Clumping factor A of Staphylococcus aureus interacts with AnnexinA2 on mammary epithelial cells

Staphylococcus aureus is one of major pathogens that can cause a series of diseases in different hosts. In the bovine, it mainly causes subclinical and contagious mastitis, but its mechanisms of infection are not fully understood. Considering the fact that virulence factors play key roles in interactions between the bacterium and host cells, this study aimed to identify if a binding partner of S. aureus clumping factor A (ClfA) exists on the bovine mammary epithelial cells. The ClfA protein was used as a bait to pull down lysates of cultured bovine mammary epithelial cells (MAC-T cells). One pull-down protein was identified through use of mass spectrometry and bioinformatics analyses as bovine AnnexinA2. The Western blot and in vitro binding assay confirmed that the full A domain of ClfA was necessary to bind to AnnexinA2. In addition, the interaction between ClfA and AnnexinA2 was validated biochemically by ELISA with a KD value of 418+/-93 nM. The confocal microscopy demonstrated that ClfA and AnnexinA2 partially co-localized in the plasma membrane and that the majority of them were transported into cytoplasm. Taken together, the results demonstrate that ClfA binds with AnnexinA2 and this interaction could mediate S. aureus invasion into bovine mammary epithelial cells.