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The role of staphylococcal Hsp60 in the internalization of S. aureus inside immortalized human keratinocytes Godin, Patrice

Abstract

S. aureus is a gram positive commensal which can be found on the skin and in the nose of the majority of healthy individuals. Its abilities to cause a wide range of life threatening diseases, to cause recurrent infections and to easily acquire antibiotic resistance have made S. aureus undeniably one of the most important pathogens everywhere in the world. Although this pathogen is certainly not new to the scientific community, S. aureus recently became increasingly important as we realized that we are quickly running out of effective therapies to treat these infections. Interestingly, it has been found within the last few years that S. aureus is able to invade and survive within a variety of human cells. In vitro studies have shown that this intracellular location allows S. aureus to resist to most known antibiotics in vitro. Further studies revealed intracellular reservoirs of S. aureus have been found in patients with chronic infections. These suggest that this intracellular phenomenon is an important neglected aspect of S. aureus pathogenesis, and could partly explain how S. aureus evades the immune system and causes recurrent infections. As this pathogen is predominantly associated with skin infections, we decided to study the capacity of S. aureus to internalize inside human skin keratinocytes (HaCaT). We have examined the dynamics of infection as well as the role of S. aureus Hsp60 protein, which has been shown to mediate the adherence and internalization of various other pathogens. Our study has helped to further our understanding of S. aureus internalization by showing that this pathogen can survive at least 76 hrs inside HaCaT cells, and that the infection can easily be monitored by flow cytometry using fluorescein isothyocyanate (FITC) labelled bacteria. We have also shown that this internalization is proportional to the starting multiplicity of infection up to 100 CFU per cell. At this infection ratio, we have demonstrated using fluorescence activated cell sorter (FACS) and confocal microscopy analyses that nearly up to 8% of HaCaT cells were found to be infected. Although the internalization could be blocked using the actin polymerization inhibitor cytochalasin D, we did not notice any significant decrease in the internalization levels of S. aureus after the addition of exogenous staphylococcal Hsp60 proteins or anti-Hsp60 antisera and F(ab)₂ fragments as an attempt to block this internalization. Our studies on the membrane localization of Hsp60 on the surface of S. aureus using antibody staining and membrane separation also failed to prove that this protein is associated on the membrane of this bacterium to a high extent. We therefore showed that staphylococcal Hsp60 does not appear to play a role in the internalization of S. aureus inside human HaCaT keratinocytes.

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