Molecular Pathogenesis

Mutation of the Maturase Lipoprotein Attenuates the Virulence of Streptococcus equi to a Greater Extent than Does Loss of General Lipoprotein Lipidation

Andrea Hamilton, Carl Robinson, Iain C. Sutcliffe, Josh Slater, Duncan J. Maskell, Nick Davis-Poynter, Ken Smith, Andrew Waller, Dean J. Harrington
DOI: 10.1128/IAI.01116-06

ABSTRACT

Streptococcus equi is the causative agent of strangles, a prevalent and highly contagious disease of horses. Despite the animal suffering and economic burden associated with strangles, little is known about the molecular basis of S. equi virulence. Here we have investigated the contributions of a specific lipoprotein and the general lipoprotein processing pathway to the abilities of S. equi to colonize equine epithelial tissues in vitro and to cause disease in both a mouse model and the natural host in vivo. Colonization of air interface organ cultures after they were inoculated with a mutant strain deficient in the maturase lipoprotein (ΔprtM138-213, with a deletion of nucleotides 138 to 213) was significantly less than that for cultures infected with wild-type S. equi strain 4047 or a mutant strain that was unable to lipidate preprolipoproteins (Δlgt190-685). Moreover, mucus production was significantly greater in both wild-type-infected and Δlgt190-685-infected organ cultures. Both mutants were significantly attenuated compared with the wild-type strain in a mouse model of strangles, although 2 of 30 mice infected with the Δlgt190-685 mutant did still exhibit signs of disease. In contrast, only the ΔprtM138-213 mutant was significantly attenuated in a pony infection study, with 0 of 5 infected ponies exhibiting pathological signs of strangles compared with 4 of 4 infected with the wild-type and 3 of 5 infected with the Δlgt190-685 mutant. We believe that this is the first study to evaluate the contribution of lipoproteins to the virulence of a gram-positive pathogen in its natural host. These data suggest that the PrtM lipoprotein is a potential vaccine candidate, and further investigation of its activity and its substrate(s) are warranted.

The group C streptococcus, Streptococcus equi, is the etiological agent of strangles, one of the most prevalent and important diseases of the horse (52). Nearly 30% of all reported equine infections worldwide may be attributable to this organism (8). Strangles is so called because of the pharyngeal constriction which occurs as a consequence of lymph node swelling (often accompanied by abscess formation) in the upper respiratory tract of the horse following the spread of infection from the nasopharynx. In approximately 5% of cases, systemic spread of the organism leads to abscess formation in other organs, resulting in the usually fatal condition known as “bastard strangles” (44). There is comparatively little information regarding the molecular basis of virulence in S. equi (22). As for other pathogenic streptococci (4, 15, 32, 38), much work has focused on the identification of secreted and surface-located components that may interact with the host (2, 17, 22, 27, 35). A major development in the study of this important veterinary pathogen has been the availability of data from the S. equi genome project (http://www.sanger.ac.uk/Projects/S_equi/).

One major mechanism by which gram-positive bacteria can retain exported proteins within their cell envelopes is lipid modification, which anchors these lipoproteins to the outer face of the plasma membrane (6, 48). Bioinformatic analyses of gram-positive bacterial genomes, including those of Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus agalactiae, have revealed that lipoproteins are a numerically significant feature (ca. 2%) of their predicted proteomes (3, 46, 47, 50, 51). Moreover, the predicted functions of these putative lipoproteins include roles in nutrient acquisition, adherence, protein maturation, and cell signaling. Thus, lipoproteins are predicted to play important roles in the interactions between pathogenic bacteria and their hosts. Three S. equi lipoproteins have been characterized to date: LppC, a lipoprotein acid phosphatase enzyme (21); MBL, a putative metal-binding lipoprotein homologous to pneumococcal PsaA that is predicted to participate in ABC transporter-mediated uptake of manganese (23); and HAP, initially identified as hyaluronate (capsule)-associated protein (10) but which is likely to act as a substrate-binding lipoprotein for ABC transporter-mediated uptake of oligopeptides (22). Bioinformatic analysis of the draft S. equi genome sequence has allowed us to identify at least 32 other putative lipoproteins (I. C. Sutcliffe and D. J. Harrington, unpublished observations), including a homologue (PrtM) of the pneumococcal vaccine candidate PpmA (33). Recently, the signal sequences of five of these putative lipoproteins were recovered in a screen for signal peptides, using a phage display technique (27).

