Functional assembly of two membrane-binding domains in listeriolysin O, the cytolysin of Listeria monocytogenes.
Listeriolysin O (LLO) is a major virulence factor secreted by the pathogenic Listeria monocytogenes and acts as pore-forming cytolysin. Based on sequence similarities between LLO and perfringolysin (PFO), the cytolysin from Clostridium perfringens of known crystallographic structure, two truncated LLO proteins were produced: LLO-d123, comprising the first three predicted domains, and LLO-d4, the last C-terminal domain. The two proteins were efficiently secreted into the culture supernatant of L. monocytogenes and were able to bind to cell membranes. Strikingly, when expressed simultaneously, the two secreted domains LLO-d123 and LLO-d4 reassembled into a haemolytically active form. Two in-frame linker insertions were generated in the hinge region between the d123 and d4 domains. In both cases, the insertion created a major cleavage site for proteolytic degradation and abolished cytolytic activity, which might suggest that the region connecting d123 and d4 participates in the interaction between the two portions of the monomer.
Dubail
Iharilalao
I
Laboratoire de Microbiologie, INSERM U-411, Faculté de Médecine Necker-Enfants Malades, 156 rue de Vaugirard, 75730 Paris Cedex 15, France1.
Autret
Nicolas
N
Laboratoire de Microbiologie, INSERM U-411, Faculté de Médecine Necker-Enfants Malades, 156 rue de Vaugirard, 75730 Paris Cedex 15, France1.
Beretti
Jean-Luc
JL
Laboratoire de Microbiologie, INSERM U-411, Faculté de Médecine Necker-Enfants Malades, 156 rue de Vaugirard, 75730 Paris Cedex 15, France1.
Kayal
Samer
S
Laboratoire de Microbiologie, INSERM U-411, Faculté de Médecine Necker-Enfants Malades, 156 rue de Vaugirard, 75730 Paris Cedex 15, France1.
Berche
Patrick
P
Laboratoire de Microbiologie, INSERM U-411, Faculté de Médecine Necker-Enfants Malades, 156 rue de Vaugirard, 75730 Paris Cedex 15, France1.
Charbit
Alain
A
Laboratoire de Microbiologie, INSERM U-411, Faculté de Médecine Necker-Enfants Malades, 156 rue de Vaugirard, 75730 Paris Cedex 15, France1.
D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D003603:Cytotoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000378:metabolism; D005057:Eukaryotic Cells / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006460:Hemolysin Proteins; D006461:Hemolysis; D006801:Humans; D008089:Listeria monocytogenes / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D016254:Mutagenesis, Insertional; D017510:Protein Folding; D017422:Sequence Analysis, DNA; D013329:Structure-Activity Relationship
D001427:Bacterial Toxins; D003603:Cytotoxins; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; C020452:Clostridium perfringens theta-toxin; C038705:hlyA protein, Listeria monocytogenes