Figure 3.

Expression of ScaB in Escherichia coli is sufficient to mediate adherence to mammalian cells. (A) Immunofluorescence microscopy using an anti-scaB antibody revealed the presence of ScaB on the surface of the recombinant E. coli (lower panel). Preimmune serum did not detect the recombinant protein (upper panel). Scale bars, 5 μm. (B) Expression of O. tsutsugamushi ScaB on the surface of E. coli. Immunoblot analysis of outer membrane fractions of induced E. coli harboring the empty vector (lane 1), ScaB (lane 2), and recombinant ScaB_23–372 protein was performed using an anti-ScaB serum. (C) E. coli transformed with the pET28a vector or with ScaB domain was induced with isopropyl β-D-thiogalactoside (IPTG) and incubated with HeLa cells. Confluent monolayers of HeLa cells were infected for 30 min at 37°C, washed repeatedly with phosphate-buffered saline (PBS), and then stained with an anti-E. coli antibody (red), Phalloidin (gray), and ToPro-3 for nuclear staining (blue). Scale bars, 50 μm. (D) Colony-forming unit (CFU)-based quantification of adherent E. coli transformed with the vector (black bars) or ScaB (red bars) was performed for different host cells (HeLa, A549, and ECV304 cell lines). ^*p < 0.05 and ^****p < 0.0001. The data presented are representative of at least three independent assays for each cell line. Error bars represent the SD of each data set.