Bacterial lipoproteins are synthesized with distinctive type II signal peptides that direct them into the Sec pathway for protein export (6, 18) and thence into a unique modification pathway which requires a minimum of two specific enzymes. First, prolipoprotein diacylglyceryl transferase (Lgt) transfers a diacylglycerol moiety from membrane phospholipid substrates onto a critical cysteine residue in the cleavage region (“lipobox”) of the type II signal peptide (36, 42). The lipid-modified prolipoprotein is then acted upon by a dedicated lipoprotein signal peptidase (Lsp) which cleaves the signal sequence preceding the thioether-modified cysteine, thereby leaving the lipid anchor unit at the N terminus of the mature lipoprotein (41, 54). These two steps are sufficient for lipoprotein anchoring and appear to be the extent of the pathway in many gram-positive bacteria (46, 47).

This pathway for lipid modification of bacterial proteins is an attractive target for antimicrobial drug development, as both the Lgt and Lsp enzymes are unique to prokaryotes. Likewise, as lipoproteins are likely to play important roles in host colonization and bacterial virulence, individual lipoproteins have potential as targets for novel therapeutic or prophylactic (vaccine) strategies (28). Consistent with this are the findings that, although Lgt is not apparently an essential enzyme during in vitro growth of gram-positive bacteria (12, 29, 34, 39), an lgt mutant strain of S. pneumoniae was attenuated for virulence in a mouse model of infection (34) and the normal functions of Bacillus subtilis lipoproteins in protein secretion, sporulation, and germination processes are impaired in lgt mutants (12, 25, 29, 39). Moreover, an Lgt mutant of Staphylococcus aureus that grew normally in rich media exhibited growth defects in minimal media, consistent with defects in lipoprotein-mediated nutrient uptake systems, notably ABC transporters (45). Similarly, although lsp is dispensable for the growth of gram-positive bacteria in vitro (13, 37, 40, 53, 55), studies using cell culture or animal models of infection show that lsp is required for full virulence of Listeria monocytogenes (37), Mycobacterium tuberculosis (40), and S. aureus (11, 30). In contrast, inactivation of lsp in Streptococcus suis did not appear to lead to an attenuation in virulence in a cocolonization model of piglet infection (13).

We hypothesized that the lipid modification pathway is essential for full virulence of S. equi. As the Lgt enzyme catalyzes the first and committed step on this path, we initially studied the contribution of this enzyme to the physiology and virulence of S. equi. In parallel, we investigated the deletion of a single, specific lipoprotein, namely the putative maturase lipoprotein (PrtM). We believe that this is the first study to evaluate the contribution of lipoproteins to the virulence of a gram-positive bacterial pathogen in its natural host.

MATERIALS AND METHODS

Bacterial strains and culture conditions.S. equi strain 4047 was originally isolated in 1990 from a submandibular abscess of a New Forest pony and has been maintained in the culture collection of the Animal Health Trust, Newmarket, United Kingdom. This strain is the subject of the S. equi genome sequencing project. Escherichia coli TG1 repA+, which allows the stable replication of the plasmid pG+host9 at 37°C, was kindly supplied by Emmanuelle Maguin (Institut Nationale de la Recherche Agronomique, Jouy en Josas, France). S. equi was cultured at 37°C (unless otherwise stated). Liquid cultures were grown in Todd-Hewitt broth (THB) plus 0.2% (wt/vol) yeast extract in an atmosphere containing 5% CO2. Semisolid cultures were grown on Todd-Hewitt agar (THA) or Columbia base agar containing 5% defibrinated horse blood in an atmosphere containing 5% CO2. Mutant S. equi strains containing recombinant plasmids were grown on THA containing erythromycin at 0.5 μg ml−1 (THAE) or in THB containing erythromycin at 1.0 μg ml−. E. coli strains were cultured in Luria-Bertani (LB) broth or agar at 37°C.

