Sortase B, a new class of sortase in Listeria 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 DeltasrtB 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.

FIG. 1.

srtB loci in L. monocytogenes and S. aureus. (A) Genetic organization of the srtB locus in S. aureus (34) and in L. monocytogenes (21). ORFs are designated according to the L. monocytogenes genome nomenclature (lmo). The srtB gene corresponds to lmo2181. lmo2182, lmo2183, and lmo2184 are homologous to ABC iron transporter-encoding genes; svpA (lmo2185) and lmo2186 display homologies with isdC in S. aureus. lmo2179 and lmo2178 encode LPXTG proteins of unknown function that are unrelated to the staphylococcal IsdA and IsdB LPXTG proteins. Bent arrows indicate promoters; circles, putative transcription terminators. Fur boxes are indicated. Solid arrows flanking dotted lines, below the svpA locus of L. monocytogenes, indicate the positions of the primers used in the RT-PCR analysis (see panel D). The ΔsrtB and ΔsrtA ΔsrtB mutants were constructed from L. monocytogenes EGD-e by homologous recombination using a thermosensitive vector carrying two short, blunt-end-ligated PCR fragments (hatched rectangles), which were produced with primers SB1-SB2 and SB3-SB4. The in-frame deletion was confirmed by PCR analysis using primers flanking (SB1, SB4, SB10, SB12) (see panel E) or inside (SB11) srtB. (B) Schematic representation of IsdC from S. aureus and of Lmo2186 and SvpA from L. monocytogenes. The N-terminal peptide signal and the C-terminal hydrophobic domain and charged tail are shown. The percentage of identity (id) between the SvpA repeats and Lmo2186 is shown. (C) Sequence alignment of the C-terminal regions of IsdC, Lmo2186, and SvpA. Hydrophobic domains are italicized. The NPQTN cleavage motif in IsdC is boldfaced; putative SrtB cleavage motifs in SvpA and Lmo2186 are underlined. (D) Tris-acetate-EDTA-agarose gel electrophoresis of transcripts amplified by RT-PCR. Numbers (in kilobases) with arrows on the left correspond to sizes on the DNA ladder. Numbers above each lane correspond to those at the bottom of panel A: 1, srtB; 2, lmo2182 and srtB; 3, srtB and lmo2180; 4, svpA and lmo2184; 5, lmo2186 and svpA. (E) In-frame deletions in mutants were genetically verified by PCR analysis using primers flanking srtB (SB1-SB12) or srtA (SA5-SA7). DNA fragments were separated on an ethidium bromide-stained agarose gel.

FIG. 2.

Roles of SrtA and SrtB in anchoring Listeria surface proteins to the PG. (A) Cultures of wild-type (wt), ΔsrtA, ΔsrtB, and ΔsrtA ΔsrtB EDGe strains were fractionated into membrane (M) and highly pure PG fractions, and proteins were detected by immunoblotting with anti-InlA, -p60, or -InlB antibodies. InlA is missorted in the ΔsrtA and ΔsrtA ΔsrtB mutants. Membrane-associated p60 and InlB proteins do not appear in PG fractions. (B) Immunofluorescence analysis of surface-bound proteins recognized by the polyclonal antiserum 839, which was raised against purified macromolecular L. monocytogenes PG. The labeling is mostly polar (arrows). A lack of SrtA notably reduces the amount and number of proteins that associate with PG, whereas a lack of both SrtA and SrtB fully suppresses the detection of these proteins. (C) Immunoblots of highly pure PG and supernatant (S) fractions, prepared from wild type, ΔsrtA, ΔsrtB, ΔsrtA ΔsrtB, ΔsrtA ΔsrtB/pP1srtA, and ΔsrtA ΔsrtB/pP1srtB EDG-e strains and probed with an anti-PG antiserum as previously described (5). The 60- to 70-kDa polypeptide is present in ΔsrtA and absent in ΔsrtA ΔsrtB strains. MW, molecular weight (in thousands).

FIG. 3.

Display of SvpA on the surfaces of listeriae grown in BHI. The EDGe wild type, ΔsrtA, ΔsrtB, and svpA strains were analyzed by phase-contrast and immunofluorescence staining with the SvpA-specific polyclonal antibody. The merge image shows the phase contrast in blue and the SvpA labeling in green. SvpA is polarly (arrows) or laterally (arrowheads) localized at cell surfaces in the wild-type bacterial population. Inactivation of srtB abolished SvpA surface association, and complementation of the mutation restored it.

FIG. 4.

Localization of SvpA in bacterial compartments. Cytoplasmic (C), membrane (M), PG, and supernatant (Sp) fractions of wild-type (wt) and svpA EGD-e strains were analyzed by Western blotting with an anti-SvpA polyclonal antibody. A major polypeptide of ∼66 kDa is specifically detected in extracts from the wild-type strain. The Sp fraction is 50-fold diluted with respect to the other fractions.

FIG. 5.

SrtB is required for SvpA anchoring to PG. (A) PG fractions (as in Fig. 2) of wild-type EDG-e, svpA, ΔsrtA, ΔsrtB, ΔsrtA ΔsrtB, ΔsrtA/pP1srtB, ΔsrtB/pP1srtB, and ΔsrtA ΔsrtB/pP1srtB strains were analyzed by Western blotting with anti-SvpA (top) or anti-InlA (bottom) antibodies by using the Tris-Tricine electrophoresis system to visualize proteins from high to low molecular weights. Inactivation of srtB abolishes the anchoring of SvpA to PG. MW, molecular weight (in thousands). (B) Supernatant fractions of the same strains were analyzed by Western blotting with the anti-SvpA antibody by using the Tris-glycine system to separate proteins in the 90- to 40-kDa range. The SvpA forms present in the samples of ΔsrtB and ΔsrtA ΔsrtB strains (labeled as SvpA*) run with higher mobility than those detected in all other strains (labeled as SvpA), which express SrtB. A 60-kDa band (indicated by the star) that appears in all strains, including the svpA mutant, is nonspecific.

FIG. 6.

SrtB-mediated anchoring of SvpA is not required for bacterial growth in macrophages. RAW 264.7 macrophages were exposed to wild-type EGD-e bacteria (squares) or to the ΔsrtA (circles), ΔsrtB (triangles), or svpA (diamonds) mutant for 15 min at 4°C. After 45 min of internalization at 37°C, gentamicin was added to kill extracellular bacteria, and bacterial survival was monitored for 8 h postinfection. In contrast to that of the svpA mutant, the intracellular multiplication of the ΔsrtB mutant was not affected.