Adherence of Staphylococcus intermedius to canine corneocytes involves a protein–protein interaction that is sensitive to trypsin but resistant to cold
Authors: Simou C. , Forsythe P. J. , Hill P. B. , Thoday K. L. 
Published: August 2004

Adherence is a prerequisite for canine cutaneous bacterial infection that is potentially affected by a large number of variables. The aim of this study was to explore the nature of this interaction by investigating the effects of long‐term storage, cold temperatures and trypsin on staphylococcal adherence to canine corneocytes. Sheets of corneocytes were collected using double‐sided tape from the ventral abdomen of healthy dogs at 5, 3 and 2 months before use and stored at –80°C, 4°C and room temperature (approximately 20°C) in dry and dark conditions; corneocytes were also collected on the day of each experiment. Staphylococcus intermedius froma case of bacterial pyoderma was prepared in phosphate‐buffered saline (PBS) and applied in triplicate to the canine corneocyte‐covered tapes using PBS as the negative control. After incubation, rinsing and staining with crystal violet, quantification of the adherent bacteria was carried out blindly by computerised image analysis of 15 fields. All the experiments were processed in two duplicate, identical experiments. There were no significant differences in the adherence of S. intermedius to fresh corneocytes and to those that had been stored at –80°C (P = 0.3703) or 4°C (P = 0.1129) at any time point and by subanalysis (P > 0.05 in each case). By contrast, the adherence to corneocytes stored at room temperature showed significant variation (P = 0.0079), with the values for fresh samples being higher than those stored for 5 months; however, subanalysis did not reveal significant differences between individual time points (P > 0.05). These results indicate that the mechanisms of adherence are not affected by even prolonged storage at cold temperatures. Such storage greatly facilitates the performance and comparison of adherence studies. To determine if adherence involved interaction of proteins on the surface of the organism with those on corneocytes, trypsin, a potent broad‐spectrum proteinase, was added to the incubation buffers. Preliminary experiments showed that trypsin concentrations of 1, 10–2, 10–4 and 10–6% did not affect the viability of S. intermedius in incubations lasting up to 4 h. Subsequent adherence assays were therefore conducted using a 4‐h incubation period and the above concentrations. Adherence of S. intermedius to canine corneocytes was completely abolished following incubation of the organism with trypsin at concentrations ranging from 1 to 10–2%, but adherence still occurred at the lower concentrations, demonstrating a dose–response phenomenon. These results suggest that a protein receptor on the surface of the organism was digested, thus eliminating the capacity for adherence. Paradoxically, pretreatment of the corneocytes with trypsin resulted in a significant enhancement in adherence of the organism (P < 0.0035). This may result from exposure of surface receptors after digestion of surface proteins. Alternatively, trypsin may damage the corneocyte envelope and expose cytoplasmic keratin fibres and matrix which express greater affinity for the bacterium. These studies provide evidence that adherence of S. intermedius to canine corneocytes involves a specific protein–protein interaction. Elucidation of the precise proteins involved could provide molecules that might be amenable to therapeutic targeting. Funding: Self‐funded.