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. 2010 Sep 17;76(21):7332–7337. doi: 10.1128/AEM.00928-10

Functionality of Sortase A in Lactococcus lactis▿

Yakhya Dieye 1,†,‡, Virginie Oxaran 1,†, Florence Ledue-Clier 1, Walid Alkhalaf 1,§, Girbe Buist 2,, Vincent Juillard 1, Chang Won Lee 3, Jean-Christophe Piard 1,*
PMCID: PMC2976267  PMID: 20851967

Abstract

Lactococcus lactis IL1403 harbors a putative sortase A (SrtA) and 11 putative sortase substrates that carry the canonical LPXTG signature of such substrates. We report here on the functionality of SrtA to anchor five LPXTG substrates to the cell wall, thus suggesting that SrtA is the housekeeping sortase in L. lactis IL1403.

The GRAS (generally recognized as safe) status of lactic acid bacteria (LAB) has catalyzed a myriad of promising applications using these bacteria as a vehicle for in situ delivery of bioactive proteins such as antigens or digestive enzymes in the gastrointestinal tract of the human host (4, 26). In the context of therapeutic applications of LAB, a major fundamental goal is to determine whether they can be engineered to deliver bioactive proteins to the right bacterial and host locations. We previously designed a protein-targeting system in LAB that addressed proteins to the desired bacterial site (i.e., cytoplasm, cell wall, or external medium), as validated using a model protein reporter and various antigens (14, 15). Studies investigating the use of LAB as vaccine delivery vehicles suggested that the cell-wall-anchored protein form may possess superior ability to induce a strong immune response (3, 14). Among the various surface display systems described in Gram-positive bacteria (13), a dedicated surface protein anchoring system catalyzed by sortases was first described and characterized in Staphylococcus aureus (29). It covalently anchors proteins via their C-terminal cell wall anchor (CWA) domain to the bacterial peptidoglycan. SrtA-like sortases process proteins bearing an LPXTG C-terminal motif and are considered to be the housekeeping sortase that anchors most proteins harboring a sorting signal (32). Other sortases were subsequently shown to anchor proteins bearing the same or other motifs (11, 16).

Surprisingly, while the roles of sortases and LPXTG proteins are well documented in pathogens, few reports have examined these functions in other bacteria. A report suggests a relationship between sortase activity and adhesion of the LAB Lactobacillus salivarius, although direct involvement of sortase was not demonstrated (47). Recently, sortase activity was correlated to assembly of pili and adhesion properties in Lactobacillus rhamnosus (21). To further characterize sortase in LAB, we chose an industrially important member of this bacterial group, Lactococcus lactis, to study sortase A functionality in anchoring its putative substrates on the cell wall.

L. lactis IL1403 harbors an expressed sortase A gene.

The L. lactis IL1403 genome was searched by using BLAST (www.ncbi.nlm.nih.gov/BLAST) and with SrtA protein (NP_735398.1) from a phylogenetically close Streptococcus agalactiae strain as the seed. Two putative sortases were identified: one class A, subfamily I, sortase, YlcC (NP_267269); and one class C sortase, YhhA (NP_266915) (11, 16). We focused on the function of YlcC of L. lactis IL1403 (SrtA) in anchoring lactococcal proteins to the cell wall. SrtA is annotated as a putative 287-amino-acid protein.