Plasmids and primers.The plasmids and primers used in this study are shown in Table 1.

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TABLE 1.

Oligonucleotide primers and plasmids used in this study

Construction of in-frame-deleted lgt and prtM alleles.In order to generate Lgt-deficient and PrtM-deficient mutants of S. equi 4047 by allelic replacement, copies of the S. equi lgt and prtM genes containing in-frame deletions were constructed. The design for the Lgt mutant PCR primers (SELGTAR 1U, 2L, 3U, and 4L) (Table 1) was based upon sequences found within the lgt gene and adjacent sequences. The 22 nucleotides at the 5′ end of primer SELGTAR 3U were designed to complement the SELGTAR 2L primer sequence. PCR using Pfu polymerase (Promega), S. equi 4047 chromosomal DNA, and primers SELGTAR 1U and SELGTAR 2L generated the expected 526-bp DNA fragment. A second PCR with the SELGTAR 3U and SELGTAR 4L primers generated the expected 496-bp DNA fragment. The PCR products from each reaction were diluted, mixed, and allowed to anneal via their overlapping, complementary ends. A third PCR was then carried out with these annealed DNA fragments as a template and with primers SELGTAR 1U and SELGTAR 4L, again using the Pfu polymerase. The product of this reaction was a DNA fragment of 1,022 bp containing the 5′ 189 base pairs and 3′ 96 base pairs (plus upstream and downstream sequences) but lacking the central 495 bp of the lgt gene. The fragment was digested with the restriction endonucleases ApaI and SacII and cloned into the corresponding restriction sites of the pG+Host9 vector to give the recombinant plasmid pAH08. To generate a PrtM mutant, a copy of the prtM gene that lacked bases 411 to 639, which include the sequence encoding most of the parvulin-like domain of the protein, was constructed. Sequences flanking the deletion were generated by PCR using Vent DNA polymerase (New England Biolabs) with the primer pairs 5′PRTM/PRTM-NDEL and 3′PRTM/PRTM-CDEL (Table 1). The corresponding 342-bp and 376-bp PCR products were then digested with the restriction endonucleases EcoRI and EcoRV (5′ product) and SalI and EcoRV (3′ product), and the digested products were ligated into the EcoRI- and SalI-digested pG+Host9:ISS1 plasmid in a three-way ligation to form the deletion construct pGprtMΔ. Engineering of an EcoRV site into primers as part of the cloning strategy results in the introduction of a non-prtM DNA sequence encoding the amino acids aspartic acid and isoleucine at the site of the deletion. Plasmids pAH08 and pGprtMΔ were transformed into E. coli TG1repA+, and transformants were selected at 37°C on LB plates containing erythromycin (150 μg ml−1).

Allelic-replacement mutagenesis.Transformation of the encapsulated S. equi strain 4047 with plasmids pAH08 and pGprtMΔ was achieved using a modification of the method described by Simon and Ferretti (43). Briefly, an overnight culture of S. equi 4047 grown in THB containing hyaluronidase (30 μg ml−1) was diluted 20-fold in 200 ml of the same medium and grown to an optical density at 595 nm of 0.125. Bacterial cells were harvested by centrifugation and washed three times in 10-ml volumes of ice-cold 0.5 M sucrose. After the final wash, the pellet was resuspended in 1 ml of ice-cold 0.5 M sucrose, and 100-μl aliquots of the competent cells were used in transformation reactions. The transformation reactions were performed with 1 to 5 μg plasmid DNA using a Gene Pulser electroporater (Bio-Rad, United Kingdom) with pulse settings of 2.5 kV cm−1, 200Ω, and 25 μF, typically giving a pulse time of 4 to 6 ms. Ice-cold THB was added to the transformed cells, which were then incubated at 37°C for 3 h to allow cell recovery. Transformants were selected by plating serial dilutions of the cells on THAE, followed by incubating them overnight at 28°C (the permissive temperature) to allow plasmid replication.

