The cell wall of Gram-positive bacteria is a major cellular component responsible for maintaining the structural stability, providing a barrier to osmotic pressures, facilitating interactions with the surrounding environment and essential for its survival. These interactions in Gram-positive bacteria are facilitated by a vast array of macromolecular structures which include teichoic acid, lipoteichoic acids, exopolysaccharides, Surface (S) layer proteins, enzymes, and cell adhesion molecules (Marraffini et al., 2006; Weidenmaier and Peschel, 2008).The ecological niche of the microbes often dictates the mosaic-like surface display of macromolecules. In pathogenic bacteria, the surface proteins such as internalin A in Listeria monocytogenes and protein A in Staphylococcus aureus, play a major role in establishing pathogenicity and infection, whereas in probiotic microbes, these surface structures play a vital role in eliciting the health benefits upon the host (Cabanes et al., 2002; Clancy et al., 2010; Mazmanian et al., 2000).
Bacteria possess two major secretory pathways: a general secretion pathway (Sec-dependent pathway) and Twin-Arginine translocation pathway (TAT-dependent pathway), through which surface proteins are anchored or targeted to the cell exterior. The Sec-dependent pathway recognizes the secretion of unfolded proteins that are folded after the secretion. These proteins contain an N-terminal leader peptide, a hydrophobic core, and a C-terminal sequence that promotes binding of Sec machinery, whereas, TAT- dependent pathway is used for the translocation of folded proteins (Call and Klaenhammer, 2013). Furthermore, the surface proteins are anchored on the cell surface by transmembrane domains (Tjalsma et al., 2004), through lipid modifications (Tjalsma et al., 2000), noncovalently by cell wall binding repeats (Fernández-Tornero et al., 2001) or by covalent interactions (Navarre and Schneewind, 1994).
The covalent attachment of the surface proteins to the cell wall of peptidoglycan after sec targeting are executed by sortase enzymes that are almost exclusively found in Gram-positive bacteria(Comfort, 2004; Pallen et al., 2001).Targeted proteins recognized by sortases contains a C-terminal cell wall sorting signal, pentapeptide recognition motifs, a hydrophobic membrane-spanning region of ?20 amino acids and a positively charged lysine/arginine tail (Fischetti et al., 1990). After the secretion of target proteins via the Sec apparatus of the general secretory pathway, the corresponding sortases recognizes the cell wall sorting signal and allow the target for cleavage (Kline et al., 2009).
The different classes of sortase share a common ping-pong bi-bi transpeptidation reaction mechanism, according to which the sortase first binds the 5-amino acid recognition motif located in the C-terminal region of the substrate protein (Frankel et al., 2005, 2007).The sortases then form thioacyl?enzyme intermediates between the catalytic cysteine and the substrate threonine, which are resolved by a nucleophilic attack by components of the bacterial cell wall. Since, after the discovery of different classes of sortases, Staphylococcus aureus sortase A (SrtA) has been the prototype for understanding the mechanism of action of these enzymes (Mazmanian et al., 1999). In Staphylococcus aureus, sortase A (SrtA) recognizes the pentaglycine sequence on the surface proteins, which are being secreted through the cytoplasmic membrane. The pentaglycine sequence contains an LPXTG motif at the C-terminus of the protein. The SrtA cleaves the scissile bond between threonine and glycine residues to form an acyl-enzyme intermediate which subsequently transfers the carboxyl of threonine which is amide-linked to the pentaglycine cross-bridge of lipid II (Marraffini et al., 2006). Finally, the lipid II- surface protein complex gets incorporated into the peptidoglycan by means of transglycosylation and transpeptidation reactions (Paterson and Mitchell, 2004; Spirig et al., 2011). The sortase enzyme accepts the nucleophiles which might vary in different Gram-positive bacteria as the composition of peptidoglycan layers in the cell envelope vary from strain to strain. For example, Diaminopimelic acid which cross-bridges the peptidoglycan in Bacillus anthracis is thought to be the point of attachment for the sortase substrate proteins in Bacillus strains.14–16 In absence of a dedicated nucleophile, the acyl-enzyme intermediate complex was shown to be hydrolyzed, resulting in cleavage of the recognition sequence without formation of a new peptide bond.12
Many Gram-positive bacteria contain multiple sortases within their genome which function non-redundantly to sort distinct proteins to the cell surface by recognizing their class-specific sorting signals. Sortase transpeptidase can be generally categorized into different classes, A?F, based on their phylogeny, predicted substrate preference and their functions (Bradshaw et al., 2015). Sortase super families possess a conserved TLXTC motif at its active site. Class A sortases are present in many Gram-positive bacteria and play a housekeeping role in anchoring a large number of proteins to the cell envelope. Class B sortases have been implicated in iron homeostasis by attaching the iron acquisition proteins to the cell envelope (Mazmanian and al., 2003). Class C sortases are responsible for catalyzing the transpeptidation reactions by catalyzing the polymerization of pilin subunits in bacteria. Class D sortases anchor proteins that are involved in sporulation. Sortases of class E and F are found in Actinobacteria, where class E anchors proteins to the cell surface that function to promote aerial hyphae formation (Kattke et al., 2016) and functions of class F remains unknown.