*2.1.1. LPXTG motif containing anchors*

The most widely used surface display systems of LAB are based on cell wall anchored proteins that contain an LPXTG motif. These proteins are first synthesized as a preprotein containing an N-terminal signal peptide and a C-terminal cell wall anchor domain. The anchor domain starts at its C-terminus with a short tail of positively charged residues (five to seven amino acids) that remain in the cytoplasm. Upstream of the cytoplasmic domain, a stretch of approximately 30 hydrophobic amino acids is preceded by the highly conserved pentapeptide LPXTG. The charged tail and hydrophobic domain are thought to function as a temporary stop to position the LPXTG motif for proteolytic cleavage. Correct positioning results in cleavage between the threonine and glycine residues followed by amid linkage of the threonine residue to the peptide crossbridge in the peptidoglycan of cell wall, by the action of a sortase (Navarre & Schneewind, 1994). The amino acid composition of the peptide crossbridge varies among the different LAB species and is flexible with respect to the sorting reaction (Strauss et al., 1998; Ton-That et al., 1998). The anchor domain is preceded by a wall associated region of about 50 to 125 residues and is characterized by a high percentage of proline/glycine and/or threonine/serine residues (Fischetti et al., 1990). In surface display genetic constructs, secretion signal peptides are fused to the N-termini of target proteins to transport them to the outside of cell and the LPXTG motif containing anchors are fused to the C-termini of target proteins in order to attach them to the cell surface. The cell surface associated proteinases of *Lactococcus lactis* and *Lactobacilli casei*  (PrtPs), M6 protein of *Streptococcus pyogenes*, and Protein A of *Staphylococcus aureus* (SpA) are among LPXTG type anchors, which have mainly been used for the construction of surface display systems in LAB (Maassen et al., 1999; Norton et al., 1996; Piard et al., 1997; Pouwels et al., 1996; Steidler et al., 1998).

Slight deviations from the LPXTG consensus sequence is found in some LAB. Recently Kleerebezem (2003) found that sortase substrates of *Lactobacillus plantarum* contain LPQTXE motifs instead of the LPXTG motifs. In addition, in the carboxy end of cell surface proteinase of *Lactobacillus delbrueckii* (PrtB), a degenerated LPKKT motif is surrounded by two imperfect repeats of 59 residues, which are lysine rich. However, downstream of the LPKKT motif, there is no hydrophobic domain and no charged tail at the extreme C-terminus. It was shown that the C-terminal region of PrtB was able to attach to the cell wall of *L. lactis* and the capacity of attachment was drastically reduced by absence of the duplicated sequences. The high content of total positive charges in the anchoring region of PrtB suggests interactions of the anchor with the negatively charged teichoic acids of the cell wall. The mechanism of PrtB attachment to the cell wall probably implicates electrostatic forces (Germond et al., 2003).

### *2.1.2. Lipoprotein anchors*

Lipoproteins are lipid modified proteins produced as secretory precursors with a signal peptide linked to their amino termini. The C-terminal region of their signal peptide contains

a well conserved lipobox motif of four amino acid residues and invariably, the last residue is cysteine. The covalent binding of lipoproteins to the cell membrane is generally achieved via diacylglyceryl modification of the indispensible cysteine residue in the lipobox by a diacylglyceryl transferase. Lipidation of the cysteine residue is a perquisite for cleavage of the signal peptide by a lipoprotein specific signal peptidase (SPase II)(Yamaguchi et al., 1988; Venema et al., 2003). The lipoprotein anchors should be fused at their C-termini to Ntermini of target proteins in order to display them on the cell surface. Poquet (1998) identified four lactococcal lipoprotein anchors using nuclease of *S. aureus* as an export specific reporter enzyme. The nuclease activity was shown to require an extracellular location in *L. Lactis* demonstrating its suitability to report the protein export. The enzyme activity was detected in a plate test by the presence of pink halos. Fusions of the lipoprotein anchors to the nuclease expressed in *L. Lactis* were associated with the cell fraction and the recombinant lactococcal cells showed strong nuclease activities indicating the cell surface anchoring function of the lipoproteins. For one of the anchors (NlpI), the surface location of the fusion protein was also confirmed by proteinase K treatment of *L. lactis* cells (Poquet et al., 1998). Basic membrane protein A (BmpA) of *L. Lactis* is a putative lipoprotein that has been used for the protein display on the surface of lactococci (Berlec et al., 2011).

