Abstract
Purpose: :
When the metabolic enzyme gylceraldehyde phosphate dehydrogenase (GAPDH) of S. aureus is located on the cell surface rather than in the cytoplasmic compartment, it has been shown to have functions other than those described for its metabolic activities. We have previously found that a clinical isolate from a case of S. aureus keratitis produces a 5-fold excess of surface GAPDH compared to the standard strain S. aureus 8325-4. This finding correlates with increased virulence in a mouse model of microbial keratitis. However, the role of GAPDH in bacterial virulence and the establishment of infection in the cornea remain to be fully determined.
Methods: :
The effects of inhibiting the actions of GAPDH using polyclonal antibodies generated against a 15 amino acid sequence from staphylococcal GAPDH on the ability of S. aureus strains 38 and 8235-4 to adhere to and invade human corneal epithelial cells (HCE) were examined in vitro. Further, the effects of opsonisation of S. aureus using these antibodies on the gross appearance, bacterial load and levels of neutrophil infiltration of corneas during infection with S. aureus in a mouse model.
Results: :
Association of S-38 pre-incubated with polyclonal antibodies to GAPDH with HCE was significantly reduced by approximately 23-fold (p =0.001) while invasion of HCE also showed a 20-fold reduction (p= 0.03) compared to controls. However, pre-incubation of strain 8325-4 with the polyclonal antibodies did not affect either association or invasion. Opsonisation of S-38 with polyclonal antibodies to GAPDH resulted in a significantly improved clinical outcome (p=0.04) which corresponded to a 35-fold reduction in bacterial load and an approximately 2-fold reduction in number of infiltrating leukocytes (=0.04).
Conclusions: :
GAPDH is inplicated in facilitating the binding of S. aureus to corneal cells and promoting internalization. While pre-exposure to antibodies to GAPDH resulted in an improvement in outcome of corneal infection which may result from both reduction in adhesion and internalization by corneal epithelial cells as well as opsonisation facilitating phagocytosis, this may not be a useful intervention as GAPDH is highly conserved.
Keywords: Staphylococcus • microbial pathogenesis: experimental studies • keratitis