Figuración y representación en el Tractatus.

The complex surface architecture of Gram-positive bacteria serves as a scaffold to display different extracellular proteins. After translocation across the cytoplasmic membrane of Gram-positive bacteria, proteins are either released into the medium or anchored on the surface of bacterial cells through various mechanisms, covalently or non-covalently.

1.5.2.1 Proteins containing transmembrane helices

Not all SecA-dependent extracellular proteins have a signal peptidase cleavage site in the

“C” region of their signal peptides. Thus, after these preproteins are translocated across the cytoplasmic membrane, they are not released from the membrane. Instead, they are retained in the cytoplasmic membrane through the N-terminal signal peptide anchor. In lactobacilli, the N-terminally anchored proteins are involved in diverse extracellular functions such as transport, cell-wall metabolism and signalling [191]. Alternatively, some proteins with a C-region cleavage site can be anchored in the cytoplasmic membrane through their C-terminal end that contains a hydrophobic transmembrane helix as a membrane anchor, often called the “stop-transfer” signal or membrane anchor. The functions of the C-terminally anchored proteins in lactobacilli are largely unknown.

1.5.2.2 Covalently attached membrane proteins

Another group of cytoplasmic membrane anchored proteins are lipoproteins. These proteins carry a type II signal sequence that shares the similar tripartite organisation as type-I signal peptides except that the H region is shorter and C region has a signature lipobox motif (L-x-x-C). The cysteine residue in the lipobox motif is modified by the lipoprotein diacylglyceryl transferase. After the cleavage, the mature protein is covalently anchored in the cytoplasmic membrane through thioether linkage via the N-terminal Cys residue. Lactobacillus genomes encode various numbers of lipoproteins, which often play

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1.5.2.3 Proteins containing a C-terminal cell-wall-anchor motif

In addition to the cytoplasmic membrane of Gram-positive bacteria, many extracellular proteins are covalently anchored to the PG layers. This type of proteins typically contains a cleavable N-terminal signal peptide as well as a C-terminal conserved peptidoglycan- anchoring motif, LPxTG, followed by a hydrophobic region and positively charged residues. The LPxTG motif is recognised by a transpeptidase, called sortase A (SrtA), which cleaves this motif between threonine and glycine residues and links the mature protein to the glycine residue of PG through the threonine residue [81]. Sortase-dependent peptidoglycan-anchored proteins are commonly present in lactobacilli and are involved in adhesion.

1.5.2.4 Cell wall-bound proteins

Many bacterial surface-attached proteins do not have a membrane anchor or LPxTG motif. Instead, these proteins contain one or more cell wall-binding domains (CWBDs) that non- covalently bind to the surface structures of Gram-positive bacteria. CWBDs include LysM, SLH, WxL, choline-binding domains and SH3.

LysM domain is found in many eukaryotic and prokaryotic proteins. In bacteria, LysM domain-containing proteins are often cell wall enzymes, such as hydrolases, peptidases, chitinases and esterases [202]. A LysM domain usually contains several LysM motifs (Pfam PF01476) each containing 44-65 residues. The optimal number of LysM motifs determines the function of these enzymes. Binding of LysM usually occurs at the specific site of peptidoglycan chains, most likely GlcNAc moiety[203]. Depending on the protein, the LysM motif can be located anywhere along a protein sequence, but is predominantly N- or C-terminal [191].

The SLH domains are mainly found in bacterial S-layer proteins that undergo self- assembly to form the tightly-knit S-layer on bacterial surface. Typically, Gram-positive S-layer proteins consist of three SLH domains that each contains 50-70 residues. These domains mediate the anchoring of the S-layer proteins to the bacterial surface [79, 80]. However, in Lactobacillus species, S-layer proteins do not have such SLH domains but

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carbohydrate-binding motifs of clostridial toxins and streptococcal glucosyltransferases [80]. It is proposed that the S-layer proteins of Lactobacillus species bind to the cell

surface through secondary cell wall polymers, LTAs, TAs or neutral PSs [80, 204].

C-terminally located Wxl domain was initially discovered by in silico analysis of

predicted proteins in genome sequences of lactobacilli, enterococci and listeria and

located at the C-terminus of these proteins [205, 206]. The Wxl-domain-containing proteins were experimentally demonstrated to non-covalently bind to the cell wall of E. faecalis. Several Lactobacillus genomes, such as L. plantarum, L. sakei, L. casei, and L. coryniformis, are found to encode Wxl domain-containing proteins [63]. However, the

functions of these proteins are unknown. In L. plantarum, a Wxl-containing protein is

proposed to form a cell surface protein complex with other extracellular proteins, involved in carbon acquisition [205].

Bacterial SH3b domain (subfamilies SH3_3, SH3_4 and SH3_5) is the prokaryotic counterpart of the well-known eukaryotic SH3 domain and was shown to bind to the bacterial cell wall. The SH3b domain seems to bind to the various sites on the peptidoglycan in different bacteria. For example, in Staphylococcus species, the SH3_5

domain of lysostaphin ALE-1 binds to the cross-bridge of the cell wall [207, 208]. The binding is also dependent on the amino acid composition and length of the cross-bridge. On the other hand, SH3_5 domain from Acm2 protein of L. plantarum binds to the

GlcNAc-containing glycan chains and targets this protein to the septum of dividing cells [209, 210]. In lactobacilli, the SH3b-containing proteins are proposed to function mainly in cell wall metabolism [191].

The choline-binding domain (Pfam PF01473) consists of multiple tandem copies of twenty-amino acid sequences that are rich in aromatic amino acids and glycines. Proteins carrying this domain are often enzymes, such as autolysins and cell wall hydrolases, which bind to the choline residues of LTA or TA of Gram-positive bacteria [191]. In lactobacilli, choline-binding domains are only found in few Lactobacillus species, such

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