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*Lactobacillus brevis* ATCC 8287 (SlpA). The C-terminal regions of SA and CbsA showed the cell surface anchoring function and the N-terminal regions were involved in the self assembly process. In contrast, the self assembly domain of SlpA was located in the Cterminal region and its cell wall binding domain was found in the N-terminal region. The (lipo)teichoic acids were identified as the cell wall ligands of SA and CbsA. Moreover, the specific cell wall component that interacts with SlpA was shown to be the neutral polysaccharide moiety of the cell wall (Antikainen et al., 2002; Avall-Jaaskelainen et al., 2008; Smit et al., 2002). Avall-Jaaskelainen (2002) decribed the construction of recombinant *L. brevis* strains expressing poliovirus epitope VP1 of 10 amino acid residues inserted in the *slpA* gene. Insertion sites in the *slpA* gene were selected on the basis of the hydrophilicity profile of the SlpA protein. The four most hydrophilic parts of the SlpA protein were selected for testing because it was expected that parts of them were likely to be sites where the epitope would be accessible to the cell surface. One of the insertion sites was at the Nterminus of SlpA and the others were at its C-terminus. Only one site at the C-terminus showed strong colour response in whole cell enzyme linked immunosorbent assay (ELISA) using anti epitope antibody demonstrating that the epitope was accessible on the surface of the recombinant *L. brevis*. In another study, the C-terminal region of SLP of *L. crispatus* K2-4- 3 (LcsB) isolated from the chicken intestine was used for the construction of surface display systems. Green fluorescent protein (GFP) was fused to the N-terminus of LcsB. The fusion protein (GFP-LcsB) was expressed in *Escherichia coli.* It was then purified and mixed with various LAB. The binding of the fusion protein to LAB cells was viewed by the fluorescence microscopy. GFP-LcsB was associated with the cell surface of various LAB including *L. delbrueckii*, *L. brevis*, *Lactobacillus helveticus*, *Lactobacillus johnsonii*, *L. crispatus*, *Streptococcus thermophilus*, *L. lactis* and *Lactobacillus salivarius*. GFP alone did not bind to the cells. These results indicated that binding of GFP to the surface of LAB cells is directed by LcsB. However, the fusion protein could not attach to the cell surface of *L. casei*. The reason for this observation requires further studies to elucidate the target ligand of LcsB on the cell surface

The lysine motif (LysM) was first discovered in the lysozyme of *Bacillus* phage ø29 as a Cterminal repeat composed of 44 amino acids separated by 7 amino acids. The cell wall attachment of several bacterial proteins in both gram positive and gram negative organisms occurs through LysMs, often repeated several times in the protein sequence. Many LysM containing proteins are cell wall hydrolases (Buist et al., 2008). The C-terminal region of peptidoglycan hydrolase (AcmA) of *L. lactis* MG 1363 (CpH) contains three 44 amino acid residue lysMs separated by stretches of 21 to 31 amino acids rich in the serine, threonine, and asparagine residues. CpH is able to bind to the cell surface of lactococci and several strains of lactobacilli. Moreover, CpH is able to bind both to the cell surface of LAB treated with sodium dodecyl sulfate (SDS) to remove cell wall associated proteins and LAB treated with trichloroacetic acid (TCA) to remove carbohydrates and (lipo) teichoic acids. These findings suggest that peptidoglycan is the binding ligand of the CpH domain (Buist et al.,

of LAB (Hu et al., 2011).

*2.2.2. Lysin motif containing anchors* 

### *2.1.3. Transmembrane anchors*

The strategy to insert target amino acid sequences in the exterior loop between transmembrane spanning domains (TMSs) can limit the insert size in order not to disturb the membrane protein topology. Therefore, a fusion approach is often preferred in which a target protein is simply linked at its N-terminus to one or more TMSs of a cytoplasmic membrane protein. *L. Lactis* bacteriocin transport accessory protein (LcnD) and *Bacillus subtilis* poly-γ-glutamate synthetase A protein (PgsA) are transmembrane proteins, which were fused to the N-termini of target proteins in order to display them on the cell surface of *L. Lactis* and *L. casei*, respectively (Franke et al., 1996; Narita et al., 2006). In addition, in the same random procedure as described above for the lipoprotein anchors Poquet (1998) identified seven lactococcal gene fragments encoding TMSs that function as membrane anchors in *L. Lactis*.

