Serum and phagocytosis resistance assays. (A) Difference in serum resistance between NTUH-K2044, MGH-78578, NTUH-K9, and mutants 1–20. The assays were performed sequentially three times. A high degree of consistency was observed between experiments. (B) Restoration of serum resistance by magA complementation in a magA
− mutant, in comparison with magA
− mutant, magA
− mutant–carrying vector only, wild-type–carrying vector only, and wild type. (C) Western blot analysis of complement C3 deposition showing that magA
− mutant (lanes 2 and 5) and cps
− mutant (lanes 3 and 6) have much higher levels of complement C3 deposition than the wild type (lanes 1 and 4). 1-min exposure to serum (lanes 1–3). 5-min exposure to serum (lanes 4–6). (D) Phagocytosis assay showing wild-type NTUH-K2044 is less associated with mouse macrophages than magA
− mutant or cps
− mutant. Complementation with magA-carrying plasmid partially restores the phenotype. (E and F) Fluorescence microscopy images (original magnification, 400). Wild-type NTUH-K2044 (E) is less likely to be ingested or attached by human neutrophils than a magA
− mutant (F). (G) Confocal fluorescence microscopy showing that magA
− mutants were ingested and disintegrated by human neutrophils.