triggered high or low innate immune responses in macrophage cells. Some of these mutants were further studied and published as described below. Among the mutants that failed to activate the innate immune system we identified a secDF mutant. SecDF is a component of the Sec secretion system of bacteria. We specifically found that this protein plays a major role in the secretion of several critical L. monocytogenes virulence factors. A ΔsecDF mutant exhibited impaired membrane translocation of factors that mediate L. monocytogenes phagosomal escape and spread from cell to cell. This impaired translocation was monitored by accumulation of the factors on the bacterial membrane and by reduced activity upon secretion. This defect in secretion was shown to be associated with a severe intracellular growth defect of the ΔsecDF mutant in macrophage cells and a less virulent phenotype in mice, despite normal growth in laboratory medium. We found that SecDF is up-regulated when the bacteria reside in macrophage phagosomes and that it is necessary for efficient phagosomal escape. We concluded that SecDF plays a role as a chaperone that facilitates the translocation of L. monocytogenes virulence factors during infection.
In a search for bacterial determinants that work together with the MDRs in the induction of Type I interferon, we performed a second genetic screen for L. monocytogenes mutants that modulate Type I interferon response in the background of L. monocytogenes strain that over-express MdrM (i.e. ΔmarR mutant), the major MDR that was shown to affect Type I interferon response in macrophage cells. In this screen we looked for mutants that suppress or enhance the induction of IFN-β upon macrophages infection. We found that mutants defected in L. monocytogenes lipoteichoic acid (LTA) synthesis (i.e. ΔlafA mutant) led to enhanced activation of the type I interferon responses in infected macrophage cells. This innate immune response required the MDR transporters and was recapitulated by exposing macrophage cells to culture supernatants derived from the LTA mutant bacteria. These results indicated that there are small molecules, possibly substrates of the MDRs, which are activating the innate immune system. Interestingly, we found that the MDR transporters themselves are required for full production of LTA, an observation that links for the first time MDR transporters to LTA synthesis. In light of our findings, we propose that the MDR transporters play a role in regulation of LTA synthesis, possibly via c-di-AMP transport, a physiological function that triggers the host innate immune system.
In addition we performed a broad study searching for physiological conditions that might require the function of the MDRs transporters, in attempt to understand the mechanism by which they affect the Type I interferon response in mammalian cells.
First, we found that it is not MdrM alone, but a cohort of MDR transporters that together induce type I interferons during infection. In a search for a physiological function of these transporters we identified a role in cell wall stress responses. A mutant deleted of four transporters, ΔmdrMTAC, was sensitive to sub-lethal concentration of vancomycin due to an inability to produce and shed peptidoglycan under this stress. Remarkably, c-di-AMP was involved with this phenotype, as over-expression of the c-di-AMP phosphodiesterase (PdeA) resulted in increased susceptibility of ΔmdrMTAC to vancomycin, whereas over-expression of the c-di-AMP diadenylate cyclase (DacA) reduced its susceptibility to this drug. These observations demonstrated a physiological association between c-di-AMP and the MDR transporters, supporting the premise that MDR transporters mediate c-di-AMP secretion to regulate peptidoglycan synthesis in response to cell wall stress.
Based on the above studies we have used ΔmdrMTAC and ΔlafA mutants (low and high interferon inducers, respectively) to isolate active fractions of bacterial culture supernatants that activate IFN-β. We are now establishing a new collaboration with METABOLOMIC DISCOVERIES GmbH company in Germany to identify the nature of these molecules.
The results of our studies will facilitate the understanding of the mechanisms by which L. monocytogenes modulates immune responses and will contribute greatly to the study of other bacterial pathogens. Over all isolating L. monocytogenes mutants that induce altered innate immune responses may represent novel vaccine platforms and may eventually comprise more potent vaccines. The contribution of the expected outcome of this research to society is primarily by facilitating our basic understanding of the complex interactions between immune-cells and pathogens and might increase our arsenal for vaccine development and immunotherapy.