In silico analysis of the srtA locus using the gene prediction server FGENE SB (Softberry) (http://linux1.softberry.com/berry.phtml?topic=fgenesb&group=programs&subgroup=gfindb) indicated a start codon located 117 bp downstream of that indicated in the original genome annotation (http://www.ncbi.nlm.nih.gov/protein/15673095?report=fasta) and yielding a protein of 248 residues, devoid of 39 N-terminal amino acids predicted by the annotated sequence. This is in agreement with the SrtA orthologs found in the two other sequenced lactococci, namely, L. lactis MG1363 (YP_001032740) and SK11 (YP_809171), whose sortase A is also devoid of those 39 amino acids. The srtA locus contains the following gene cluster: lepA-ylbE-ylcA-gyrA-apbE-srtA-ylcD-ylcE. To analyze srtA expression, total RNAs from L. lactis IL1403 were extracted with the Biofidal kit (Themis, Villeurbanne, France). Synthesis of cDNA was performed using Thermoscript reverse transcriptase (Invitrogen, Cergy Pontoise, France) and srtA-specific primer (5′-CCGCAGCAAAATCAAAACTCACAT-3′) or ylcE-specific primer (5′-GCCAGGATAATCGGTAGCCC-3′). Reverse transcription (RT)-PCR analyses were performed on both cDNAs using primer couples ylbE2 (5′-ACTTATATGGGGCTGTCAAACTCATG-3′) and PrSor2d (5′-CCGCAGCAAAATCAAAACTCACAT-3′) for ylbE-to-srtA amplification or lepA1 (5′-GGAGCAATATTAGTTGTTGATGCCG-3′) and gyrA3 (5′-CCAACGGCAATCCCTGTGG-3′) for lepA-to-gyrA amplification. This indicated that srtA is cotranscribed along with upstream genes up to lepA and with downstream genes down to ylcE at least. Analysis of the sequence upstream and downstream of srtA by using the Prokaryotic Promoter Prediction server (http://bioinformatics.biol.rug.nl/websoftware/ppp/ppp_start.php) showed the presence of several promoter and terminator sequences in the srtA locus (not shown). Thus, the srtA gene can be transcribed under the control of the lepA extended promoter (5′-ATGCTATAAT-3′), but other putative alternative promoters upstream of srtA, gyrA, and ylcA might also be involved. Altogether, these results showing that the L. lactis ylcC-encoded sortase is a class A sortase and is in close vicinity to the gyrA gene suggest that it could be the housekeeping sortase of L. lactis (11).

L. lactis IL1403 harbors 11 putative cell-wall-anchored substrates, some of which have adhesion domains.

Eleven putative sortase substrate genes were identified in the L. lactis IL1403 genome (7), none of which had a predicted function (Table 1). The most frequently found cell wall anchoring motif in the encoded proteins was LPKTG(E/D). A signal peptide was predicted for nine proteins. These potential sortase substrates could be divided in two groups, depending on the type of the amino acid following the glycine residue of the LPXTG motif. Six substrates harbor a negatively charged residue (Asp or Glu) after LPXTG, while the remaining five substrates contain small or hydrophobic amino acids (Ala, Val, Thr, or Gly) at the same position. The effect of such a difference in substrate recognition by SrtA was investigated (see below). Interestingly, collagen- or mucin-binding domains were identified in YbeF, YndF, YwfG, and YreF, while a von Willebrand factor A domain initially identified as involved in blood clotting (37) and recently shown to be involved in adhesion of S. agalactiae (23) was detected in YvcC. These domains suggest possible functions in adhesion to epithelial or possibly other cells (43). Note that some but not all of those putative sortase substrates are present in the two other sequenced lactococci (Table 1). Also, three additional LPXTG proteins (PrtP, NisP, and CluA) that are absent in L. lactis IL1403 have been previously characterized in other lactococcal strains (18, 45, 48). Their structural genes are present on mobile genetic elements.

TABLE 1.

Putative sortase substrates in the deduced proteome of Lactococcus lactis IL1403

graphic file with name zam9991015020004.jpg
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a Gray shading indicate that the corresponding protein has been confirmed as an SrtA substrate in this study.

b LPXTG consensus motifs and the following amino acid are single underlined, transmembrane domains predicted using the TMHMM server (24) are double underlined, and cationic residues are in boldface.

c SP, signal peptides were predicted using SignalP3.0 (2). +, present; −, absent.

d +, present; −, absent; *, C-terminal-truncated protein; **, pseudogene.

Construction of an srtA deletion mutant and of reporter plasmids harboring cell wall anchor regions of two putative sortase substrates. (i) Deletion of L. lactis IL1403 gene srtA.

