Olaf Schneewind

Exogenous and endogenous glycolipid antigens activate NKT cells during microbial infections

CD1d-restricted natural killer T (NKT) cells are innate-like lymphocytes that express a conserved T-cell receptor and contribute to host defence against various microbial pathogens. However, their target lipid antigens have remained elusive. Here we report evidence for microbial, antigen-specific activation of NKT cells against Gram-negative, lipopolysaccharide (LPS)-negative alpha-Proteobacteria such as Ehrlichia muris and Sphingomonas capsulata. We have identified glycosylceramides from the cell wall of Sphingomonas that serve as direct targets for mouse and human NKT cells, controlling both septic shock reaction and bacterial clearance in infected mice. In contrast, Gram-negative, LPS-positive Salmonella typhimurium activates NKT cells through the recognition of an endogenous lysosomal glycosphingolipid, iGb3, presented by LPS-activated dendritic cells. These findings identify two novel antigenic targets of NKT cells in antimicrobial defence, and show that glycosylceramides are an alternative to LPS for innate recognition of the Gram-negative, LPS-negative bacterial cell wall.

Sortases and the Art of Anchoring Proteins to the Envelopes of Gram-Positive Bacteria

SUMMARY The cell wall envelopes of gram-positive bacteria represent a surface organelle that not only functions as a cytoskeletal element but also promotes interactions between bacteria and their environment. Cell wall peptidoglycan is covalently and noncovalently decorated with teichoic acids, polysaccharides, and proteins. The sum of these molecular decorations provides bacterial envelopes with species- and strain-specific properties that are ultimately responsible for bacterial virulence, interactions with host immune systems, and the development of disease symptoms or successful outcomes of infections. Surface proteins typically carry two topogenic sequences, i.e., N-terminal signal peptides and C-terminal sorting signals. Sortases catalyze a transpeptidation reaction by first cleaving a surface protein substrate at the cell wall sorting signal. The resulting acyl enzyme intermediates between sortases and their substrates are then resolved by the nucleophilic attack of amino groups, typically provided by the cell wall cross bridges of peptidoglycan precursors. The surface protein linked to peptidoglycan is then incorporated into the envelope and displayed on the microbial surface. This review focuses on the mechanisms of surface protein anchoring to the cell wall envelope by sortases and the role that these enzymes play in bacterial physiology and pathogenesis.

Sorting of protein a to the staphylococcal cell wall

The cell wall of gram-positive bacteria can be thought of as representing a unique cell compartment, which contains anchored surface proteins that require specific sorting signals. Some biologically important products are anchored in this way, including protein A and fibronectin binding protein of Staphylococcus aureus and streptococcal M protein. Studies of staphylococcal protein A and Escherichia coli alkaline phosphatase show that the signal both necessary and sufficient for cell wall anchoring consists of an LPXTGX motif, a C-terminal hydrophobic domain, and a charged tail. These sequence elements are conserved in many surface proteins from different gram-positive bacteria. We propose the existence of a hitherto undescribed sorting mechanism that positions proteins on the surface of 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.

Conservation of a hexapeptide sequence in the anchor region of surface proteins from gram-positive cocci.

A highly conserved hexapeptide sequence (both at the protein‐and DNA level) has been identified within the C‐terminal end of all 11 known surface proteins from Gram‐positive cocci. The hexapeptide, with the consensus sequence LPXTGE, is located about 9 amino acids N‐terminal from the C‐terminal hydrophobic domain which is found in all these surface molecules. The conservation of the hexapeptide, despite sequence variation within the regions flanking it, suggests that it is important for the attachment of these proteins within the cell.

Vaccine protection against Staphylococcus aureus pneumonia.

Staphylococcus aureus pneumonia causes significant mortality in hospitalized or healthy individuals, and recent increases in morbidity are attributed to the rapid spread of methicillin-resistant S. aureus (MRSA) strains, which are often not susceptible to antibiotic therapy. α-Hemolysin (Hla), a secreted pore-forming toxin, is an essential virulence factor of MRSA in a mouse model of S. aureus pneumonia. We show that the level of Hla expression by independent S. aureus strains directly correlates with their virulence. Active immunization with a mutant form of Hla (HlaH35L), which cannot form pores, generates antigen-specific immunoglobulin G responses and affords protection against staphylococcal pneumonia. Moreover, transfer of Hla-specific antibodies protects naive animals against S. aureus challenge and prevents the injury of human lung epithelial cells during infection. Thus, Hla vaccination or immunotherapy may prevent S. aureus pneumonia in humans.

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.

Staphylococcus aureus sortase mutants defective in the display of surface proteins and in the pathogenesis of animal infections

Many gram-positive bacteria covalently tether their surface adhesins to the cell wall peptidoglycan. We find that surface proteins of Staphylococcus aureus are linked to the cell wall by sortase, an enzyme that cleaves polypeptides at a conserved LPXTG motif. S. aureus mutants lacking sortase fail to process and display surface proteins and are defective in the establishment of infections. Thus, the cell wall envelope of gram-positive bacteria represents a surface organelle responsible for interactions with the host environment during the pathogenesis of bacterial infections.

Sortase-catalysed anchoring of surface proteins to the cell wall of Staphylococcus aureus

Many surface proteins of Gram‐positive bacteria are anchored to the cell wall envelope by a transpeptidation mechanism, requiring a C‐terminal sorting signal with a conserved LPXTG motif. Sortase, a membrane protein of Staphylococcus aureus, cleaves polypeptides between the threonine and the glycine of the LPXTG motif and catalyses the formation of an amide bond between the carboxyl‐group of threonine and the amino‐group of peptidoglycan cross‐bridges. S. aureus mutants lacking the srtA gene fail to anchor and display some surface proteins and are impaired in the ability to cause animal infections. Sortase acts on surface proteins that are initiated into the secretion (Sec) pathway and have their signal peptide removed by signal peptidase. The S. aureus genome encodes two sets of sortase and secretion genes. It is conceivable that S. aureus has evolved more than one pathway for the transport of 20 surface proteins to the cell wall envelope.

Assembly of pili on the surface of Corynebacterium diphtheriae

Pili of Gram‐negative pathogens are formed from pilin precursor molecules by non‐covalent association within the outer membrane envelope. Gram‐positive microbes employ the cell wall peptidoglycan as a surface organelle for the covalent attachment of proteins, however, an assembly pathway for pili has not yet been revealed. We show here that pili of Corynebacterium diphtheriae are composed of three pilin subunits, SpaA, SpaB and SpaC. SpaA, the major pilin protein, is distributed uniformly along the pilus shaft, whereas SpaB is observed at regular intervals and SpaC seems positioned at the pilus tip. Assembled pili are released from the bacterial surface by treatment with murein hydrolase, suggesting that the pilus fibres may be anchored to the cell wall envelope. All three pilin subunit proteins are synthesized as precursors carrying N‐terminal signal peptides and C‐terminal sorting signals. Some, but not all, of the six sortase genes encoded in the genome of C. diphtheriae are required for precursor processing, pilus assembly or cell wall envelope attachment. Pilus assembly is proposed to occur by a mechanism of ordered cross‐linking, whereby pilin‐specific sortase enzymes cleave precursor proteins at sorting signals and involve the side chain amino groups of pilin motif sequences to generate links between pilin subunits. This covalent tethering of adjacent pilin subunits appears to have evolved in many Gram‐positive pathogens that encode sortase and pilin subunit genes with sorting signals and pilin motifs.