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Incubation of intact Salmonella typhi Ty21a, Salmonella enterica serovar Typhimurium (Salmonella typhimurium) aroA or Escherichia coli DH5 alpha with membrane vesicles (MVs) derived from either Shigella flexneri M90T or Pseudomonas aeruginosa dsp89 resulted in a significant incorporation of vesicle antigens into the outer membrane of the bacteria; each recipient strain possessed a surface mosaic of new Shigella and Pseudomonas antigens intermixed with the native antigens of the Salmonella or Escherichia strains. Electron microscopy of preparations during the integration of vesicle antigens revealed that the MVs rapidly fused with the outer membrane of the host strains. Western blot analysis of host bacteria confirmed the integration of foreign antigens. Quantitative analysis for binding and fusion of antigens using an ELISA showed that approximately 78.7 +/- 12.8 ng of the Pseudomonas and 67.5 +/- 13.8 ng of the Shigella LPSs (microgram host protein)-1 were integrated into the Sal. typhimurium strain. Similar integrations of the Shigella or Pseudomonas vesicles were found with the E. coli or Sal. typhi strains. There was no loss of viability in the recipient bacteria after incorporation of the MVs, although vesicle antigens became diluted during continued growth as daughter cells shared the vesicle antigens. The new antigens were highly stable after being incorporated into recipient strains, being able to withstand storage of several months at 4 degrees C as well as several cycles of freezing and thawing. Since the recipient bacteria are common vaccine strains, the procedure described here offers a simple efficient means of introducing exogenous surface antigens, in their native form, into the outer membranes of Gram-negative bacteria for possible vaccine use.

Membrane vesicles derived from Pseudomonas aeruginosa and Shigella flexneri can be integrated into the surfaces of other Gram-negative bacteria