Exploring the role of the CTL epitope region of listeriolysin O in the pathogenesis of Listeria monocytogenes.
Listeria monocytogenes is a facultative intracellular bacterial pathogen responsible for severe opportunistic infections in humans and animals. The secreted cholesterol-dependent cytolysin, listeriolysin O (LLO), mediates phagosomal escape and allows bacterial growth in the cytosol of infected cells. In order to identify new LLO determinants participating in bacterial pathogenesis, this study focused on a major target of LLO proteolytic cleavage in vitro, the CTL epitope region (residues 91-99). Mutations were generated by site-directed mutagenesis in the epitope or in the two clusters of positive charges flanking the epitope. Two LLO mutants (a single mutation K103A and a double mutation R89G, K90G) were normally and stably secreted by L. monocytogenes. In contrast, a mutant carrying four amino acid substitutions in the epitope itself (Y92K, D94A, E97K, Y98F) was highly susceptible to proteolytic degradation. While these three LLO mutant proteins showed a reduced haemolytic activity, they all promoted efficient phagosomal escape and intracellular multiplication in different cell types, and were non-cytotoxic. The deletion of the epitope (Delta91-99), as well as the substitution of two, three or four of the four lysine residues (K103 to K106) by alanine residues did not lead to the production of a detectable protein. These results confirm the lack of correlation between haemolytic activity and phagosomal membrane disruption. They reveal the importance of the 91-99 region in the production of a stable and functional LLO. LD(50) determinations in the mouse model suggest a possible link between LLO stability and virulence.
Lety
Marie-Annick
MA
Faculté de Médecine Necker-Enfants Malades, INSERM U-570, 156, rue de Vaugirard, 75730 Paris Cedex 15, France.
Frehel
Claude
C
Faculté de Médecine Necker-Enfants Malades, INSERM U-570, 156, rue de Vaugirard, 75730 Paris Cedex 15, France.
Raynaud
Catherine
C
Faculté de Médecine Necker-Enfants Malades, INSERM U-570, 156, rue de Vaugirard, 75730 Paris Cedex 15, France.
Dupuis
Marion
M
Faculté de Médecine Necker-Enfants Malades, INSERM U-570, 156, rue de Vaugirard, 75730 Paris Cedex 15, France.
Charbit
Alain
A
Faculté de Médecine Necker-Enfants Malades, INSERM U-570, 156, rue de Vaugirard, 75730 Paris Cedex 15, France.
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D004195:Disease Models, Animal; D018984:Epitopes, T-Lymphocyte / Q000235:genetics / Q000276:immunology*; D005260:Female; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006460:Hemolysin Proteins; D006461:Hemolysis; D007928:Lethal Dose 50; D008089:Listeria monocytogenes / Q000235:genetics / Q000472:pathogenicity*; D008088:Listeriosis / Q000382:microbiology; D008264:Macrophages / Q000382:microbiology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010588:Phagosomes / Q000382:microbiology; D017384:Sequence Deletion; D016019:Survival Analysis; D037521:Virulence Factors / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
D001427:Bacterial Toxins; D018984:Epitopes, T-Lymphocyte; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D037521:Virulence Factors; C038705:hlyA protein, Listeria monocytogenes