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.

Evolutionary pathway to increased virulence and epidemic group A Streptococcus disease derived from 3,615 genome sequences

“…The molecular events included phage-mediated virulence gene acquisition, single nucleotide variant accumulation, and horizontal transfer of the 36-kb region encoding the virulence factors Nga and Slo (61)(62)(63)(64)(65)(66)(67)(68)(69)(70)(71)(72)(73), likely mediated by generalized transduction (24). It is now clear from our analysis that a complex multistep (9).…”

Disease Manifestations and Pathogenic Mechanisms of Group A Streptococcus

“…Finally, a number of these antigens exhibit toxic effects in the host or possess enzymatic activity, including SOF, ScpA, SpeB, Sse, Sib35, SpyCEP, SLO, ADI, and TF. The incorporation of the active forms of these antigens into GAS vaccine preparations may raise safety concerns, and consequently, inactive forms of these antigens have been engineered by employing site-directed mutagenesis for use in vaccine studies, including SOF (581), ScpA (571), SpeB (575), SpyCEP (578,582), SLO (582,583), and ADI (584).…”

Four-Component Staphylococcus aureus Vaccine 4C-Staph Enhances Fcγ Receptor Expression in Neutrophils and Monocytes and Mitigates S. aureus Infection in Neutropenic Mice

“…Among them, an "air-pouch" murine model has been extensively used to study inflammation (7)(8)(9). This model was adapted further to evaluate the effect of fibrinogen depletion on S. aureus infections (10), as well as to study the host responses to group A Streptococcus infection, and the reduction of infection in mice immunized with specific antigens (11)(12)(13). The model is based on two dorsolateral subcutaneous injections of air to generate a "pouch" in which bacteria are subsequently inoculated, mimicking a skin infection.…”