To replace the wild-type lgt and prtM genes with their respective in-frame-deleted alleles, transformants containing either pAH08 or pGprtMΔ were subjected to two rounds of homologous recombination as previously described by Biswas et al. (5). The first recombination event, leading to the integration of pAH08 or pGprtMΔ into the strain 4047 chromosome, was achieved by growing transformants at 28°C overnight and then increasing the temperature to 37°C for 3 h. Integrants were selected following growth on THAE overnight at 37°C. The integrants were then inoculated into THBE and grown at 37°C overnight, followed by dilution into THB and incubation at 28°C for a further 48 h. Incubation at the permissive temperature allows plasmid replication and facilitates the second recombination event. The bacteria were plated on THA and grown at 37°C to ensure excision of the free plasmid. Putative mutant colonies were subcultured onto fresh THA and THAE plates to confirm their erythromycin sensitivity. The presence of the mutant allele in the chromosome of putative mutants was determined by PCR, using the primers SELGTAR 1U and SELGTAR 4L for the lgt mutants and the 5′PRTM and 3′PRTM primers for the prtM mutants. PCR products, representing the deletion derivatives of each allele, were generated using proofreading DNA polymerases, and the predicted deletions were confirmed by DNA sequencing. DNA sequencing was performed by the University of Newcastle Central Facility for Molecular Biology using an ABI Prism 377 DNA sequencer or at the Animal Health Trust using an ABI3100 DNA sequencer with BigDye fluorescent terminators. A representative mutant strain for each deleted allele was chosen for subsequent studies and designated Δlgt190-685 or ΔprtM138-213.

Analysis of the presence and localization of lipoproteins.Lack of Lgt activity in the Δlgt190-685 mutant was confirmed by radiolabeling lipoproteins. Radiolabeling of S. equi lipoproteins was performed as previously described by Sutcliffe et al. (49).

In order to demonstrate the presence of surface-located lipoproteins in the S. equi 4047 and S. equi Δlgt190-685 strains, transmission electron microscopy was performed as described by Dixon et al. (14). Western blotting was used to indicate the presence of lipoproteins in either cell extract or secreted protein profiles. The preparation of bacterial cell extracts, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Western blotting were all performed as previously described (21). SDS-solubilized cell extracts contained a mixture of both soluble and membrane-associated proteins. The primary anti-LppC antibody used in this study was kindly provided by Horst Malke and used at the recommended dilution. Cell-associated and supernatant acid phosphatase activities of the wild-type and mutant strains were determined spectrophotometrically as previously described (21).

Investigation of the virulence of S. equi mutants in an in vitro colonization model.Air interface respiratory organ cultures were constructed using equine upper respiratory tract tissues (nasal turbinate, guttural pouch, and trachea) and methods described for human (26) and canine (1) tissues with some modifications. Tissues were obtained from an abattoir and washed in Dulbecco's modified Eagle's medium (DMEM) containing antibiotics (penicillin, 100 U ml−1; streptomycin, 50 μg ml−1; gentamicin, 100 μg ml−1; amphotericin, 2.5 μg ml−1) for 4 h to remove commensal flora. Following further washing in DMEM to remove residual antibiotics and amphotericin, the tissues were dissected into pieces of approximately 5 mm2 and mounted at an air interface on agarose platforms surrounded by 2.5 ml DMEM in 6-well cell culture plates. Organ cultures were maintained in a humidified 5% CO2 incubator. Viability of the air interface organ cultures was assessed using polystyrene bead clearance (1). Contamination was monitored by running a bacteriology loop around all four edges of the culture pieces and streaking onto horse blood agar plates. Any tissue pieces in which contamination was detected were discarded. Organ culture pieces were infected with a 10-μl suspension containing 1 × 105 CFU of wild-type S. equi 4047, Δlgt190-685, or ΔprtM138-213 or were mock-infected with THB. Colonization of organ culture pieces was assessed by measuring viable counts (six organ culture pieces per time point) of adherent bacteria at 4 h and 24 h postinfection (p.i.). Organ culture pieces were vortexed for 15 s in phosphate-buffered saline to remove nonadherent bacteria and were then homogenized before being plated for 10-fold serial dilutions onto THA for enumeration of the colonies. Changes in the surface features of organ culture pieces (two per time point) in response to infection with wild-type S. equi or the two mutants at 24 h p.i. were assessed by morphometric analysis of scanning electron microscopy (SEM) images of the epithelial surface. The tissues were processed and surface morphometry was carried out using standard methods (26). The percentage of the epithelial surface covered with mucus was recorded. The data represent the means and standard deviations of the results of six independent experiments using tissues from different horses. Differences in colonization and surface morphometry data were tested for statistical significance using Mann-Whitney U tests and are reported at the 5% level.

