Abstract
Streptococcus pyogenes is a major human pathogen worldwide, responsible for both local and systemic infections. These bacteria express the subtilisin-like protease SpyCEP which cleaves human IL-8 and related chemokines. We show that localization of SpyCEP is growth-phase and strain dependent. Significant shedding was observed only in a strain naturally overexpressing SpyCEP, and shedding was not dependent on SpyCEP autoproteolytic activity. Surface-bound SpyCEP in two different strains was capable of cleaving IL-8. To investigate SpyCEP action in vivo, we adapted the mouse air pouch model of infection for parallel quantification of bacterial growth, host immune cell recruitment and chemokine levels in situ. In response to infection, the predominant cells recruited were neutrophils, monocytes and eosinophils. Concomitantly, the chemokines KC, LIX, and MIP-2 in situ were drastically increased in mice infected with the SpyCEP knockout strain, and growth of this mutant strain was reduced compared to the wild type. SpyCEP has been described as a potential vaccine candidate against S. pyogenes, and we showed that surface-associated SpyCEP was recognized by specific antibodies. In vitro, such antibodies also counteracted the inhibitory effects of SpyCEP on chemokine mediated PMN recruitment. Thus, α-SpyCEP antibodies may benefit the host both directly by enabling opsonophagocytosis, and indirectly, by neutralizing an important virulence factor. The animal model we employed shows promise for broad application in the study of bacterial pathogenesis.
Highlights
The Gram-positive bacterium Streptococcus pyogenes is a strictly human pathogen
interleukin 8 (IL-8) treated with culture supernatants from SpyCEP-expressing S. pyogenes showed a reduced capacity to induce polymorphonuclear cells (PMN) transmigration in vitro [3]
The growth of S. pyogenes was monitored by optical density at 600 nm (OD600) using an Ultrospec 10 cell density meter (Amersham Biosciences)
Summary
The Gram-positive bacterium Streptococcus pyogenes is a strictly human pathogen. It causes a wide range of local and systemic infections, ranging from relatively common and trivial diseases such as tonsillitis and erysipelas, to life-threatening conditions such as streptococcal toxic shock syndrome, septicemia, and necrotizing fasciitis. SpyCEP (S. pyogenes cell envelope protease, called ScpC) was originally described in a clinical isolate from a case of necrotizing fasciitis, where broth culture supernatants contained interleukin 8 (IL-8)-degrading activity [2]. IL-8 treated with culture supernatants from SpyCEP-expressing S. pyogenes showed a reduced capacity to induce PMN transmigration in vitro [3]. In soft-tissue infection models, histopathology showed increased PMN infiltration following infection with SpyCEP mutant strains when compared to the wild type (w.t.) strain [4,7]. Hypervirulent clinical isolates with CovRS mutations show overexpression of SpyCEP [11]
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