Expression of Virulence-Associated Protein A (VapA) of Rhodoccocus equi by infected macrophages.

Abstract

Rhodococcus equi is a Gram-positive bacterium known to cause disease because of its ability to survive and replicate inside macrophages. Macrophages also act as antigen-presenting cells: peptides from bacteria that have been engulfed and processed by macrophages are presented on the external surface of the macrophages to immune cells, primarily lymphocytes. Virulent R. equi contain an 85-kb plasmid that encodes for a virulence-associated protein A (VapA) that has been shown to be required for clinical disease in foals. It is unknown whether macrophages infected with virulent R. equi express VapA peptides on their surface. Thus, the objective of this experiment was to infect J774.A1 murine macrophages with virulent and avirulent strains of R. equi and to determine whether VapA was expressed on the surface of the infected macrophages. The macrophages were first infected with a virulent strain of R. equi that were labeled with a fluorochrome (pHrodo™; Invitrogen. Carlsbad, CA, USA), and tested using flow cytometry to verify that phagocytosis of R. equi by the macrophages occurred efficiently. After confirming that phagocytosis occurred efficiently and consistently, macrophages were co-cultured with the isogenic virulent and avirulent R. equi. Macrophages infected with virulent but not avirulent strains of R.equi expressed VapA on their surface.