Identification of Candidate Carrier Proteins for Surface Display on Lactococcus lactis by Theoretical and Experimental Analyses of the Surface Proteome

Abstract

Lactococcus lactis is a lactic acid bacterium of proven safety for use in human oral applications. For this purpose, surface display of recombinant proteins is important, and new approaches for it are being sought. Analysis of the bacterial surface proteome is essential in identifying new candidate carrier proteins for surface display. We have made two different predictions of surface-associated proteins of L. lactis MG1363 by using Augur and LocateP software, which yielded 666 and 648 proteins, respectively. Surface proteins of L. lactis NZ9000, a derivative of MG1363, were identified by using a proteomics approach. The surface proteins were cleaved from intact bacteria, and the resulting peptides were identified by mass spectrometry. The latter approach yielded 80 proteins, 34 of which were not predicted by either software. Of the 80 proteins, 7 were selected for further study. These were cloned in frame with a C-terminal hexahistidine tag and overexpressed in L. lactis NZ9000 using nisin-controlled expression. Proteins of correct molecular weight carrying a hexahistidine tag were detected. Their surface localization was confirmed with flow cytometry. Basic membrane protein A (BmpA) was exposed at the highest level. To test BmpA as a candidate carrier protein, the hexahistidine tag was replaced by the B domain of staphylococcal protein A in the genetic construct. The B domain was displayed on the surface with BmpA as a carrier. The advantage of covalent BmpA binding was demonstrated. BmpA was thus shown to be a suitable candidate for a carrier protein in lactococcal surface display.Lactic acid bacteria (LAB) are among the most intensively exploited microorganisms in the dairy industry. They are used in the fermentation of foodstuffs and as probiotics on account of their health benefits (11). Lactococcus lactis is a model LAB with industrially important applications. The genomes of several L. lactis strains have been sequenced, but strains MG1363 (33) and IL1403 (4) are most commonly used in laboratories.Various biotechnological applications of surface display of recombinant proteins in bacteria have been suggested, with vaccine delivery being the most popular in LAB (20,34). Other uses include the display of enzymes (35) as whole-cell biocatalysts, display of binding molecules such as antibodies or affibodies (35) (in diagnostics as biosensors, in therapy for pathogen or toxin removal, and in bioremediation for heavy metal binding [36]), and display of proteins for protein engineering by using combinatorial libraries and in vitro selection (8).The majority of surface display techniques have been developed for Gram-negative bacteria, with autodisplay being probably the most efficacious technique (16). However, several other approaches for surface display in Gram-positive bacteria have also been described. Transmembrane proteins, lipoproteins, LPXTG-like proteins, and cell wall-binding proteins have been displayed on the surfaces of Gram-positive bacteria (10). Two of these display types have been commonly explo...

Surface Proteome Analysis of a Natural Isolate of Lactococcus lactis Reveals the Presence of Pili Able to Bind Human Intestinal Epithelial Cells

“…On the basis of MS/MS data, YhgE2 pilin appears expressed at rather high level in TIL448. In contrast, the chromosomally encoded pilin YhgE was not detected in previous studies using a shaving approach in the plasmid-free L. lactis NZ9000 (40). These results are coherent with the absence of detection of pili-like structures by electron microcopy in the present study for MG1363 (Fig.…”

A review on Lactococcus lactis: from food to factory

“…In this method, the anchoring mechanism relies on the sortase activity as this membrane-anchored enzyme cleaves the sorting signal of the target protein at its pentapeptide motif (LPXTG) and promotes covalent anchoring of the target protein to the cell wall [29, 30]. However, non-covalent binding of cell surface proteins using lysin motifs (LysM) are also alternatively used, with the LysM of the autolysin AcmA being the most common [31]. More interestingly, non-covalent binding of antigens/proteins using AcmA has been shown to allow trans surface display, where proteins are displayed from the outside of L. lactis host cells, as we have previously shown [32].…”

Properties of probiotic bacteria explored by proteomic approaches

“…For L. lactis, this information could be used to design proteins useful as a stalk for antigen presentation. In that perspective, a recent study used the shaving approach to identify surface proteins of L. lactis that could be suitable carrier proteins [49 ]. This approach led to the identification of 80 proteins among which 40 displayed the features of surface proteins (signal peptide, transmembrane domain, LPxTG motif), highlighting the specificity of this approach toward surface proteins.…”