Identifying Staphylococcus haemolyticus surface proteins. Development of a novel method for detection of bacterial surface proteins expressed during colonisation of human keratinocytes

Abstract

The aim was to develop a method for identification of expressed surface proteins of Staphylococcus haemolyticus when colonising human keratinocytes. S. haemolyticus and HaCaT cells were grown together and separated with FACS prior to surface protein shaving using the LPI™FlowCell technology (Nanoxis Consulting AB). TMT-tags were used to investigate up- and downregulation of proteins comparing S. haemolyticus incubated and not incubated with HaCaT cells prior to surface shaving. Surface shaving of S. haemolyticus was done twice (initial and optimized experiment). Method optimizations increased number of bacteria adhering to HaCaT cells and retrieval of bacteria after FACS. 319 proteins were identified by MS in total: 18 strongly upregulated and 41 slightly upregulated. Six adhesion and/or virulence proteins were detected among the 18 strongly upregulated proteins. Twelve cell wall and eight extracellular proteins were identified among the 319 proteins. 66% of the proteins were predicted to be cytoplasmic. An alternative approach where surface shaving of S. haemolyticus in overnight cultures of used and unused cell culture medium was also performed and 794 proteins were identified. Up- and downregulation of proteins were not directly comparable to the up- and downregulation of proteins in the HaCaT colonisation model, even though some detected proteins were similar. A higher number of proteins and a higher rate of cytoplasmic proteins were found using the alternative approach, possibly indicating a higher degree of bacterial lysis.