The srtA gene deletion was achieved using the pG+host-based allelic exchange method (6). Standard molecular biology techniques and procedures were performed as previously described (5, 25, 34, 38). The chromosomal region downstream of the srtA gene was PCR amplified with primers SrtA1 (5′-CACTCCGTTAGAGAAAGCAAA-3′) and SrtA2 (5′-GCACTAAACGCTTGACCATAATATTGGT-3′). The product was treated with NsiI and cloned into the NsiI-EcoRV sites of pG+host9 (27) to yield the pDelA1 plasmid. The region upstream of srtA was amplified using primers SrtA3 (5′-GAAGCTGCAGTTGAAAAGGCTCTAAAA-3′) and SrtA4 (5′-CCGCAGCAAAATCAAAACTCACAT-3′); the product was cloned into the pCRII Blunt TOPO vector (Invitrogen) and then recovered on a NotI-PmeI fragment that was cloned into the NotI-SmaI sites of plasmid pDelA1 to yield pDelA. Plasmid pDelA was established in L. lactis IL1403, and this strain was used to obtain a stable double crossing-over deletion, using the methodology described previously (6). The strain in which srtA was deleted was confirmed by PCR and Southern blot analyses (not shown). The srtA deletion mutant showed bacterial morphology and growth kinetics in brain heart infusion (BHI) medium similar to those of the parental L. lactis IL1403 strain (data not shown).

(ii) Construction of reporter plasmids.

We constructed two reporter plasmids expressing fusion of the Staphylococcus aureus nuclease (NucA) with the CWA regions of YhgE and YndF (cwaYhgE and cwaYndF), respectively (each belonging to one of the two groups of substrates described above). The sequence encoding the C-terminal CWA region of YhgE (Table 1) was PCR amplified with primers ancE1 (5′-CCGTTATCTAGACGGCTTGAACTTGGTTGATA-3′) and ancE2 (5′-CCGTTATGATCAGCCATCATCCCCTCCTAA-3′). The XbaI-BclI-digested amplicon was ligated with the 3,624-bp NheI-BamHI fragment from pVE5251 (15) to yield plasmid pAncE1. The NdeI-NotI 1,527-bp fragment of pAncE1 that harbored an spUsp45::nucA::cwaYhgE fusion (where “sp” represents signal peptide) was ligated with the 4,626-bp NdeI-EaeI fragment from plasmid pIL253 (40) to yield pAncE2. To place the spUsp45::nucA::cwaYhgE fusion under the control of the strong constitutive lactococcal P59 promoter previously used for protein cell wall anchoring in L. lactis (15, 46), the 5,429-bp EcoRV-SphI fragment from pAncE2 was ligated with the 1,240-bp EcoRV-NspI fragment from pVE5523 (15) to yield plasmid pNucA-CWAYhgE (Fig. 1A). To obtain a corresponding plasmid in which cwaYhgE was replaced by cwaYndF, the sequence encoding the C-terminal CWA region from the yndF gene (Table 1) was PCR amplified with primers ancF1 (5′-CCGTTATGATCACAACCATTGCCCCTCCTTT-3′) and ancF2 (5′-CCGTTATCTAGATGGTAATGCCTCTGGCCAAT-3′), and the product was digested with XbaI and BclI. This digested amplicon was ligated with the 3,624-bp NheI-BamHI fragment from pVE5251 (15) to yield plasmid pAncF1 (spUsp45::nucA::cwaYndF). The SpeI-NruI 1,043-bp cwaYndF-containing fragment of pAncF1 was ligated with the 5,653-bp SpeI-NruI fragment from pNucA-CWAYhgE with cwaYhgE deleted to produce pNucA-CWAYndF (Fig. 1A). Both pNucA-CWAYhgE and pNucA-CWAYndF were established in L. lactis IL1403 and in the isogenic srtA mutant. For complementation studies of the srtA mutant, the srtA gene along with its predicted 5′ promoter sequence and 3′ transcription terminator region was PCR amplified using the 5′-srtA (5′-CACACCTGGACTGAAAGACAAA-3′) and 3′-srtA (5′-CATCTTCCTTCTTTTGCAATCGCTAT-3′) primers. The resulting amplicon was cloned in EcoRV-digested pIL2608 (J. Anba, INRA, Jouy-en-Josas, France) to generate psrtA.

FIG. 1.

FIG. 1.

