(6hn) Engineering a Nano Display Platform from Bacterial Spore Coat Proteins

Bacterial endospores are encased in an assembly of coat proteins that protect the genetic material within from a number of environmental insults, including extreme heat, desiccation, caustic chemicals, and UV radiation.  In addition, the protein-based coat provides remarkable structural integrity.  We propose reconstituting the basement layer of the coat assembly, consisting of the proteins SpoVM and SpoIVA, onto spherical membranes to create synthetic spore-like particles that may be used in applications such as drug delivery or environmental remediation.  Towards this goal, we have coated spherical micron-sized silica beads with a lipid bilayer to first characterize the localization behavior of protein SpoVM, then demonstrate that the structural protein SpoIVA can bind and polymerize atop of the SpoVM-coated beads.

During sporulation in Bacillus subtilis, SpoVM is one of the first proteins to localize to the forespore and was shown to localize via a mechanism previously unobserved in prokaryotes: recognition of convex membrane curvature.  To understand the underlying mechanism of membrane curvature recognition, we use lipid membranes supported on silica beads of different radii of curvature.  In a competition between 2um-diameter beads and 8um beads, microscopy shows that fluorescently labeled SpoVM-FITC preferentially binds to 2um beads, as the radius of curvature is closest to that of the Bacillus forespore.  In contrast, the mutant SpoVM^P9A-FITC, previously shown to bind promiscuously to all membranes in vivo, binds equally well to both 2um and 8um beads in vitro.  To quantify the binding of SpoVM-FITC to the beads, we use flow cytometry to analyze the fluorescence from the 2um and 8um beads separately from the mixed population.  When normalized per surface area, binding of SpoVM-FITC at low concentrations is up to eight fold greater in the 2um beads compared to the 8um beads.  Near saturation concentrations, fluorescence per surface area is nearly equal.  In contrast, SpoVM^P9A–FITC has equal fluorescence per surface area even at low concentrations, due to the promiscuity of the mutant protein.  Interestingly, at low concentrations of SpoVM-FITC, flow cytometry reveals two distinct population of 2um beads  – one population low in fluorescence, and the other population high in fluorescence.  These two populations are also observed with fluorescence microscopy, and indicates a cooperative binding behavior of SpoVM.  Monte Carlo simulations show that binding cooperativity, coupled with a small difference in the rate of association of SpoVM between the 2um and 8um beads, is sufficient to achieve the preferential binding to 2um beads at equilibrium.  Indeed in a kinetic association assay using flow cytometry, the k_on of SpoVM was higher for 2um beads than for 8um beads.  Furthermore, evidence of cooperatively is again observed in the emergence of two distinct populations at intermediate timepoints, while later timepoints show one population of high fluorescence.  Taken together, these results highlight a mechanism for curvature-sensing driven by cooperative binding of SpoVM and differences in the association kinetics between surfaces with different curvature.

We then show that the silica beads can be coated with SpoIVA.  Using fluorescence microscopy, 2um beads coated with SpoVM and GFP-SpoIVA show an even distribution of fluorescence the bead, while a control in which the beads are incubated with GFP-SpoIVA in the absence of SpoVM shows fluorescence as punctate on one side of the spherical bead.  This punctate fluorescence of GFP-SpoIVA is also observed in-vivo in sporulating B. subtilis cells in the absence of SpoVM.  SEM of SpoVM/SpoIVA-coated beads show that SpoIVA has polymerized on the surface of the beads when compared to a SpoIVA variant with a mutation preventing ATP-hydrolysis, and thus polymerization.  Finally, we have demonstrated that this platform can be used to decorate the surface of these beads using click chemistry.  Here, we have incorporated one partner of a click reaction to SpoIVA via a C-terminal cysteine, with the reciprocal partner of the click reaction to the fluorophore Cy3, and confirmed the ligation via fluorescence microscopy.    We then demonstrate that two different fluorophores can be displayed on the surface, independently of each other, using two orthogonal sets of click chemistry reaction pairs.

Taken together, these results demonstrate that bacterial endospore coat proteins can be used to engineer a nano display platform.