Expression of green fluorescent protein and its application in pathogenesis studies of serotype 2 Streptococcus suis

We investigated the interaction between type 2 Streptococcus suis and swine phagocytes during infection of the natural host, by using green fluorescent protein (GFP) as a specific marker to observe the challenge organism. We compared the strength of the S. suis sly promoter (SP332) and the synthetic promoter (CP25) in driving GFP expression. Two GFP alleles, gfpP11 and gfpmut3*, were also compared. The two promoters and two alleles were efficiently compared using three different promoter-GFP gene combinations on a shuttle vector, which were transformed into S. suis strains SX332, SX932 or M2. Plasmid pSL6.81 has SP332 with gfpP11, pSL5.24 has SP332 with gfpmut3*, and pSL5.28 has CP25 with gfpmut3*. The transformants were fluorescent with green light when viewed with an epifluorescence microscope or during flow cytometry. The signal was also detected using a laser scanning confocal microscope. The GFP expression level varied and CP25 with gfpmut3* led to greatest expression. For optimizing GFP detection, fluorescence-based cell sorting was applied to SX332(pSL5.28) and the mean fluorescence intensity increased 25.9% after optimization. Fluorescence activated cell sorting (FACS)-based phagocytosis assay showed that, without opsonization, phagocytosis rates of SX332, SX932 and M2 by both neutrophils and monocytes were similar and low. After opsonization, the phagocytosis of M2 increased 10-fold while phagocytosis of SX332 and SX932 did not change. GFP-labeled S. suis was identified in fresh pig tonsil tissue 18 h after infection. The results of this study indicated that GFP was expressed in type 2 S. suis and GFP labeled S. suis could be used in phagocytosis and pathogenesis studies.