Staphylococcus aureus is a major human pathogen and a member of the human skin microbiota. It is capable of infecting nearly every human tissue to cause a wide spectrum of disease that includes skin and soft tissue infection, infective endocarditis, toxic shock syndrome, necrotizing pneumonia, and osteomyelitis. The success of S. aureus as a pathogen can be attributed to the vast array of virulence factors that enable it to colonize and invade human tissues. Host-specific exotoxins including leukocidins and cytolysins contribute directly to disease by causing inflammation, immune evasion, and tissue damage. The host-specific nature of these factors has made it difficult in translating findings in animals to humans. There is currently a need for alternative models that can be used to better understand S. aureus host-pathogen interactions. We propose the use of human skin explants as an alternative to the currently used in vitro and in vivo models. The skin accounts for the highest incidence of infections caused by S. aureus and human skin colonization has been identified as a risk factor for invasive infections. The first path of this study involves the characterization of human skin explant model. We assessed the viability of skin explants under our experimental conditions. Result revealed well-preserved tissue structure and viability over a period of 7 days. More so, tissue-resident skin dendritic cells and macrophages were phagocytic during this period as these cells were able to ingest E. coli particles injected into the skin. We also isolated and characterized the skin-resident T cell population. FACs analysis revealed the majority of these cells express the memory marker, CD45RO. These cells also produced various types of cytokines in response to polyclonal stimulation with staphylococcal enterotoxin B. The second path of this study involves the validation of human skin explant model. Staphylococcal α-hemolysin (Hla) is unarguably the most studied staphylococcal exotoxin and was therefore used to validate this model. Its role in diseases and mechanism of action has been well studied. Panton-Valentine leukocidin (PVL) was used as a control virulence factor as its role in S.aureus infection still remains unclear. Using purified toxins we showed that Hla was able to induce significant toxicity on human skin while PVL induced a milder but significant toxicity. The combination of both toxins resulted in increased tissue toxicity as compared with the individual toxins alone. Treatment of the skin with these toxins also resulted in the decrease of immune cells in skin epidermis. In addition, both toxins were able to elicit the release of proinflammatory cytokines and chemokines. Hla antibodies were able to block the activity of the toxin in a concentration-dependent manner. In the third part of this work, we tried to understand if Hla plays a significant role during infection and colonization of human skin. Colonization, superficial wound, and deep wound models were used for this purpose. We were able to show that although both wildtype and its isogenic hla mutants were able to induce toxicity and tissue damage, the former induced a significantly higher damage. Wildtype strains also show a significant ability to invade human skin during colonization. Recovered bacteria from each infection model revealed Hla plays no significant role in the ability of S. aureus to colonize and grow on human skin. Fhud2 and Hla, two important virulent factors that are differentially expressed by S. aureus, were used to validate the model for gene expression analysis. Results indicate a 28-fold increase in the expression of hla while fhud2 expression was increased by 2 fold. In sum, we were able to use the human skin explant model to delineate the contributions of key S. aureus virulence factors to human skin tissue pathology and evaluate the protective efficacy of Hla specific antibodies. These studies indicate human skin explant could serve as a replacement or complement for the already existing in vitro and in vivo models currently used to study this pathogen.
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|Titolo:||Human skin explant model for Staphylococcus aureus vaccine research|
|Autore:||Olaniyi, REUBEN OLAYINKA|
|Appare nelle tipologie:||8.1 Tesi Dottorato|