Construction and validation of EGFP-expressingStaphylococcus aureusclinical strains for adhesion and internalization assays on epithelial cells

Abstract

ABSTRACTBackgroundStaphylococcus aureusis both a major pathogen and a commensal bacterium in humans. It is able to adhere at the surface of epithelial cells of the anterior nares and can trigger its internalization inside these non-professional phagocytic cells. To better understand the interactions of clinical isolates with keratinocytes in the anterior nares, we developed and validated a one-step protocol expressing enhanced green fluorescent protein (EGFP) inS. aureusclinical strains with the aim to study adhesion to and internalization into mammalian cells.MethodsTwentyS. aureusclinical isolates belonging to clonal complexes 5, 8, 30, 45, 398 were selected for one-step transformation protocol with the EGFP-encoding plasmid pBSU101. EGFP expression was analysed by flow cytometry and confocal microscopy. Wild type and isogenic EGFP-expressing strains were compared for adhesion and internalization levels by using the HaCaT cell model.ResultsTransformation was achieved in all theS. aureusstrains regardless of their genetic background. The flow cytometry analysis showed that the mean proportion of EGFP-expressing bacteria was 97.2% (± 2.1) after 4h of incubation. Adhesion and internalization levels were similar in wild-type and isogenic EGFP-expressingS. aureusstrains. Confocal laser scanning microscopy confirmed that EGFP-expressingS. aureusbacteria could be easily identified inside HaCaT keratinocytes.ConclusionThis study reports an efficient protocol for expressing EGFP inS. aureusclinical strains and demonstrates that these EGFP-expressing strains are suitable for adhesion and internalization assays using HaCaT cells, which allows to perform static and dynamicin vitrostudies ofS. aureuscolonization.