Cell Wall Sorting Signal Research Articles

The YSIRK-G/S motif of staphylococcal protein A and its role in efficiency of signal peptide processing.

Many surface proteins of pathogenic gram-positive bacteria are linked to the cell wall envelope by a mechanism requiring a C-terminal sorting signal with an LPXTG motif. Surface proteins of Streptococcus pneumoniae harbor another motif, YSIRK-G/S, which is positioned within signal peptides. The signal peptides of some, but not all, of the 20 surface proteins of Staphylococcus aureus carry a YSIRK-G/S motif, whereas those of surface proteins of Listeria monocytogenes and Bacillus anthracis do not. To determine whether the YSIRK-G/S motif is required for the secretion or cell wall anchoring of surface proteins, we analyzed variants of staphylococcal protein A, an immunoglobulin binding protein with an LPXTG sorting signal. Deletion of the YSIR sequence or replacement of G or S significantly reduced the rate of signal peptide processing of protein A precursors. In contrast, cell wall anchoring or the functional display of protein A was not affected. The fusion of cell wall sorting signals to reporter proteins bearing N-terminal signal peptides with or without the YSIRK-G/S motif resulted in hybrid proteins that were anchored in a manner similar to that of wild-type protein A. The requirement of the YSIRK-G/S motif for efficient secretion implies the existence of a specialized mode of substrate recognition by the secretion pathway of gram-positive cocci. It seems, however, that this mechanism is not essential for surface protein anchoring to the cell wall envelope.

Characterization of novel LPXTG-containing proteins of Staphylococcus aureus identified from genome sequences.

Twenty-one genes encoding surface proteins belonging to the LPXTG family have been identified by in silico analysis of six Staphylococcus aureus genome sequences. Eleven genes encode previously described proteins, while 10 have not yet been characterized. Of these, eight contain the cell-wall sorting signal LPXTG responsible for covalently anchoring proteins to the cell-wall peptidoglycan. The remaining two, SasF and SasD, harbour a single residue variation in the fourth position of the LPXTG motif (LPXAG). Western blotting of lysostaphin-solubilized S. aureus cell-wall proteins demonstrated the release of SasF in the cell-wall fraction, indicating that proteins carrying LPXAG are sorted normally. Analysis of primary sequences of the Staphylococcus aureus surface (Sas) proteins indicated that several share a similar structural organization and a common signal sequence with previously characterized LPXTG proteins of S. aureus and other Gram-positive cocci. Protein SasG has 128 residue B repeats that are almost identical at the DNA level. PCR analysis indicated that recombinants with repeat length variations are present in the bacterial population whereas they are not detectable in the B-repeat-encoding region of sdrD. The sasG and sasH genes are significantly associated with invasive disease isolates compared to nasal carriage isolates. Several IgG samples purified from patients recovering from S. aureus infections had higher titres against Sas proteins than control IgG, suggesting that expression occurred during infection in some patients.

Expression of cbsA Encoding the Collagen-Binding S-Protein of Lactobacillus crispatus JCM5810 in Lactobacillus casei ATCC 393 T

The cbsA gene encoding the collagen-binding S-layer protein of Lactobacillus crispatus JCM5810 was expressed in L. casei ATCC 393(T). The S-protein was not retained on the surface of the recombinant bacteria but was secreted into the medium. By translational fusion of CbsA to the cell wall sorting signal of the proteinase, PrtP, of L. casei, CbsA was presented at the surface, rendering the transformants able to bind to immobilized collagens.

Identification of dipeptide repeats and a cell wall sorting signal in the fimbriae-associated adhesin, Fap1, of Streptococcus parasanguis.

