Abstract
BackgroundThe bacterial endospore (spore) has recently been proposed as a new surface display system. Antigens and enzymes have been successfully exposed on the surface layers of the Bacillus subtilis spore, but only in a few cases the efficiency of expression and the effective surface display and have been determined. We used this heterologous expression system to produce the A subunit of the urease of the animal pathogen Helicobater acinonychis. Ureases are multi-subunit enzymes with a central role in the virulence of various bacterial pathogens and necessary for colonization of the gastric mucosa by the human pathogen H. pylori. The urease subunit UreA has been recognized as a major antigen, able to induce high levels of protection against challenge infections.ResultsWe expressed UreA from H. acinonychis on the B. subtilis spore coat by using three different spore coat proteins as carriers and compared the efficiency of surface expression and surface display obtained with the three carriers. A combination of western-, dot-blot and immunofluorescence microscopy allowed us to conclude that, when fused to CotB, UreA is displayed on the spore surface (ca. 1 × 103 recombinant molecules per spore), whereas when fused to CotC, although most efficiently expressed (7-15 × 103 recombinant molecules per spore) and located in the coat layer, it is not displayed on the surface. Experiments with CotG gave results similar to those with CotC, but the CotG-UreA recombinant protein appeared to be partially processed.ConclusionUreA was efficiently expressed on the spore coat of B. subtilis when fused to CotB, CotC or CotG. Of these three coat proteins CotC allows the highest efficiency of expression, whereas CotB is the most appropriate for the display of heterologous proteins on the spore surface.
Highlights
The bacterial endospore has recently been proposed as a new surface display system
The anti-UreA antibody failed to react against proteins extracted from Bacillus subtilis or Escherichia coli, indicating that the recognition of UreA of H. acinonychis and H. pylori is specific and that the signal observed with E. coli expressing ureA of H. acinonychis is exclusively due to the heterologous protein
Immunological experiments will be needed to assess whether the surface display of an antigen is an essential requisite for inducing a protective immune response or whether it is preferable to have the highest possible number of recombinant molecules on the spore coat layers even though these are not exposed on the spore surface
Summary
The bacterial endospore (spore) has recently been proposed as a new surface display system. Antigens and enzymes have been successfully exposed on the surface layers of the Bacillus subtilis spore, but only in a few cases the efficiency of expression and the effective surface display and have been determined. We used this heterologous expression system to produce the A subunit of the urease of the animal pathogen Helicobater acinonychis. With respect to systems based on the use of phages or bacterial cells, the spore-display system provides additional advantages, such as high stability and safety due to the unusual properties of this peculiar cell form [8]. The commercial use of spores of various species of the Bacillus genus as probiotics or for the oral prophylaxis of gastrointestinal disorders, clearly proves the safety of spores of these species [10]
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