Bp-likeCPS confers multiple Bp-associated phenotypes to BtE555. (a) Immunofluorescence (IF) analysis of BtE555 and an isogenic strain disrupted in the Bp-liksCPS region (CPS KO). Strains were stained with DAPI (to identify nuclei) and the anti-Bp CPS monoclonal antibody. Wild-type BtE555 exhibits a clear distinctive halo of Bp-likeCPS expression around nuclei, while CPS KO strains exhibit either severely attenuated or absent Bp-likeCPS expression. (b) Colony morphologies of strains on ashdown media. BtE264 colonies (top left) are relatively round, with smooth contours, convex and glossy. In contrast, Bp K96243 colonies (bottom left) exhibit a wrinkled colony phenotype (white arrows). The BtE555 strain exhibits a mixture of smooth and wrinkled colonies (top right, white arrows), and the Bp-likeCPS KO strain (bottom right) develops small round violet colonies with no wrinkling. All strains were assayed after incubation at 37°C for 5 days. (c) Complement deposition assay. Strains were assayed for their ability to avoid human complement C3b deposition on cell surfaces (red staining; see Materials and methods). BtE264 is associated with abundant C3b deposition, while BtE555 exhibits minimal C3b accumulation. However, C3b deposition is clearly observed in the Bp-likeCPS KO strain. (d) Macrophage survival assay. Strains were assayed for their ability to survive and replicate in RAW macrophages, from 2 h to 8 h post-infection. BtE555 exhibits a highly significant ability to survive and replicate in macrophages compared to the Bp-likeCPS KO strain (P = 0.002). BtE555 also exhibits a statistically significant enhancement for survival and replication compared to BtE264 (P = 0.049).