Investigation of the virulence of S. equi mutants in a mouse model of strangles.Mice were challenged intranasally as described by Chanter et al. (9). Briefly, 30 3- to 4-week-old female BALB/c mice were challenged with 4 × 106 CFU of fresh cultures of wild-type 4047, ΔprtM138-213, or Δlgt190-685 S. equi strains, and clinical signs of disease, including weight loss and sneezing, were compared with those of a group of 10 unchallenged controls over a period of 5 days. At the end of this period, mice were euthanized and examined for signs of S. equi infection (measured as viable S. equi cell counts) and pathology by histological examination of lymph nodes and tissues of the head and neck. The extent of pathology in each mouse was then graded on the basis of pathological features most pertinent to S. equi infection, using the following scoring system: lymphadenitis, 1; lymph node abscess, 5; rhinitis, 1; marked rhinitis, 5; pharyngitis, 3; meningitis, 5; otitis media, 3; lung lesions, 5; and splenic lesions, 5.

Investigation of the virulence of S. equi mutants in a pony challenge study.Groups of five naïve, male yearling Welsh Mountain ponies were challenged with either ΔprtM138-213 or Δlgt190-685, and a similar control group of four male ponies were challenged with S. equi 4047. Each group was housed separately throughout the challenge period with strict infection control measures in place to ensure there was no cross-contamination between the groups.

Fresh cultures of each strain were grown in THB supplemented with 10% fetal calf serum (THB10) at 37°C with 5% CO2 to an optical density at 600 nm of 0.3. Previous studies have shown that this density of bacteria corresponds to approximately 2 × 108 CFU ml−1 of S. equi 4047 (unpublished observations). At this point, the cultures were diluted 1:8 in fresh prewarmed and pregassed THB10, and 2 ml of challenge inoculum was administered via both nostrils, using a flexible tube and spray nozzle, in order to administer approximately 1 × 108 CFU/pony. Clinical signs of disease, including fever, swelling of the lymph nodes, and nasal discharge were monitored daily for up to 3 weeks. The ponies were considered to be pyrexic when their temperatures exceeded 39.0°C. Clinical scores were calculated according on the scoring system presented in Table 2. Blood samples were collected to enable monitoring of the neutrophil levels present in the challenged ponies. Normally, these range from 3 to 6.5 × 106 ml−1 in healthy ponies but frequently exceed 1 × 107 ml−1 during S. equi infection. At the end of the study period, all of the ponies were euthanized and the extent of their disease was quantified on postmortem examination, using the following scoring system: abscess in a lymph node, 15; microabscess in a lymph node, 10; enlarged lymph node, 1; empyema of the guttural pouch, 5; follicular hyperplasia of the guttural pouch, 1. Samples of lesions at postmortem were used to reisolate the challenge organisms in order to confirm their identity by PCR of the lgt and prtM genes.

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TABLE 2.

