Modification of the signal sequence cleavage site of listeriolysin O does not affect protein secretion but impairs the virulence of Listeria monocytogenes.
Listeriolysin O (LLO, hly-encoded), a major virulence factor secreted by the bacterial pathogen Listeria monocytogenes, is synthesized as a precursor of 529 residues. To impair LLO secretion, the four residues of the predicted signal sequence cleavage site (EA-KD) were deleted and the mutant LLO protein was expressed in a hly-negative derivative of L. monocytogenes. Unexpectedly, the mutant protein was secreted in normal amounts in the culture supernatant and was fully haemolytic. N-terminal sequencing of the secreted LLO molecule revealed that N-terminal processing of the preprotein occurred three residues downstream of the natural cleavage site. L. monocytogenes expressing this truncated LLO showed a reduced capacity to disrupt the phagosomal membranes of bone marrow macrophages and of hepatocytes; and the mutant strain showed a 100-fold decrease in virulence in the mouse model. These results suggest that the first N-terminal residues of mature LLO participate directly in phagosomal escape and bacterial infection.
Laboratoire de Microbiologie, INSERM U-570, Faculté de Médecine Necker, 156 rue de Vaugirard, 75730 Paris Cedex 15, France.
Marie-Annick
MA
Lety
Laboratoire de Microbiologie, INSERM U-570, Faculté de Médecine Necker, 156 rue de Vaugirard, 75730 Paris Cedex 15, France.
Claude
C
Frehel
Laboratoire de Microbiologie, INSERM U-570, Faculté de Médecine Necker, 156 rue de Vaugirard, 75730 Paris Cedex 15, France.
Jean-Luc
JL
Beretti
Laboratoire de Microbiologie, INSERM U-570, Faculté de Médecine Necker, 156 rue de Vaugirard, 75730 Paris Cedex 15, France.
Patrick
P
Berche
Laboratoire de Microbiologie, INSERM U-570, Faculté de Médecine Necker, 156 rue de Vaugirard, 75730 Paris Cedex 15, France.
Alain
A
Charbit
D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins; D001483:Base Sequence; D004195:Disease Models, Animal; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006460:Hemolysin Proteins; D006801:Humans; D008089:Listeria monocytogenes / Q000235:genetics / Q000472:pathogenicity*; D008088:Listeriosis / Q000382:microbiology / Q000503:physiopathology*; D008264:Macrophages / Q000382:microbiology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009154:Mutation; D010588:Phagosomes / Q000382:microbiology; D021382:Protein Sorting Signals / Q000235:genetics*; D014407:Tumor Cells, Cultured; D014774:Virulence
D001427:Bacterial Toxins; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D021382:Protein Sorting Signals; C038705:hlyA protein, Listeria monocytogenes