Labeling Corynebacterium pseudotuberculosis using superfolder green fluorescent protein to evaluate its infection in macrophages.

To develop a visual detection method for Corynebacterium pseudotuberculosis (CP), a Gram-positive and facultative intracellular bacterium that causes chronic suppurative diseases in humans and animals, we constructed a superfolder green fluorescent protein (sfGFP) expression plasmid, pXMJ19-sfGFP, which constitutively expresses sfGFP in CP. The effects of pXMJ19-sfGFP on the biological characteristics of CP were evaluated, and no significant effects on the morphology, growth, or pathogenicity of CP were noticed following sfGFP expression. The stability of pXMJ19-sfGFP was determined through sequential propagation and infection tests in macrophages and mice. pXMJ19-sfGFP was stable within CP in the presence of chloramphenicol; however, certain degree of plasmid loss was noticed without antibiotic selection pressure. The rates of loss of pXMJ19-sfGFP in CP recovered from the macrophages infected with sfGFP-labeled strains of different pathogenicity were similar, indicating the feasibility of sfGFP-labeled CP monitoring during infection of macrophages by bacteria. The rate of macrophage infection with sfGFP-labeled CP with or without phospholipase D (pld) mutation was evaluated using flow cytometry, and the results showed that the rate of macrophage infection by CP was significantly higher than that by mutant CP. Therefore, sfGFP expression in CP is a valuable tool for visual analysis of macrophage infection by CP.