Figure 3.

Schematic representation illustrating the preparation of L. lactis GEM particles displaying Y. pestis LcrV. (a) GEM particles are obtained by heat-acid treatment of L. lactis followed by extensive washing. The resulting bacterial “shell” (GEM) particles have an appearance and dimensions similar to those of the living organism. L. lactis contains an extracellular cell-wall degrading enzyme (hydrolase AcmA), which in its natural form allows separation of daughter cells by cleaving the PGN wall during bacterial division. This enzyme is naturally exported and attaches (from the outside) to the bacterial cell wall though a C-terminal binding domain that has high affinity for the L. lactis PGN. The use of this PGN binding domain allows for a strong non-covalent binding of the fusion proteins to GEM particles. (b) The AcmA locus, containing the enzyme active site next to the PGN binding domain and preceded by a signal peptide (SP), was cloned into an expression plasmid (pPA3). LcrV was inserted in lieu of the AcmA active site on pPA3 so that the LcrV sequence is in tandem with the PGN binding domain. The resulting plasmid, pPA281, was introduced into live L. lactis, which then produced the LcrV-PGN binding domain fusion as a recombinant protein and released it into the culture medium. The producer cells were removed by centrifugation, and the antigen was concentrated and used for particle coating (no additional purification is needed). (c) GEM particles and LcrV-PGN binding domain fusion protein were mixed, resulting in immediate non-covalent binding of LcrV to the PGN layer. (d) Fluorescence microscopy showing GEM particles displaying LcrV using an LcrV-specific monoclonal antibody; no staining was observed with GEM alone (shown on a light microscopy image). GEM, Gram-positive Enhancer Matrix; PGN, peptidoglycan; PA, PGN protein anchor domain.