Scoring system used to quantify disease burden in ponies

Animal ethics.These studies were performed under a Home Office project license after ethical review and following strict welfare guidelines.

RESULTS

Identification of the lgt gene and construction of an Lgt-deficient allelic-replacement mutant.Our initial studies allowed the amplification of a 261-bp internal fragment of the S. equi 4047 lgt gene (GenBank accession number AJ403973), using degenerate primers based upon conserved amino acid sequences in the Lgt proteins of S. mutans, S. pneumoniae, and S. pyogenes. The sequence was completed by subsequent PCR experiments and verified by reference to an early release of the S. equi 4047 genome project. Putative promoter and ribosome binding site sequences were identified upstream of the lgt gene, which is located downstream of the hprK gene as in several other gram-positive bacteria (7, 24). The lgt gene of S. equi 4047 encodes a 259-amino-acid protein with a molecular mass of approximately 29.8 kDa. The derived protein sequence contains the Lgt Prosite motif G-R-X-[GA]-N-F-[LIVMF]-N-X-E-X(2)-G (PS01311/PDOC01015) and matches the Pfam profile (PF01790) for Lgt. An overlap-deletion PCR strategy was used to create a mutant lgt allele with a 495-bp in-frame deletion which removed this Prosite motif and was thus predicted to produce a nonfunctional Lgt enzyme. Replacement of the wild-type allele with the in-frame deletion derivative in S. equi Δlgt190-685 was confirmed by PCR and sequencing.

Radiolabeling of lipoproteins in S. equi 4047 and S. equi Δlgt190-685.To confirm the absence of Lgt activity in the allelic-replacement mutant, S. equi 4047 and S. equi Δlgt190-685 were grown in the presence of [14C]palmitate. Palmitate is incorporated into endogenous membrane lipids which are used as the substrate for lipid modification of preprolipoproteins by Lgt, thereby resulting in the radiolabeling of mature lipoproteins. Electrophoresis of cell extract proteins of the parent strain 4047 revealed the presence of at least 10 distinct, radiolabeled lipoproteins following autoradiography (Fig. 1, lane 1). In contrast, there was an absence of labeled protein bands in equivalent cell extracts of the mutant strain (Fig. 1, lane 2). Intensive labeling at the bottom of each lane indicated comparable incorporation of the labeled palmitate into bacterial lipids (Fig. 1). This result confirmed the absence of functional Lgt activity in the mutant.

FIG. 1.
FIG. 1.

Labeling of lipoproteins from S. equi 4047 and the Δlgt190-685 mutant with [14C]palmitic acid. SDS extracts of cells grown in the presence of radiolabeled [14C]palmitic acid were separated by SDS-PAGE. The dried gel was exposed to X-ray film for 24 h before being developed. Lane 1, S. equi 4047 extract; lane 2, S. equi Δlgt190-685 extract. The positions of molecular mass standards (in kDa) are shown on the left.