Fap1, a fimbriae-associated protein, is involved in fimbriae assembly and adhesion of Streptococcus parasanguis FW213 (Wu et al., 1998). In this study, the sequence of the fap1 gene was resolved using a primer island transposition system. Sequence analysis indicated that fap1 was composed of 7659 nucleotides. The predicted Fap1 protein contains an unusually long signal sequence (50 amino acid residues), a cell wall sorting signal and two repeat regions. Repeat regions I and II have a similar dipeptide composition (E/V/I)S, composed of 28 and 1000 repeats respectively. The two regions combined accounted for 80% of the Fap1 coding region. The experimental amino acid composition and isoelectric point (pI) of Fap1 were similar to that predicted from the deduced Fap1 protein. Results of Northern analyses revealed that the fap1 open reading frame (ORF) was transcribed as a 7.8 kb monocistronic message. Insertional inactivation at the 3' end, downstream of the fap1 ORF, did not affect Fap1, fimbrial expression or bacterial adhesion. Insertional inactivation of fap1 immediately upstream of the repeat region II abolished expression of Fap1 and fimbriae, and was concurrent with a diminution in adhesion of FW213. Inactivation of the cell wall sorting signal of fap1 also eliminated long fimbrial formation and reduced the ability of FW213 to bind to SHA. Fap1 was no longer anchored on the cell surface. Large quantities of truncated Fap1 were found in the growth medium instead. These results suggest that the fap1 ORF alone is sufficient to support Fap1 expression and adhesion, and demonstrate that anchorage of Fap1 on the cell surface is required for long fimbriae formation. These data further document the role of long fimbriae in adhesion of S. parasanguis FW213 to SHA.

Surface Proteins of Gram-Positive Bacteria and Mechanisms of Their Targeting to the Cell Wall Envelope

The cell wall envelope of gram-positive bacteria is a macromolecular, exoskeletal organelle that is assembled and turned over at designated sites. The cell wall also functions as a surface organelle that allows gram-positive pathogens to interact with their environment, in particular the tissues of the infected host. All of these functions require that surface proteins and enzymes be properly targeted to the cell wall envelope. Two basic mechanisms, cell wall sorting and targeting, have been identified. Cell well sorting is the covalent attachment of surface proteins to the peptidoglycan via a C-terminal sorting signal that contains a consensus LPXTG sequence. More than 100 proteins that possess cell wall-sorting signals, including the M proteins of Streptococcus pyogenes, protein A of Staphylococcus aureus, and several internalins of Listeria monocytogenes, have been identified. Cell wall targeting involves the noncovalent attachment of proteins to the cell surface via specialized binding domains. Several of these wall-binding domains appear to interact with secondary wall polymers that are associated with the peptidoglycan, for example teichoic acids and polysaccharides. Proteins that are targeted to the cell surface include muralytic enzymes such as autolysins, lysostaphin, and phage lytic enzymes. Other examples for targeted proteins are the surface S-layer proteins of bacilli and clostridia, as well as virulence factors required for the pathogenesis of L. monocytogenes (internalin B) and Streptococcus pneumoniae (PspA) infections. In this review we describe the mechanisms for both sorting and targeting of proteins to the envelope of gram-positive bacteria and review the functions of known surface proteins.

Influence of Lif, the lysostaphin immunity factor, on acceptors of surface proteins and cell wall sorting efficiency in Staphylococcus carnosus.

Proteins harboring a C-terminal cell wall sorting signal are covalently linked to pentaglycine acceptors within the staphylococcal peptidoglycan. This pentaglycine was modified when the lysostaphin immunity factor (Lif) of Staphylococcus simulans was expressed in Staphylococcus carnosus, likely by the exchange of two glycine residues for serine residues. A reporter protein was efficiently linked to the modified acceptor, indicating that the sorting reaction is not strictly dependent on the wild-type structures of the acceptors.

In vivo immobilization of enzymatically active polypeptides on the cell surface of Staphylococcus carnosus.

