Abstract
Invasive outcomes of Group A Streptococcus (GAS) infections that involve damage to skin and other tissues are initiated when these bacteria colonize and disseminate via an open wound to gain access to blood and deeper tissues. Two critical GAS virulence factors, Plasminogen-Associated M-Protein (PAM) and streptokinase (SK), work in concert to bind and activate host human plasminogen (hPg) in order to create a localized proteolytic environment that alters wound-site architecture. Using a wound scratch assay with immortalized epithelial cells, real-time live imaging (RTLI) was used to examine dynamic effects of hPg activation by a PAM-containing skin-trophic GAS isolate (AP53R+S−) during the course of infection. RTLI of these wound models revealed that retraction of the epithelial wound required both GAS and hPg. Isogenic AP53R+S− mutants lacking SK or PAM highly attenuated the time course of retraction of the keratinocyte wound. We also found that relocalization of integrin β1 from the membrane to the cytoplasm occurred during the wound retraction event. We devised a combined in situ-based cellular model of fibrin clot-in epithelial wound to visualize the progress of GAS pathogenesis by RTLI. Our findings showed GAS AP53R+S− hierarchically dissolved the fibrin clot prior to the retraction of keratinocyte monolayers at the leading edge of the wound. Overall, our studies reveal that localized activation of hPg by AP53R+S− via SK and PAM during infection plays a critical role in dissemination of bacteria at the wound site through both rapid dissolution of the fibrin clot and retraction of the keratinocyte wound layer.
Highlights
Group A Streptococcus (Streptococcus pyogenes or GAS) is a Gram-positive bacterial pathogen responsible for common human disorders, including pharyngitis, cellulitis, and impetigo, in addition to invasive and severe diseases, such as rheumatic fever, toxic shock syndrome, and necrotizing fasciitis [1,2,3]
In order to investigate the role of human plasminogen (hPg) activation by GAS in cellular wound remodeling, we established a keratinocyte-based scratch assay wound system to observe the effects of GAS infection on epithelial wound interfaces
Direct addition of hPm resulted in similar wound retraction events as early as 5 min post-hPm addition
Summary
Group A Streptococcus (Streptococcus pyogenes or GAS) is a Gram-positive bacterial pathogen responsible for common human disorders, including pharyngitis, cellulitis, and impetigo, in addition to invasive and severe diseases, such as rheumatic fever, toxic shock syndrome, and necrotizing fasciitis [1,2,3]. A critical determinant for skin-trophic strains of GAS dissemination during infection is mediated by two GAS virulence factors, human plasminogen (hPg)-binding M-protein (PAM) and streptokinase (SK2b) [6,7,8,9,10]. Cell-based studies have provided important details regarding the manner in which PAM and SK contribute to invasive GAS disease, real-time dynamics of how these virulence factors manipulate cell and tissue architecture during infection have not been critically examined. We sought to determine the specific role of hPg activation by GAS in the context of wound remodeling and clot dissolution, using a standardized scratch-based keratinocyte wound infection model to dynamically visualize the progress of GAS infection under epithelial wound conditions. Coupling the rate of fibrinolysis during GAS infection, together with the rate of keratinocyte wound remodeling, allowed the development of a unique fibrin clot-inwound model for in situ-based cellular modeling during GAS infection in real-time
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