### **2.2. Noncovalent anchors**

### *2.2.1. S- layer protein anchors*

Some LAB strains possess a surface layer (S-layer) of proteins as the outermost structure of the cell envelope. These S-layers are composed of regularly arranged subunits of a single protein (SLP) and may constitute up to 20% of the total cell protein content. S-layers self assemble in entropy driven process during which multiple noncovalent interactions between individual SLPs and the underlying cell surface take place. These two types of interactions in SLPs can be assigned to two separate domains including the self assembly domain and the cell wall binding domain. These domains have been characterized in SLPs of *Lactobacillus acidophilus* ATCC 4356 (SA), *Lactobacillus crispatus* JCM 5810 (CbsA), and *Lactobacillus brevis* ATCC 8287 (SlpA). The C-terminal regions of SA and CbsA showed the cell surface anchoring function and the N-terminal regions were involved in the self assembly process. In contrast, the self assembly domain of SlpA was located in the Cterminal region and its cell wall binding domain was found in the N-terminal region. The (lipo)teichoic acids were identified as the cell wall ligands of SA and CbsA. Moreover, the specific cell wall component that interacts with SlpA was shown to be the neutral polysaccharide moiety of the cell wall (Antikainen et al., 2002; Avall-Jaaskelainen et al., 2008; Smit et al., 2002). Avall-Jaaskelainen (2002) decribed the construction of recombinant *L. brevis* strains expressing poliovirus epitope VP1 of 10 amino acid residues inserted in the *slpA* gene. Insertion sites in the *slpA* gene were selected on the basis of the hydrophilicity profile of the SlpA protein. The four most hydrophilic parts of the SlpA protein were selected for testing because it was expected that parts of them were likely to be sites where the epitope would be accessible to the cell surface. One of the insertion sites was at the Nterminus of SlpA and the others were at its C-terminus. Only one site at the C-terminus showed strong colour response in whole cell enzyme linked immunosorbent assay (ELISA) using anti epitope antibody demonstrating that the epitope was accessible on the surface of the recombinant *L. brevis*. In another study, the C-terminal region of SLP of *L. crispatus* K2-4- 3 (LcsB) isolated from the chicken intestine was used for the construction of surface display systems. Green fluorescent protein (GFP) was fused to the N-terminus of LcsB. The fusion protein (GFP-LcsB) was expressed in *Escherichia coli.* It was then purified and mixed with various LAB. The binding of the fusion protein to LAB cells was viewed by the fluorescence microscopy. GFP-LcsB was associated with the cell surface of various LAB including *L. delbrueckii*, *L. brevis*, *Lactobacillus helveticus*, *Lactobacillus johnsonii*, *L. crispatus*, *Streptococcus thermophilus*, *L. lactis* and *Lactobacillus salivarius*. GFP alone did not bind to the cells. These results indicated that binding of GFP to the surface of LAB cells is directed by LcsB. However, the fusion protein could not attach to the cell surface of *L. casei*. The reason for this observation requires further studies to elucidate the target ligand of LcsB on the cell surface of LAB (Hu et al., 2011).

### *2.2.2. Lysin motif containing anchors*

430 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

*2.1.3. Transmembrane anchors* 

anchors in *L. Lactis*.

**2.2. Noncovalent anchors** 

*2.2.1. S- layer protein anchors* 

a well conserved lipobox motif of four amino acid residues and invariably, the last residue is cysteine. The covalent binding of lipoproteins to the cell membrane is generally achieved via diacylglyceryl modification of the indispensible cysteine residue in the lipobox by a diacylglyceryl transferase. Lipidation of the cysteine residue is a perquisite for cleavage of the signal peptide by a lipoprotein specific signal peptidase (SPase II)(Yamaguchi et al., 1988; Venema et al., 2003). The lipoprotein anchors should be fused at their C-termini to Ntermini of target proteins in order to display them on the cell surface. Poquet (1998) identified four lactococcal lipoprotein anchors using nuclease of *S. aureus* as an export specific reporter enzyme. The nuclease activity was shown to require an extracellular location in *L. Lactis* demonstrating its suitability to report the protein export. The enzyme activity was detected in a plate test by the presence of pink halos. Fusions of the lipoprotein anchors to the nuclease expressed in *L. Lactis* were associated with the cell fraction and the recombinant lactococcal cells showed strong nuclease activities indicating the cell surface anchoring function of the lipoproteins. For one of the anchors (NlpI), the surface location of the fusion protein was also confirmed by proteinase K treatment of *L. lactis* cells (Poquet et al., 1998). Basic membrane protein A (BmpA) of *L. Lactis* is a putative lipoprotein that has

been used for the protein display on the surface of lactococci (Berlec et al., 2011).

