Characterization of recombinant-expressed <i>L. rhamnosus</i> GG SpaCBA pili in <i>L. lactis</i>.

<p>(A) The coding region of the <i>L. rhamnosus</i> GG <i>spaCBA</i> pilus operon and a summary of the nisin promoter-regulated (P<i>nisA</i>) WT and pilin-deleted SpaBCA-piliated recombinant constructs in <i>L. lactis</i> are depicted schematically. Genes for the pilin subunits (<i>spaA</i>, <i>spaB</i>, and <i>spaC</i>) and pilin-specific sortase (<i>srtC1</i>), including those genes that are deleted (×) in the recombinant constructs, are indicated. Identities for the various SpaCBA-piliated lactococcal constructs (GRS1185, GRS1195, GRS1211, and GRS1223), including the <i>spaCBA</i> pilus operon-containing plasmids they each propagate (pKTH5391, pKTH5497, pKTH5413, and pKTH5440, respectively), are indicated. (B) Immunoblot analysis of cell wall-extracted proteins recovered from <i>L. rhamnosus</i> GG (lane 2), the empty vector (pKTH5080)-carrying GRS1052 <i>L. lactis</i> construct (lane 3), and the nisin-induced WT SpaCBA-piliated GRS1085 <i>L. lactis</i> construct (lane 4) probed with the various pilin-specific antisera (anti-SpaA, anti-SpaB, or anti-SpaC, as indicated). Asterisks on the right of each immunoblot indicate the presumed location of monomeric SpaA, SpaB, and SpaC pilin proteins. The compressed and laddered region of high-molecular-weight (HMW) protein bands representing various lengths of pili is indicated on the left. Molecular weight markers (lane 1) and their positions are indicated on the left. (C) Immunogold double-labeling electron microscopy of WT SpaCBA-piliated lactococcal cells (GRS1185) using SpaA antiserum with protein A-10-nm gold particles as well as SpaC antiserum with protein A-5-nm gold particles is shown. The location of 5-nm (white arrow) and 10-nm (black arrow) gold particles on the pilus structure are indicated. (D) Immunoblot analysis of cell wall-extracted proteins recovered from <i>L. rhamnosus</i> GG (lane 1) and the empty vector (pKTH5080)-carrying GRS1052 <i>L. lactis</i> construct (lane 2), as well as the nisin-induced WT (GRS1085) (lane 3), SpaB-deleted (GRS1195) (lane 4), SpaC-deleted (GRS1211) (lane 5), and SpaB- and SpaC-deleted (GRS1223) (lane 6) piliated <i>L. lactis</i> constructs probed with anti-SpaA, anti-SpaB, or anti-SpaC sera, each of which is indicated on the left of the respective immunoblot. Positions of monomeric SpaA (∼31 kDa), SpaB (∼21 kDa), and SpaC (∼91 kDa) protein bands are marked by an asterisk on the right of corresponding immunoblots.