Figure 5: Cell-surface display of BR187–385 and BR*120–378 promotes eDNA-dependent bacterial aggregation of Staphylococcus carnosus.
(A) The domain organization for surface display of BR187–378 and BR*120–378 on the surface of S. carnosus. PP - propeptide from S. hyicus lipase; His6 – hexahistidine tag; 3C - recognition sequence for protease 3C; X and M - regions from the SpA gene for covalent anchoring of the proteins to the peptidoglycan cell wall. (B) Supernatants of 3C-protease incubated staphylococcal cells were spotted on nitrocellulose membranes. Staining using polyclonal anti-serum against BR187–385 confirmed the expression of both BR*120–378 and BR187–378, and the absence and presence of signal for the BR143–156 region allowed for an unambiguous discrimination between BR*120–378- and BR187–378-expressing cells, respectively. As expected, signals from HRP-coupled anti-His antibodies were detected for all three constructs. (C) The BR187–378 and BR*120–378 displaying cells were stained using fluorescently labeled secondary antibodies directed against primary polyclonals against BR187–385. Singlets, duplets and higher aggregates are highlighted in white, bronze and pink, respectively. (D) Phase-like contrast microscopy images revealed the formation of cellular aggregates of S. carnosus displaying BR187–378 and BR*120–378, but not of cells displaying the DUF domain alone. (E) The two-dimensional aggregates sizes of staphylococcal cells were quantitatively analyzed from an area of about 0.05 mm2, which corresponds to approximately 64 times the area shown in panel D. The derived particle areas are plotted as normalized density histogram distributions, with the Scar-DUF control and the corresponding samples in black and red, respectively. The histogram represents the particle distribution obtained from a single experiment. The results from three independent experiments are summarized in the q-q plot shown in panel G. (F) Incubation of Scar-BR187–378 with DNaseI reduced the formation of aggregates, compared to the same sample not incubated with DNaseI (panel D). (G) The mean particle area sizes of quantiles with cut-off values at 25%, 50%, 75%, 85%, and 95% were calculated from three independent experiments, and summarized in a quantile-quantile plot (q-q plot). After incubation with DNaseI, both q-q plot curves of Scar-BR187–378 and Scar-BR120–378 are downward-shifted (light and dark green, respectively).
