Abstract
The presentation of microbial protein antigens by Major Histocompatibility Complex (MHC) molecules is essential for the development of acquired immunity to infections. However, most biochemical studies of antigen processing and presentation deal with a few relatively inert non-microbial model antigens. The bacterial pore-forming toxin listeriolysin O (LLO) is paradoxical in that it is cytotoxic at nanomolar concentrations as well as being the source of dominant CD4 and CD8 T cell epitopes following infection with Listeria monocytogenes. Here, we examined the relationship of LLO toxicity to its antigenicity and immunogenicity. LLO offered to antigen presenting cells (APC) as a soluble protein, was presented to CD4 T cells at picomolar to femtomolar concentrations- doses 3000–7000-fold lower than free peptide. This presentation required a dose of LLO below the cytotoxic level. Mutations of two key tryptophan residues reduced LLO toxicity by 10–100-fold but had no effect on its presentation to CD4 T cells. Thus there was a clear dissociation between the cytotoxic properties of LLO and its very high antigenicity. Presentation of LLO to CD8 T cells was not as robust as that seen in CD4 T cells, but still occurred in the nanomolar range. APC rapidly bound and internalized LLO, then disrupted endosomal compartments within 4 hours of treatment, allowing endosomal contents to access the cytosol. LLO was also immunogenic after in vivo administration into mice. Our results demonstrate the strength of LLO as an immunogen to both CD4 and CD8 T cells.
Highlights
LLO is an immunological enigma: it is both a major virulence determinant and a major immunogen following L. monocytogenes infection, yet it is a highly cytotoxic protein
Cytotoxicity of LLO We examined the antigenicity of soluble LLO as well as of LLO having two key tryptophans mutagenized to alanines [24]. These residues are part of a highly conserved undecapeptide sequence (483-ECTGLAWEWWR-493) involved in the pre-pore to pore transition when cholesterol-dependent cytolysin (CDC) family members bind to membranes [25]
The dose of LLOWT required for cell death coincided with the drop in the T cell response detected in our T cell assays
Summary
LLO is an immunological enigma: it is both a major virulence determinant and a major immunogen following L. monocytogenes infection, yet it is a highly cytotoxic protein. Intracellular L. monocytogenes requires LLO to escape the phagosome and survive in infected cells. LLOdeficient L. monocytogenes (Dhly) are not pathogenic and are poorly immunogenic even at high doses [9,10,11,12]. LLO production is carefully controlled by the microbe, through both transcriptional and post-translational mechanisms to prevent the early destruction of the infected cell [13,14,15]. L. monocytogenes engineered to have uncontrolled LLO activity are less virulent because they destroy their protective host niche [16]
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