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Cell wall anchoring of fusion protein in the L. lactis IL1403 WT, srtA deletion mutant, and srtA deletion mutant complemented using psrtA. (A) Fusions used to analyze cell wall anchoring. P59 is a strong constitutive lactococcal promoter (46), “sp” refers to the signal peptide of Usp45 (44), nucA to the A form of staphylococcal nuclease (30), t1t2 to transcriptional terminators (33), and cwa to the cell wall anchor region of the protein indicated in subscript. YndF and YhgE are lactococcal proteins without assigned function yet (Table 1). The amino acid sequence of the LPXTG motifs is indicated on the right. The corresponding plasmids are indicated in parentheses. (B) Western blot analyses performed on cell wall fractions of L. lactis IL1403 (wt) and the srtA deletion mutant (srtA) harboring pNucA-CWAYhgE or pNucA-CWAYndF, using polyclonal anti-NucA antibodies. NucA-CWAYhgE and NucA-CWAYndF refer to the cell wall anchor regions of YhgE and YndF proteins that were fused to the C terminus of NucA. (C) Complementation studies by Western blotting using NucA-CWAYhgE as the reporter; wt and srtA are the same as described above; psrtA, srtA mutant complemented with plasmid psrtA.

Sortase A mediates cell wall anchoring of at least five substrates in L. lactis IL1403.

Nuclease A anchoring to the surface of L. lactis by CWAYhgE or by CWAYndF was first assessed by Western blot analyses of cell wall protein extracts obtained as described previously (42). Bands at the expected sizes were detected in the samples from the wild-type strain harboring plasmid pNucA-CWAYhgE or pNucA-CWAYndF (Fig. 1B). In contrast, these bands were lacking in the samples from the srtA mutant containing the same plasmids. These results suggest that YndF and YhgE cell wall anchor motifs are substrates of SrtA. Complementation of the L. lactis srtA deletion mutant expressing nucA-cwaYhgE with psrtA restored the appearance of cell-wall-anchored NucA (Fig. 1C). This confirms that the cell wall anchoring signals (underlined) of YhgE (LPFTGG) and YndF (LPETGD) which differ in the charge of the amino acid following the glycine residue are both recognized and processed by SrtA. Thus, while these differences in amino acid charge were suggested to be discriminating for sortase recognition in Streptococcus pyogenes (1), the variable motifs of YndF and YhgE do not interfere with L. lactis sortase A specificity.

To gain further insight into the anchoring of LPXTG-bearing proteins by SrtA, we analyzed the localization of a third reporter that corresponds to a fusion containing (i) the signal peptide and the CWA of lactococcal proteinase PrtP (39), (ii) a truncated form of the Plasmodium falciparum merozoite surface antigen 2 (MSA2) (17), and (iii) the c-myc epitope (Fig. 2A). PrtP is encoded from a plasmid that is absent in IL1403, and its CWA belongs to the same group as YndF. The spPrtP::msa2::myc::cwaPrtP fusion is harbored by the pCWS1a plasmid, which was previously described (17), and is under the control of the PnisA promoter that is induced by nisin. The L. lactis IL1403 wild-type (WT) strain and srtA mutant harboring plasmids pCWS1a and pNZ9530 for nisin expression (12) were treated as described previously (41). The wild-type strain bearing the fusion displayed fluorescence, which was located mostly at the septum and at the pole of L. lactis cells (Fig. 2B). In contrast, no fluorescence was observed in cells of the srtA mutant, indicating that the cell wall anchor region of PrtP is a substrate of SrtA. The localized fluorescence at the septum suggests that protein cell wall secretion and anchoring could occur at the site of new peptidoglycan synthesis, which is consistent with results showing similar localization of SrtA (9, 36) and studies in other bacteria (10, 35).

FIG. 2.

FIG. 2.

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Localization of Msa2::Myc::CWAPrtP fusion protein in L. lactis IL1403. (A) Fusion structure (the plasmid is indicated in parentheses): PnisA refers to the nisin promoter (12), “sp” refers to the signal peptide of PrtP (22), msa2 to a putative protective antigen from Plasmodium falciparum, myc to the human c-Myc epitope, cwaPrtP to the cell wall anchor region of PrtP, and ter to the transcriptional terminator of prtP (22). PrtP is a plasmid-encoded cell-wall-anchored proteinase involved in nitrogen nutrition in L. lactis (39). The amino acid sequence of the LPXTG motif is indicated on the right. (B) Microscopic observations of the L. lactis wild-type (wt) and srtA deletion mutant (srtA) strains expressing the above fusion gene: the upper pictures show bacterial cells observed under light microscopy, while the lower pictures show the same bacteria whose fusion protein was visualized with anti-Myc antibodies that were further detected with a fluorophore.