Many surface proteins of Gram-positive bacteria are covalently anchored to the cell wall by a ubiquitous mechanism, involving a specific, C-terminal sorting signal. To achieve cell-wall immobilization of a normally secreted enzyme in vivo, we constructed a hybrid protein consisting of Staphylococcus hyicus lipase and the C-terminal region of Staphylococcus aureus fibronectin binding protein B (FnBPB). This region comprised the authentic cell-wall-spanning region and cell-wall sorting signal of FnBPB. Expression of the hybrid protein in Staphylococcus carnosus resulted in efficient cell-wall anchoring of enzymatically active lipase. The cell-wall-immobilized lipase (approximately 10,000 molecules per cell) retained more than 80% of the specific activity, compared to the C-terminally unmodified S. hyicus lipase secreted by S.carnosus cells. After releasing the hybrid protein from the cell wall by lysostaphin treatment. Its specific activity was indistinguishable from that of the unmodified lipase. Thus, the C-terminal region of FnBPB per se was fully compatible with folding of the lipase to an active conformation. To study the Influence of the distance between the cell-wall sorting signal and the C-terminus of the lipase on the activity of the immobilized lipase, the length of this spacer region was varied. Reduction of the spacer length gradually reduced the activity of the surface-immobilized lipase. On the other hand, elongation of this spacer did not stimulate the activity of the immobilized lipase, indicating that the spacer must exceed a critical length of approx. 90 amino acids to allow efficient folding of the enzyme, which probably can only be achieved outside the peptidoglycan web of the cell wall. When the lipase was replaced by another enzyme, the Escherichia coli beta-lactamase, the resulting hybrid was also efficiently anchored in an active conformation to the cell wall of S. carnosus. These results demonstrate that it is possible to immobilize normality soluble enzymes on the cell wall of S. carnosus-without radically altering their catalytic activity-by fusing them to a cell-wall-immobilization unit, consisting of a suitable cell-wall-spanning region and a standard cell-wall sorting signal.

Cell wall sorting of lipoproteins in Staphylococcus aureus

Many surface proteins are thought to be anchored to the cell wall of gram-positive organisms via their C termini, while the N-terminal domains of these molecules are displayed on the bacterial surface. Cell wall anchoring of surface proteins in Staphylococcus aureus requires both an N-terminal leader peptide and a C-terminal cell wall sorting signal. By fusing the cell wall sorting of protein A to the C terminus of staphylococcal beta-lactamase, we demonstrate here that lipoproteins can also be anchored to the cell wall of S. aureus. The topology of cell wall-anchored beta-lactamase is reminiscent of that described for Braun's murein lipoprotein in that the N terminus of the polypeptide chain is membrane anchored whereas the C-terminal end is tethered to the bacterial cell wall.

Proteolytic cleavage and cell wall anchoring at the LPXTG motif of surface proteins in Gram‐positive bacteria

Many surface proteins are thought to be anchored to the cell wall of Gram-positive bacteria via their C-terminus. Cell wall anchoring requires a specific sorting signal, normally located at the predicted C-terminus of surface proteins. Here we show that when placed into the middle of a polypeptide chain, the sorting signal causes the specific cleavage of the precursor as well as the cell wall anchoring of its N-terminal fragment, while the C-terminal fragment remains within the cytoplasm. N-terminal sequencing of the C-terminal cleavage fragment suggests that the cleavage site is located between threonine (T) and glycine (G) of the LPXTG motif, the signature sequence of cell wall sorting signals. All surface proteins harbouring an LPXTG sequence motif may therefore be cleaved and anchored by a universal mechanism. We also propose a novel hypothesis for the cell wall linkage of surface proteins in Gram-positive bacteria.

Cell wall sorting signals in surface proteins of gram-positive bacteria.

Staphylococcal protein A is anchored to the cell wall, a unique cellular compartment of Gram-positive bacteria. The sorting signal sufficient for cell wall anchoring consists of an LPXTG motif, a C-terminal hydrophobic domain and a charged tail. Homologous sequences are found in many surface proteins of Gram-positive bacteria and we explored the universality of these sequences to serve as cell wall sorting signals. We show that several signals are able to anchor fusion proteins to the staphylococcal cell wall. Some signals do not sort effectively, but acquire sorting activity once the spacing between the LPXTG motif and the charged tail has been increased to span the same length as in protein A. Thus, signals for cell wall anchoring in Gram-positive bacteria are as universal as signal (leader) sequences.