Article Text

  1. A. T. Shannon,
  2. S. Patel,
  3. J. M. Fleckenstein
  1. Memphis, TN.


Background Enterotoxigenic Escherichia coli (ETEC) infections are a major cause of death due to diarrhea among children in developing countries. No vaccine for ETEC presently exists. We have recently described a new ETEC surface protein, EatA, a member of an autotransporter virulence family. Collectively, these molecules are characterized by three functional domains: an N-terminal signal sequence, followed by a passenger domain that encodes a serine protease motif, and a carboxy terminal β-barrel that functions as a transporter for the secreted passenger domain. To investigate the use of EatA as an antigen delivery mechanism capable of deploying chimeric proteins on the bacterial cell surface, we used a unique transposon system referred to as TnphoA/in to identify regions of the molecule that are permissive for the insertion of foreign epitopes.

Methods Recombinant E. coli CC118 bearing EatA expression plasmid pJFF001 were infected with bacteriophage TnphoA/in, and cells expressing eatA::phoA gene fusions were identified on selective media containing the alkaline phosphatase indicator XP. Plasmids isolated from these recombinants were then subjected to restriction enzyme digestion and DNA sequencing to identify the location of transposon insertions. A single plasmid bearing an insertion in the 5′ end of eatA was digested with BamHI and religated leaving an in-frame “scar” within EatA bearing a single BamHI site in place of TnphoA. A 1490 bp region of the emm24 gene encoding M24 protein from Streptococcus pyogenes was then inserted into this BamHI site creating the eatA-emm24 fusion expression plasmid pSP035.

Results Most of the active TnphoA/in gene fusions mapped to a highly constrained region at the 5′ end of EatA. Expression of the eatA::emm24 fusion resulted in significant secretion of the chimeric protein as demonstrated by immunoblotting concentrated supernatants from cultures of recombinant E. coli (pSP035).

Conclusions EatA-based chimeric antigen delivery constructs are technically feasible and this technology may be exploited in future development of vaccines to protect against ETEC and other mucosal pathogens.

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