To uncover other possible SrtA substrates, cell wall proteins were prepared from the L. lactis IL1403 WT and srtA mutant by mutanolysin treatment (31) and analyzed by two-dimensional electrophoresis (2-DE) as described previously (19). This methodology based on mutanolysin release of cell-wall-associated proteins was applied to identify cell-wall-associated proteins that are present in the WT strain and absent in the srtA mutant. These identified proteins are likely substrates of SrtA. Comparison of the 2-DE gels loaded with equivalent amounts of protein from the WT strain and from the srtA mutant extracts showed seven protein spots that were missing in the srtA deletion mutant (Fig. 3). These spots were not observed in mutanolysin-untreated extract from the WT strain (data not shown), suggesting that they correspond to proteins covalently associated to the cell wall. These seven protein spots were digested with trypsin and subjected to peptide mass fingerprinting essentially according to reference 20, using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (Voyager-DE STR biospectrometry workstation; PerSeptive Biosystems, Inc.). Three protein spots were identified as being YbeF, YwfG, and YndF, whose CWA has been studied as described above using the NucA reporter. These proteins are all LPXTG-containing proteins identified in in silico analyses as putative sortase substrates (Table 1). YhgE, whose cell wall anchor has been shown above to be a substrate of SrtA, was not identified in this experiment. It is possible that YhgE corresponds to one of the spots that were missing in the srtA mutant sample but that failed to be identified by peptide mass fingerprinting. Alternatively, the expression of YhgE might be too low under the growth conditions we used for the protein to be detected. Thus, at least five LPXTG-containing proteins, four of which (YhgE, YndF, YbeF, and YwfG) are chromosome encoded and one of which (PrtP) is plasmid encoded, are processed by SrtA in L. lactis IL1403. Although the other putative sortase substrates could not be identified or visualized in this work, this suggests that SrtA is the housekeeping sortase of L. lactis IL1403.

FIG. 3.

FIG. 3.

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Comparison by 2-DE gels of cell-wall-anchored proteins in the L. lactis IL1403 wild type (wt) and srtA mutant (srtA). Cell-wall-anchored proteins were prepared by mutanolysin digestion of purified cell walls. Equivalent amounts (60 μg) of proteins were separated by 2-DE run on IPG strips of pH 3 to 10 in the first dimension and by 7.5 to 17.5% gradient SDS-PAGE. Proteins were visualized by silver staining. Spots were cut and analyzed for protein identification by peptide mass fingerprinting. The seven protein spot groups of IL1403 wt which are absent in srtA mutant strain are marked by circles; three of them were successfully identified and are labeled by their gene product names (8).

In summary, we have identified and studied a class A sortase in L. lactis IL1403 and showed that it is responsible for the cell wall anchoring of at least five LPXTG-containing proteins. We therefore propose that SrtA is the housekeeping sortase in L. lactis. The characterization of SrtA will make it possible to increase and to control the attachment at the cell wall of L. lactis of molecules of interest such as PrtP, which allows the desired growth of lactococci in dairy products or other biotechnologically important bioactive proteins. In addition to SrtA, we have found by in silico analysis a putative class C sortase in L. lactis. Class C sortases were mainly identified in pathogenic Gram-positive bacteria. However, the only biological function shown so far to involve these sortases is formation of pili (28). Pili have never been observed in lactococci, thus raising a question about the possible role of SrtC in L. lactis. A detailed characterization of SrtC will be needed to answer this question.

ADDENDUM IN PROOF

While this manuscript was under review, Siezen et al. (J. Bacteriol. 192:2649-2650, 2010) published the complete genome sequence of the plant-associated bacterium Lactococcus lactis subsp. lactis KF147. This sequence reveals the presence of an srtA gene coding for a sortase A protein with 100% identity to that of the organism studied in this work, L. lactis IL1403.

Acknowledgments

We are grateful to Shaynoor Dramsi, Alexandra Gruss, and Oscar Kuipers for helpful and stimulating discussions and for critical reading of the manuscript.

Y.D. was the recipient of a joint grant from ID-DLO, INRA, and Fondation pour la Recherche Médicale (Paris, France). V.O. was the recipient of a doctoral grant from the French government.

Footnotes

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Published ahead of print on 17 September 2010.

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