title: Expression and display of UreA of Helicobacter acinonychis on the surface of Bacillus subtilis spores.
abstract: BACKGROUND
  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. 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.
  
  
  RESULTS
  We 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 x 10(3) recombinant molecules per spore), whereas when fused to CotC, although most efficiently expressed (7-15 x 10(3) 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.
  
  
  CONCLUSIONS
  UreA 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.
authors.affiliation: Department of Structural and Functional Biology, Federico II University of Naples, via Cinthia 4, Naples 80126, Italy.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Hinc
authors.forename: Rachele
authors.initials: R
authors.lastname: Isticato
authors.forename: Marcin
authors.initials: M
authors.lastname: Dembek
authors.forename: Joanna
authors.initials: J
authors.lastname: Karczewska
authors.forename: Adam
authors.initials: A
authors.lastname: Iwanicki
authors.forename: Grazyna
authors.initials: G
authors.lastname: Peszyńska-Sularz
authors.forename: Maurilio
authors.initials: M
authors.lastname: De Felice
authors.forename: Michał
authors.initials: M
authors.lastname: Obuchowski
authors.forename: Ezio
authors.initials: E
authors.lastname: Ricca
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D016998:Helicobacter / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013171:Spores, Bacterial / Q000235:genetics / Q000378:metabolism; D014510:Urease / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D014510:Urease