The Sortase SrtA of Listeria monocytogenes Is Involved in Processing of Internalin and in Virulence

Abstract

Listeria monocytogenes is an intracellular gram-positive human pathogen that invades eucaryotic cells. Among the surface-exposed proteins playing a role in this invasive process, internalin belongs to the family of LPXTG proteins, which are known to be covalently linked to the bacterial cell wall in gram-positive bacteria. Recently, it has been shown in Staphylococcus aureus that the covalent anchoring of protein A, a typical LPXTG protein, is due to a cysteine protease, named sortase, required for bacterial virulence. Here, we identified in silico from the genome of L. monocytogenes a gene, designated srtA, encoding a sortase homologue. The role of this previously unknown sortase was studied by constructing a sortase knockout mutant. Internalin was used as a reporter protein to study the effects of the srtA mutation on cell wall anchoring of this LPXTG protein in L. monocytogenes. We show that the srtA mutant (i) is affected in the display of internalin at the bacterial surface, (ii) is significantly less invasive in vitro, and (iii) is attenuated in its virulence in the mouse. These results demonstrate that srtA of L. monocytogenes acts as a sortase and plays a role in the pathogenicity.Gram-positive bacteria are surrounded by a cell wall envelope containing attached polypeptides and polysaccharides that may interact with host cells and play a role in the virulence of pathogenic species (34). In gram-positive bacteria, several distinct mechanisms of cell wall attachment and display of surface proteins have been recently described (reviewed in reference 5). The only surface proteins known to be covalently linked to the cell wall are the LPXTG proteins, exemplified by protein A of Staphylococcus aureus (34). In a pioneer work, Schneewind et al. described a cysteine protease of S. aureus, designated sortase, which is responsible for the covalent attachment of protein A, and identified the biochemical processes allowing the anchoring of protein A to the cell wall (27). Very recently, the three-dimensional structure of the sortase of S. aureus was determined by nuclear magnetic resonance spectroscopy, thus identifying a catalytic domain responsible for the transpeptidation reaction (17). After synthesis in the bacterial cytoplasm, surface protein precursors are translocated across the membrane and the NH 2 -proximal leader peptide is removed by leader peptidase. The COOH-terminal sorting signal is first cleaved by sortase between the threonine and glycine residues of the LPXTG motif. Then, the enzyme covalently links the carboxyl of the threonine to the cell wall peptidoglycan by amide linkage (34,35,43,44,45). The sorting signal consists of a conserved LPXTG motif followed by a membrane-spanning hydrophobic domain and a tail mostly composed of positively charged residues (18, 28, 42). Notably, it was recently shown that in the gram-positive human pathogen Listeria monocytogenes, the surface proteins were also cleaved between the threonine and the glycine residue of the LPXTG motif and amide-linked to the peptidogl...

The arsenal of virulence factors deployed byListeria monocytogenesto promote its cell infection cycle

“…The cysteine transport-associated protein, CtaP, was identified in a study aiming to detect novel proteins secreted in response to anchors LPXTG-containing proteins to the peptidoglycan (see regulation section). 49,50 The inlJ gene is only found in the genomes of L. monocytogenes strains. An inlJ deletion mutant is significantly less virulent following intragastric and intravenous infection of mice.…”

Sortase inhibitor phenyl vinyl sulfone inhibits Renibacterium salmoninarum adherence and invasion of host cells

“…Sortase catalyzes covalent anchoring of these proteins to the peptidoglycan surface during cell wall synthesis (reviewed in Navarre & Schneewind 1999, Ton-That et al 2004, Marraffini et al 2006). Elimination of sortase activity and the consequent disruption of surface protein anchoring correlates with a dramatic decrease in pathogenicity of Gram-positive bacteria in animal infections (Mazmanian et al 2000, Bolken et al 2001, Bierne et al 2002, Garandeau et al 2002, Jonsson et al 2002, Weiss et al 2004. Sortase is also involved in the assembly of cell surface pili on Gram-positive bacteria, which often aid attachment to host cells (Ton-That & Schneewind 2003).…”

A Comparative Genome Analysis Identifies Distinct Sorting Pathways in Gram-Positive Bacteria

“…An analysis of bacterial genomes indicates that this anchoring mechanism is conserved in gram-positive bacteria, since nearly all species encode SrtA homologs and proteins bearing a CWS (34, 55). Sortases may be excellent targets for new antimicrobial agents, since pathogens deficient in these enzymes exhibit reduced virulence (11,12,23,35,43,46).A large number of proteins are related to SrtA, but their functions have yet to be determined (55). Consistent with playing a role in surface protein chemistry, all SrtA homologs contain appropriately positioned active site residues (SrtA residues H120 and C184) (32) and transmembrane segments, and their genes are frequently clustered with genes encoding CWScontaining proteins.…”

“…Consistent with playing a role in surface protein chemistry, all SrtA homologs contain appropriately positioned active site residues (SrtA residues H120 and C184) (32) and transmembrane segments, and their genes are frequently clustered with genes encoding CWScontaining proteins. Moreover, several homologs have been shown to be directly involved in protein anchoring, since their elimination prevents the display of surface proteins (11,23,26,54). Although the SrtA protein recognizes the sequence LPXTG within its substrates, this motif is widely varied, and a second S. aureus sortase, called SrtB, processes proteins bearing the sequence NPQTN (46).…”

“…An analysis of bacterial genomes indicates that this anchoring mechanism is conserved in gram-positive bacteria, since nearly all species encode SrtA homologs and proteins bearing a CWS (34, 55). Sortases may be excellent targets for new antimicrobial agents, since pathogens deficient in these enzymes exhibit reduced virulence (11,12,23,35,43,46).…”