title: Targeted amino acid substitutions impair streptolysin O toxicity and group A Streptococcus virulence.
abstract: Streptolysin O is a potent pore-forming toxin produced by group A Streptococcus. The aims of the present study were to dissect the relative contributions of different structural domains of the protein to hemolytic activity, to obtain a detoxified form of streptolysin O amenable to human vaccine formulation, and to investigate the role of streptolysin O-specific antibodies in protection against group A Streptococcus infection. On the basis of in silico structural predictions, we introduced two amino acid substitutions, one in the proline-rich domain 1 and the other in the conserved undecapeptide loop in domain 4. The resulting streptolysin O derivative showed no toxicity, was highly impaired in binding to eukaryotic cells, and was unable to form organized oligomeric structures on the cell surface. However, it was fully capable of conferring consistent protection in a murine model of group A Streptococcus infection. When we engineered a streptococcal strain to express the double-mutated streptolysin O, a drastic reduction in virulence as well as a diminished capacity to kill immune cells recruited at the infection site was observed. Furthermore, when mice immunized with the toxoid were challenged with the wild-type and mutant strains, protection only against the wild-type strain, not against the strain expressing the double-mutated streptolysin O, was obtained. We conclude that protection occurs by antibody-mediated neutralization of active toxin.
  
  
  OBJECTIVE
  We present a novel example of structural design of a vaccine antigen optimized for human vaccine use. Having previously demonstrated that immunization of mice with streptolysin O elicits a protective immune response against infection with group A Streptococcus strains of different serotypes, we developed in this study a double-mutated nontoxic derivative that represents a novel tool for the development of protective vaccine formulations against this important human pathogen. Furthermore, the innovative construction of an isogenic strain expressing a functionally inactive toxin and its use in infection and opsonophagocytosis experiments allowed us to investigate the mechanism by which streptolysin O mediates protection against group A Streptococcus. Finally, the ability of this toxin to directly attack and kill host immune cells during infection was studied in an air pouch model, which allowed parallel quantification of cellular recruitment, vitality, and cytokine release at the infection site.
authors.affiliation: Novartis Vaccines and Diagnostics, Siena, Italy.
authors.forename: Emiliano
authors.initials: E
authors.lastname: Chiarot
authors.forename: Cristina
authors.initials: C
authors.lastname: Faralla
authors.forename: Nico
authors.initials: N
authors.lastname: Chiappini
authors.forename: Giovanna
authors.initials: G
authors.lastname: Tuscano
authors.forename: Fabiana
authors.initials: F
authors.lastname: Falugi
authors.forename: Gabriella
authors.initials: G
authors.lastname: Gambellini
authors.forename: Annarita
authors.initials: A
authors.lastname: Taddei
authors.forename: Sabrina
authors.initials: S
authors.lastname: Capo
authors.forename: Elena
authors.initials: E
authors.lastname: Cartocci
authors.forename: Daniele
authors.initials: D
authors.lastname: Veggi
authors.forename: Alessia
authors.initials: A
authors.lastname: Corrado
authors.forename: Simona
authors.initials: S
authors.lastname: Mangiavacchi
authors.forename: Simona
authors.initials: S
authors.lastname: Tavarini
authors.forename: Maria
authors.initials: M
authors.lastname: Scarselli
authors.forename: Robert
authors.initials: R
authors.lastname: Janulczyk
authors.forename: Guido
authors.initials: G
authors.lastname: Grandi
authors.forename: Immaculada
authors.initials: I
authors.lastname: Margarit
authors.forename: Giuliano
authors.initials: G
authors.lastname: Bensi
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D000992:Antitoxins / Q000097:blood; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology / Q000633:toxicity; D004195:Disease Models, Animal; D051379:Mice; D008958:Models, Molecular; D050505:Mutant Proteins / Q000235:genetics / Q000276:immunology / Q000633:toxicity; D013290:Streptococcal Infections / Q000276:immunology / Q000382:microbiology / Q000473:pathology / Q000517:prevention & control; D013297:Streptococcus pyogenes / Q000235:genetics / Q000276:immunology / Q000472:pathogenicity*; D013301:Streptolysins / Q000235:genetics* / Q000276:immunology / Q000633:toxicity*; D016019:Survival Analysis; D014774:Virulence; D037521:Virulence Factors / Q000235:genetics* / Q000276:immunology / Q000633:toxicity*
chemical_list: D000907:Antibodies, Bacterial; D000992:Antitoxins; D001426:Bacterial Proteins; D050505:Mutant Proteins; D013301:Streptolysins; D037521:Virulence Factors; C025964:streptolysin O