The strategy to insert target amino acid sequences in the exterior loop between transmembrane spanning domains (TMSs) can limit the insert size in order not to disturb the membrane protein topology. Therefore, a fusion approach is often preferred in which a target protein is simply linked at its N-terminus to one or more TMSs of a cytoplasmic membrane protein. *L. Lactis* bacteriocin transport accessory protein (LcnD) and *Bacillus subtilis* poly-γ-glutamate synthetase A protein (PgsA) are transmembrane proteins, which were fused to the N-termini of target proteins in order to display them on the cell surface of *L. Lactis* and *L. casei*, respectively (Franke et al., 1996; Narita et al., 2006). In addition, in the same random procedure as described above for the lipoprotein anchors Poquet (1998) identified seven lactococcal gene fragments encoding TMSs that function as membrane

Some LAB strains possess a surface layer (S-layer) of proteins as the outermost structure of the cell envelope. These S-layers are composed of regularly arranged subunits of a single protein (SLP) and may constitute up to 20% of the total cell protein content. S-layers self assemble in entropy driven process during which multiple noncovalent interactions between individual SLPs and the underlying cell surface take place. These two types of interactions in SLPs can be assigned to two separate domains including the self assembly domain and the cell wall binding domain. These domains have been characterized in SLPs of *Lactobacillus acidophilus* ATCC 4356 (SA), *Lactobacillus crispatus* JCM 5810 (CbsA), and The lysine motif (LysM) was first discovered in the lysozyme of *Bacillus* phage ø29 as a Cterminal repeat composed of 44 amino acids separated by 7 amino acids. The cell wall attachment of several bacterial proteins in both gram positive and gram negative organisms occurs through LysMs, often repeated several times in the protein sequence. Many LysM containing proteins are cell wall hydrolases (Buist et al., 2008). The C-terminal region of peptidoglycan hydrolase (AcmA) of *L. lactis* MG 1363 (CpH) contains three 44 amino acid residue lysMs separated by stretches of 21 to 31 amino acids rich in the serine, threonine, and asparagine residues. CpH is able to bind to the cell surface of lactococci and several strains of lactobacilli. Moreover, CpH is able to bind both to the cell surface of LAB treated with sodium dodecyl sulfate (SDS) to remove cell wall associated proteins and LAB treated with trichloroacetic acid (TCA) to remove carbohydrates and (lipo) teichoic acids. These findings suggest that peptidoglycan is the binding ligand of the CpH domain (Buist et al.,

1995). The C-terminus of endolysin Lyb5 of *Lactobacillus fermentum* bacteriophage øPYB5 (Ly5C) contains three LysMs. Each of LysMs is composed of 41 amino acids and they are separated by intervening sequences varying in length and composition. Ly5C fused to GFP was expressed in *E. coli*. After mixing the fusion protein with various cells *in vitro*, GFP was successfully displayed on the surfaces of *L. lactis, L. casei*, *L. brevis*, *L. plantarum*, *L. fermentum*, *L. delbrueckii*, *L. helveticus*, and *S. thermophilus* cells. Increases in the fluorescence intensities of TCA treated *L. lactis* and *L. casei* cells compared to those of nontreated cells showed that the cell wall peptidoglycan was the cell surface binding target of Ly5C. Concentration of sodium chloride and pH influenced the binding capacity of the fusion protein, and optimal conditions of these factors were determined empirically in order to obtain high fluorescence intensities of *L. lactis* and *L. casei* cells (Hu et al., 2010). N-terminus of putative muropeptidase (MurO) of *L. plantarum* also contains two LysMs composed of about 43 amino acids separated by 22 amino acid residue sequences. The LysM domain fused to GFP was expressed in *E. coli* and it was able to bind to the cell surface of *L. plantarum* after being mixed with the cells (Xu et al., 2011). Examination of supernatant fractions from broth cultures of *L. fermentum*, revealed the presence of a 27-kDa protein termed Sep. The N-terminus of Sep contains a LysM. Sep fused N-terminally to a six histidine epitope was expressed in *L. fermentum*, *Lactobacillus rhamnosus,* and *L. lactis*. The protein was found associated with the surface of the expression hosts. However, it was largely present in the supernatant of the cell cultures (Turner et al., 2004).

