Cell‐surface display of a Pseudomonas aeruginosa strain K pilin peptide within the paracrystalline S‐layer of Caulobacter crescentus

Abstract

SummaryThe paracrystalline surface (S)-layer of Caulobacter crescentus is composed of a single secreted protein (RsaA) that interlocks in a hexagonal pattern to completely envelop the bacterium. Using a genetic approach, we inserted a 12 amino acid peptide from Pseudomonas aeruginosa strain K pilin at numerous semirandom positions in RsaA. We then used an immunological screen to identify those sites that presented the inserted pilin peptide on the C. crescentus cell surface as a part of the S-layer. Eleven such sites (widely separated in the primary sequence) were identified, demonstrating for the first time that S-layers can be readily exploited as carrier proteins to display 'epitope-size' heterologous peptides on bacterial cell surfaces. Whereas intact RsaA molecules carrying a pilin peptide could always be found on the surface of C. crescentus regardless of the particular insertion site, introduction of the pilin peptide at 9 of the 11 sites resulted in some proteolytic cleavage of RsaA. Two types of proteolytic phenomena were observed. The first was characterized by a single cleavage within the pilin peptide insert with both fragments of the S-layer protein remaining anchored to the outer membrane. The other proteolytic phenomenon was characterized by cleavage of the S-layer protein at a point distant from the site of the pilin peptide insertion. This cleavage always occurred at the same location in RsaA regardless of the particular insertion site, yielding a surface-anchored 26 kDa proteolytic fragment bearing the RsaA N-terminus; the C-terminal cleavage product carrying the pilin peptide was released into the growth medium. When the results of this work were combined with the results of a previous study, the RsaA primary sequence could be divided into three regions with respect to the location of a peptide insertion and its effect on S-layer biogenesis: (i) insertions in the extreme N-terminus of RsaA either produce no apparent effect on S-layer biogenesis or disrupt surface-anchoring of the protein; (ii) insertions in the extreme C-terminus either produce no apparent effect on S-layer biogenesis or disrupt protein secretion; and (iii) insertions more centrally located in the protein either have no apparent effect on S-layer biogenesis or result in proteolytic cleavage of RsaA. These data are discussed in relation to our previous assignment of the RsaA N-and C-terminus as regions that are important for surface anchoring and secretion respectively.

S-Layer Proteins

“…and Campylobacter sp., all of those sequenced to date are produced with an N-terminal secretion signal peptide which is cleaved off after translocation through the plasma membrane. Linker peptide insertions at the C-terminal end of the S-layer protein of Caulobacter crescentus (Table 1) indicated the existence of a type I secretion signal (8,9). A similar observation was reported for the S-layer protein of Campylobacter fetus, which also reveals potential secretion signals at the C-terminal part (142).…”

Caulobacter crescentus Synthesizes an S-Layer-Editing Metalloprotease Possessing a Domain Sharing Sequence Similarity with Its Paracrystalline S-Layer Protein

“…Once RsaA is secreted, the S-layer forms by a process of self-assembly into a hexagonal array of about 40,000 interlinked protein monomers per cell (27). Anchoring of RsaA to the cell surface is dependent on a smooth lipopolysaccharide molecule and Ca 2ϩ ions (43) and appears to involve the Nterminal portion of RsaA (5,6).…”

“…This application has recently become available for general use under the trade name PurePro (Invitrogen, Carlsbad, Calif.). Current research is directed toward optimizing the Caulobacter S-layer system for the surface presentation of large heterologous insertions (50 to 250 amino acids) within the S-layer without adverse affects on S-layer biogenesis (5,6).…”

Secretion of the Caulobacter crescentus S-Layer Protein: Further Localization of the C-Terminal Secretion Signal and Its Use for Secretion of Recombinant Proteins

“…Of interest for this study were the BamHI linker insertions corresponding to the C-terminal aa 690, 785, 908, and 944. In order to better cover the region between aa 785 and 908, BamHI linkers were inserted at positions in rsaA⌬P corresponding to aa 863 and 893 using methods similar to those used previously (5,6). Plasmids bearing DNAs encoding 336, 242, 166, 134, 119, and 82 aa of the RsaA C terminus were constructed by exploiting the newly introduced 5Ј BamHI sites in rsaA and the 3Ј HindIII site in the plasmid vector pTZ18UB (Table 1; Fig.…”