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Title: Investigation of the role of Staphylococcus aureus toxins in a cartilage explant model of septic arthritis
Author: Smith, Innes Donald Mackenzie
ISNI:       0000 0004 6062 8668
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2015
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Septic arthritis has the potential to be a highly destructive joint disease. Although numerous bacterial species are capable of inducing septic arthritis, Staphylococcus aureus is most commonly implicated, accounting for up to 65% of cases. Whilst this organism is known to produce a diverse array of potential virulence factors, studies investigating a variety of S. aureus-related infections have implicated alpha(Hla)-, beta(Hlb)- and gamma(Hlg)-haemolysins as key damaging toxins, with the ‘pore-forming’ Hla considered to be the most potent. The work presented in this study focused on gaining further insight into the interaction between S. aureus toxins and in situ chondrocytes during an episode of septic arthritis. An in vitro bovine osteochondral explant model of S. aureus-induced septic arthritis was developed in this study. Utilising fluorescence-mode confocal laser scanning microscopy (CLSM), the model, which avoided the complexities of a host immune response, permitted an assessment of the following: (1) the spatial and temporal quantification of in situ chondrocyte viability following exposure to both a laboratory ‘wild-type’ (S. aureus 8325-4) and clinical strains of S. aureus; (2) the influence of Hla, Hlb and Hlg on in situ chondrocyte viability through the use of specific ‘haemolysin-knockout’ mutant strains; (3) the influence of altered culture medium osmolarity and extracellular Ca2+ on Hla-induced in situ chondrocyte death; and (4) dynamic changes in intracellular Ca2+ within in situ chondrocytes following Hla exposure. S. aureus 8325-4 and S. aureus clinical strains rapidly reduced in situ chondrocyte viability ( > 45% chondrocyte death at 40hrs). The increased acidity, observed during bacterial culture, had a minimal effect on chondrocyte viability. Chondrocyte death commenced within the superficial zone (SZ) of cartilage and rapidly progressed to the deep zone (DZ). Simultaneous exposure of SZ and DZ chondrocytes to S. aureus 8325-4 toxins (achieved with the use of subchondral bone-free explants) demonstrated that SZ chondrocytes were more susceptible to the toxins than DZ chondrocytes. When explants were cultured in the presence of a selection of isogenic S. aureus mutants, with varying Hla, Hlb and Hlg production capabilities (all originating from S. aureus 8325-4), Hla-producing mutants induced significant in situ chondrocyte death compared to toxin deficient controls (Hla-Hlb-Hlg-). In contrast, mutants producing Hlb and Hlg in the absence of Hla were unable to induce significant chondrocyte death. Hla alone was therefore identified as the key damaging toxin to in situ chondrocyte viability. Raised culture medium osmolarity had no influence on Hla-induced in situ chondrocyte death. In the absence of Hla, a high extracellular Ca2+ concentration (20mM) had no influence on chondrocyte viability during the experimental period. Hla-induced chondrocyte death increased in the presence of raised extracellular Ca2+ concentrations thereby confirming a role of Ca2+ in the chondrocyte death pathway. There was no significant difference between S. aureus growth in high and low Ca2+ culture media. Finally, when live osteochondral explants stained with the Ca2+-sensitive fluorophore Fluo-4 were cultured with an Hla-containing S. aureus supernatant (S. aureus 8325-4 (Hla+Hlb+Hlg+)) there was a significant rise in intracellular Ca2+ in comparison to those explants exposed to a non-Hla-containing supernatant (S. aureus DU5938 (Hla- Hlb-Hlg-)). The Hla-induced Ca2+ transients were always followed by chondrocyte death. Thus, it is likely that Hla-induced chondrocyte death was associated with a rise in intracellular Ca2+. These findings are of translational relevance. Firstly, toxins released by S. aureus have a rapid and fatal action on in situ chondrocytes, thereby advocating the prompt and thorough removal of bacteria and their toxins during the treatment of septic arthritis. Secondly, the identification of Hla alone as the key damaging toxin to in situ chondrocyte viability, with its destructive action being associated with a rise in intracellular Ca2+, may enable the development of future targeted therapeutic strategies in order to reduce the extent of cartilage destruction during and after an episode of septic arthritis.
Supervisor: Simpson, Hamish ; Hall, Andrew ; Amyes, Sebastian Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: cartilage ; chondrocytes ; confocal ; microscopy ; septic arthritis