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proteins. Most solute binding proteins are lipoproteins. However, BspA is not a lipoprotein and is attached to the cell envelope by electrostatic interactions. It has been used as a fusion partner to direct proteins to the cell surface of *L. fermentum* BR11. In these constructs, BspA was fused at its C-terminus to target proteins and the fusion proteins were expressed in *L. fermentum* BR11 (Turner & Giffard, 1999). The C-terminal region of cell associated dextransucrase of *Leuconostoc mesentroides* IBT-PQ (DsrP) contains five repeats of 65 amino acid residues. The domain expressed in *E. coli* was able to bind to the cell surface of *L. mesentroides* IBT-PQ cells after being mixed with the cells (Olvera et al., 2007). The carboxy end of PrtP of *Lactobacillus acidophilus* was used for the protein display on *L. acidophilus* using the internal mode. The association of this domain with the cell surface was mediated

Research in the field of surface engineering of LAB has mainly been focused on the construction of vaccine delivery vehicles but other interesting applications have also been reported. In this section, we will describe different areas of biotechnology in which surface

Vaccination represents one of the most effective public health strategies to combat infectious diseases (Mielcarek et al., 2001). One of the technologies being developed for vaccine production is the use of bacteria as live vectors for the delivery of recombinant vaccine antigens to the immune system. Such vaccines have the potential for the production of

Most infections affect or initiate infectious processes at mucosal surfaces and mucosal local immune responses can block pathogens at the portal of entry. Live bacterial vaccines can induce mucosal, as well as systemic, immune responses when delivered via mucosal routes, such as oral or intranasal administration (Mielcarek et al., 2001). The mucosal, needle free, administration of vaccines can significantly decrease the need for syringes with their inherent added cost and risk of disease transmission, and it can increase compliance, and consequently the coverage of vaccination programs (Giudice & Campbell, 2006). The first live recombinant bacterial vectors developed were derived from attenuated pathogenic microorganisms. In addition to the difficulties often encountered in the construction of stable attenuated mutants of pathogenic organisms, attenuated pathogens may retain a residual virulence level that renders them unsuitable for the vaccination of partially immunocompetent individuals such as infants, the elderly or immunocompromised patients (Curtiss, 2002). These problems can be addressed by the application of nonpathogenic food grade LAB as antigen delivery vehicles. LAB therefore represent attractive alternatives as antigen carriers and their use has mainly been focused on the construction of mucosal vaccines. The cellular location of antigens can influence the elicited immunological responses. Cell surface anchored antigens are better recognized by the immune system than

protective antigens *in vivo* and are inexpensive to manufacture (Moore et al., 2001).

by electrostatic interactions (Kajikawa et al., 2011).

**3.1. Development of vaccine delivery vehicles** 

**3. Applications of surface engineering of LAB** 

display of heterologous proteins on LAB have been investigated.

### *2.2.3. WxL anchors*

The C-terminal cell wall binding domain designated WxL was first identified in proteins of *Lactobacillus* and other LAB based on *in silico* analysis (Kleerebezem et al., 2010). WxL domain contains a WxL motif followed by a proximal well conserved YXXX(L/I/V)TWXLXXXP motif. This domain was found in gene clusters that also encode additional extracellular proteins with C-terminal membrane anchors and LPxTG motif containing anchors, suggesting that they form an extracellular protein complex (Siezen et al., 2006). The C-terminal WxL domains identified in two proteins of *Enterococcus faecalis* were fused at their N-termini to an export reporter enzyme (nuclease of *S. aureus)* and a secretion signal peptide. The fusion proteins expressed in *E. faecalis* were detected in both cell wall and supernatant fractions of the recombinant enterococci. Removal of the WxL domains from the fusion proteins nearly eliminated them in the cell wall. Treatment of the cell wall fractions with SDS disrupted binding of the fusion proteins to these fractions. These results indicated that the fusion proteins had noncovalent interactions with the cell wall of *E. faecalis*. The fusion proteins were able to attach to the cell surface of *E. faecalis* and *L. johnsonii* when they were added exogenously (Brinster et al., 2007).

### *2.2.4. Other anchors*

Basic surface protein A (BspA) is a surface located protein of *L. fermentum* BR11. Sequence comparisons have been shown that BspA is a member of family III of the solute binding proteins. Most solute binding proteins are lipoproteins. However, BspA is not a lipoprotein and is attached to the cell envelope by electrostatic interactions. It has been used as a fusion partner to direct proteins to the cell surface of *L. fermentum* BR11. In these constructs, BspA was fused at its C-terminus to target proteins and the fusion proteins were expressed in *L. fermentum* BR11 (Turner & Giffard, 1999). The C-terminal region of cell associated dextransucrase of *Leuconostoc mesentroides* IBT-PQ (DsrP) contains five repeats of 65 amino acid residues. The domain expressed in *E. coli* was able to bind to the cell surface of *L. mesentroides* IBT-PQ cells after being mixed with the cells (Olvera et al., 2007). The carboxy end of PrtP of *Lactobacillus acidophilus* was used for the protein display on *L. acidophilus* using the internal mode. The association of this domain with the cell surface was mediated by electrostatic interactions (Kajikawa et al., 2011).
