Role of Caf proteins in outer membrane vesicle release from Escherichia coli

Abstract

Outer membrane vesicles (OMVs) are nanostructures that are spontaneously produced by Gram-negative bacteria during all phases of growth and often in response to stress. Gram-negative bacteria display a variety of surface proteinaceous appendages by which they attach to objects or move in their environments. These structures mainly involve repetitive assemblies organised into filamentous polymers; one example is the F1 antigen which uses the chaperone-usher pathway to assemble an amorphous capsule (Caf1) to help Yersinia pestis evade phagocytosis. In this study, flocculated materials, consisting of OMVs trapped in a polymeric network of Caf1, were observed in Escherichia coli cultures expressing Caf1 proteins. It was demonstrated that the flocculent layer is Caf1 polymer dependent and that expression of several caf1 operon genes increases OMV formation. The deletion of the molecular usher Ca1fA was found to prevent Caf1 polymer formation at the cell surface and to cause hypervesiculation and an increase in liposaccharide (LPS) release. In addition to LPS, OMVs contain an abundance of outer membrane proteins (OMPs), such as OmpF and OmpA. To measure OMV production a range of fluorescent OmpA proteins were created. These included truncated and full-length forms of OmpA fused with GFP or mCherry or containing a tetra-cysteine motif which binds the FlAsH-EDT2 fluorescent probe. This study showed that full length OmpA-mCherry fusions are highly expressed and insert into the OM making them the most suitable probes for measuring OMV levels. It was also shown that the level of Caf1A present in the OM may not be the limiting factor in polymer production and that only a very low level of Caf1A is needed to produce flocculent. Also, these findings demonstrated for the first time that the caf1R gene directly regulates the caf1A gene. Disappointingly, overexpression of caf1A did not elevate Caf1A levels, increase Caf1 polymer production or decrease OMV formation by reducing Caf1 aggregation in the periplasm. This may be due to a limit on the amount of Caf1A that can be inserted into the OM. These results will help maximise Caf1 production for biomaterials applications and may suggest the use of OMVs for vaccine applications containing Caf1 as a protein scaffold for antigen delivery