The cell wall sorting pathway of surface proteins in Gram-positive bacteria

proposed by Olaf Schneewind from his study of the surface protein A of S. aureus [140]. A genetic screen for S. aureus mutants that failed to anchor protein A to the bacterial cell wall resulted in the identification of the sortase gene named srtA (surface protein sorting A) that codes for sortase A (SrtA) [91]. Since then, anchoring of protein A by sortase A on the cell wall is the paradigm of covalently anchoring of proteins in Gram-positive bacteria (Fig. 1.2).

Table 1.1. C-terminal sorting signals

Organism Protein Sequence of C-terminal sorting signal^a

L. monocytogenes Internalin A SLPTTGDSDNALYLLLGLLAVGTAMALTKKARASK S. aureus Protein A ALPETGEENPFIGTTVFGGLSLALGAALLAGRRREL S. aureus FnBPB ELPETGGEESTNKGMLFGGLFSILGLVLLRRNKKNHKA B. subtilis YfkN LLPDTATSMYSILLAGFLISALGTAMYLHQRRKQNRANQA

B. subtilis YhcR QLPDTSAGYYNFMVIGAAVTLSGTYLYVRRKRSASRT

aThe sorting motifs representing possible sortase cleavage sites are indicated in bold italics.

The positively charged residues that constitute the charged C-terminal tail are underlined.

Hydrophobic segments are indicated in grey shading and were predicted by the Membrane Protein Explorer (MPEx) V.2.2 from the Laboratory of Dr. Stephen White. This table was extracted partly from sortase substrate database [10].

The surface protein A of S. aureus carries two important elements for the anchoring: (i) the N-terminal signal peptide that directs protein A across the cytoplasmic membrane by the Sec pathway and (ii) the C-terminal cell wall sorting signal that encompasses a 35-residue peptide with an LPXTG sorting motif, followed by a hydrophobic domain and a positively charged tail (Table 1.1). This surface protein is synthesized as precursor in the bacterial cytoplasm bearing an N-terminal signal peptide and a C-terminal cell wall sorting signal. After translocation across the cytoplasmic membrane, the N-terminal signal peptide is removed by the signal peptidase, thereby generating the P2 precursor. The C-terminal sorting signal retains the P2 precursor species within the secretory pathway and permits substrate recognition at the LPXTG motif. The sortase, a membrane-anchored transpeptidase with an

active-site cysteine, cleaves surface proteins between the threonine (T) and the glycine (G) of the LPXTG motif generating an acyl-enzyme intermediate. The acyl-enzyme intermediate, with a thioester bond between the thiol of sortase and the carboxyl group of the threonine at the C-terminal end of surface proteins, is resolved by the nucleophilic attack of the amino group of the m-diaminopimelic acid (m-Dpm) cross-bridge of L. monocytogenes [28] or of the pentaglycine cross-brige of S. aureus [92, 158] within the lipid II precursor. Surface proteins linked to lipid II may be incorporated into the cell wall envelope by the transglycosylation and transpeptidation reactions that generate the mature cell wall (Fig. 1.2). This pathway is universal in many Gram-positive bacteria, and the functional elements, the cell wall cross bridges, sorting motif, sortase, and penicillin binding proteins are conserved.

Fig. 1.2. The cell wall sorting pathway of surface proteins in S. aureus [85]. Surface proteins are first synthesized in the bacterial cytoplasm as full-length precursor (P1) containing an N-terminal signal sequence and a C-terminal sorting signal. The signal peptide directs the translocation of the polypeptide through the Sec system followed by removal of the signal peptide. The product of this reaction, the P2 precursor harbouring the C-terminal sorting signal, is retained in the cytoplasmic membrane via its C-terminal hydrophobic domain (black box) and positively charged tail (+). Sortase cleaves the peptide bond between the threonine (T) and the glycine (G) of the LPXTG motif, generating an acyl intermediate (AI). Lipid II, the peptidoglycan biosynthesis precursor, and its pentaglycine cross bridge (Gly5) amino group attacks the acyl intermediate, linking the C-terminal threonine of the surface protein to lipid II (P3 precursor) and regenerating the active site of sortase. The P3 precursor functions as a substrate for penicillin binding proteins and is incorporated into the cell wall envelope to generate mature anchored surface protein (M), which is also displayed on the bacterial surface.

Screening of sequenced genomes of Gram-positive bacteria revealed that typically more than one sortase homologue is present; and interestingly, in the majority of genomes where sortase enzyme genes have been identified, usually multiple sortases are encoded [20, 110]. Based on sequence alignments of sixty-one sortases from completely sequence Gram-positive genomes, four groups of sortases designated SrtA, SrtB, SrtC and SrtD were suggested [31]. Each subgroup is characterized by membrane topology, genome position, and preference for substrates with specific amino acids within the cell wall sorting signal pentapeptide motif (Table 1.2). The S. aureus genome, where sortases have been detected first, codes for two enzymes, sortase A and B [20, 91, 93, 110]. While sortase A recognizes the sorting sequence containing the LPXTG sorting motif and cleaves the peptide bond between the threonine and glycine residues [92, 158], sortase B recognizes the NPQTN motif, catalyzing cleavage of the peptide bond between T and N [180]. In Streptococcus pyogenes, one LPXTG containing protein is recognized by SrtC (Spy0135) which is characterized by the presence of an additional C-terminal hydrophobic domain [6].

Table 1.2. Sortase classifications A LPkT-Ge^e N terminus Bacillus, Listeria, Staphylococcus,

Enterococcus, Lactobacillaceae, Streptococcaceae

[20, 31, 85, 138, 156]

B NPqt-nd^e N terminus Bacillus, Listeria, Staphylococcus, Streptococcaceae, Clostridia

a Classification of sortases is based on sequence, membrane topology, genomic positioning, and preference for specific amino acids within the cell wall sorting signal pentapeptide motif region of their cognate substrates [20, 31, 85]. ^b Cell wall sorting signal is normally an pentapeptide motif, in which uppercase letters represent amino acids that are absolutely conserved. ^c Membrane anchor region is based on transmembrane predictions and regions of high hydrophobicity. ^d Bacterial taxa harbour one or more sortase genes belonging to the respective sortase classification. ^d The cleavage site symbolized by a hyphen is verified experimentally.