Sortase B, a New Class of Sortase inListeria monocytogenes

Abstract

Sortases are transamidases that covalently link proteins to the peptidoglycan of gram-positive bacteria. The genome of the pathogenic bacterium Listeria monocytogenes encodes two sortases genes, srtA and srtB. The srtA gene product anchors internalin and some other LPXTG-containing proteins to the listerial surface. Here, we focus on the role of the second sortase, SrtB. Whereas SrtA acts on most of the proteins in the peptidoglycan fraction, SrtB appears to target minor amounts of surface polypeptides. We identified one of the SrtB-anchored proteins as the virulence factor SvpA, a surface-exposed protein which does not contain the LPXTG motif. Therefore, as in Staphylococcus aureus, the listerial SrtB represents a second class of sortase in L. monocytogenes, involved in the attachment of a subset of proteins to the cell wall, most likely by recognizing an NXZTN sorting motif. The ⌬srtB mutant strain does not have defects in bacterial entry, growth, or motility in tissue-cultured cells and does not show attenuated virulence in mice. SrtB-mediated anchoring could therefore be required to anchor surface proteins involved in the adaptation of this microorganism to different environmental conditions.

Listeria monocytogenesSurface Proteins: from Genome Predictions to Function

“…Both proteins share homology to S. aureus IsdC and bear as putative sorting motifs NAKTN (Lmo2185) or NKVTN or NPKSS (Lmo2186), respectively. Inactivation of srtB impairs sorting of Lmo2185 (16). Lmo2185 and Lmo2186 are the only two SrtB substrates present in the L. monocytogenes cell wall proteome, consistent with the fact that there is no other protein with an NXXTX sorting signal encoded in the L. monocytogenes genome (146).…”

“…The genes encoding SrtB and IsdC are both part of the isd locus, which is involved in bacterial heme iron uptake (121,124). L. monocytogenes has also an alternative sortase B, whose coding sequence maps to an operon containing two genes encoding putative substrates, Lmo2185 (formerly SvpA) and Lmo2186 (16). Both proteins share homology to S. aureus IsdC and bear as putative sorting motifs NAKTN (Lmo2185) or NKVTN or NPKSS (Lmo2186), respectively.…”

“…This approach has proven to be successful for LPXTG proteins and lipoproteins. An srtA mutant, in which LPXTG proteins are missorted, displays a severe attenuation phenotype following oral infection, with a more pronounced effect than an internalin mutant, pointing to the role of several LPXTG proteins in pathogenesis (16,17). Similarly, interfering with the processing of lipoproteins affects intracellular survival of L. monocytogenes (13,148).…”

Association of ActA to Peptidoglycan Revealed by Cell Wall Proteomics of Intracellular Listeria monocytogenes

“…Intracellular L. monocytogenes-Fractions containing cytosolic, membrane, and cell wall proteins of L. monocytogenes grown overnight at 37°C in BHI or IMM media were obtained as described previously (29). Peptidoglycan-associated proteins were obtained by two methods depending on the sample as follows: muramidase (Cellosyl) treatment of previously purified peptidoglycan material (30) or, alternatively, mutanolysin treatment on intact bacteria (17).…”

“…The details of this immunization will be published elsewhere. Protein separation by SDS-PAGE and Western immunoblotting were performed as described previously (29).…”