Investigation of the effect of the Lgt mutation on the processing of a known S. equi lipoprotein.In order to determine the effect of the Lgt mutation on the processing of an individual lipoprotein, the presence of the S. equi LppC acid phosphatase (21) was investigated in the wild-type and mutant strains by Western blot analysis. As expected, a single cross-reacting band representing the mature form of LppC was seen in cell extracts of the parent 4047 strain probed with an antibody to the Streptococcus dysgalactiae subsp. equisimilis acid phosphatase LppC (Fig. 2A, lane 2). When a cell extract of S. equi 4047 which had been treated with globomycin was analyzed, a second cross-reacting band representing the prolipoprotein-LppC (pro-LppC) form of the protein was seen (Fig. 2A, lane 1). The appearance of this additional, higher-molecular-weight band is consistent with the inhibition of lipoprotein signal peptidase II by globomycin (21). A cross-reacting doublet was seen in cell extracts of S. equi Δlgt190-685 (Fig. 2A, lane 3), although the cross-reacting bands were considerably less intense for this strain, despite a total protein load equivalent to that of the wild type. Moreover, neither of the bands corresponded in molecular weight with the pro-LppC form seen in the globomycin-treated culture, suggesting that the preprolipoprotein-LppC (prepro-LppC) form of the protein, which is unlipidated but retains its signal peptide, migrates faster than the pro-LppC form. The lower amount of cell-associated LppC observed for S. equi Δlgt190-685 could be explained by a reduced retention of prepro-LppC in the cell membrane as a consequence of the inability of the mutant strain to modify this protein with lipid. Consequently, we investigated the release of unlipidated LppC by performing Western blotting on concentrated culture supernatants obtained from the cultures from which the cell extracts had been derived. There was a minor but detectable cross-reacting protein in the supernatant of S. equi 4047 but not in the supernatant of S. equi Δlgt190-685 (data not shown). It was also noticeable that the band detected in the supernatant of S. equi 4047 was smaller than the mature form of the protein seen in cell extracts of the same strain, suggesting that a proportion of the membrane-anchored LppC is released by proteolytic processing in the parent strain. Whole-cell acid phosphatase assays were also performed for each strain. As previously observed for S. equi strain 9682 (21), a peak of acid phosphatase activity at a pH optimum of 5 was readily detectable for S. equi 4047, but this activity was significantly reduced in the mutant strain S. equi Δlgt190-685 (Fig. 2B). However, acid phosphatase activity was undetectable in the culture supernatants of both strains (data not shown), suggesting that the protein detected in Western blots of S. equi 4047 culture supernatants is probably not active. Further confirmation of a reduced level of LppC in the cell envelope of the mutant compared to that of the wild type came from LppC-specific immunogold-labeling experiments. Single cocci of S. equi 4047 and Δlgt190-685 (n = 10 for each) were labeled with 234 ± 20 and 54 ± 20 gold particles, respectively. Cumulatively, these data suggested that there was a significant defect in LppC localization within the cell envelope of S. equi Δlgt190-685 compared to that of the parent strain.

FIG. 2.
FIG. 2.

Changes in the nature (A) and the activity (B) of an acid phosphatase (LppC) in the S. equi Δlgt190-685 mutant. (A) Proteins in SDS extracts prepared from cells of the parent (4047) and mutant strain (Δlgt190-685) were separated by SDS-PAGE and transferred to nitrocellulose. Immunoblotting was carried out using a polyclonal antibody raised to the LppC acid phosphatase of S. equisimilis. Lane 1, globomycin-treated S. equi 4047; lane 2, S. equi 4047; lane 3, S. equi Δlgt190-685. (B) Whole-cell acid phosphatase activity was determined for strain 4047 (⧫) and the Δlgt190-685 mutant (▪) across a range of pH values by spectrophotometric changes associated with the release of p-nitrophenol from the substrate p-nitrophenol phosphate. Results are representative of three different experiments.

Construction of a PrtM-deficient allelic-replacement mutant.The S. equi Δlgt190-685 mutant had been shown to be defective in the processing of lipoproteins generally (Fig. 1). To gain further insight into the significance of specific lipoproteins in S. equi, we created a S. equi mutant strain defective in the function of the putative maturase lipoprotein PrtM. The PrtM sequence was identified from the S. equi genome project and, in addition to its homology to pneumococcal PpmA (33), it exhibits significant homologies to the maturase proteins of other gram-positive bacteria (16, 19, 56, 57). This family of sequences belongs to the parvulin family of PpiC-type peptidyl-prolyl cis-trans isomerases (PPIase). A S. equi mutant (ΔprtM138-213) was constructed with an in-frame internal deletion in the prtM coding sequence corresponding to the central (parvulin-like) PPIase domain (57). This mutant is predicted to synthesize a nonfunctional PrtM protein, although the absence of an in vitro assay for PrtM function precludes experimental confirmation of this. Growth of both the S. equi Δlgt190-685 and the ΔprtM138-213 mutants in nutrient-rich broth was comparable to that of wild-type S. equi (data not shown).

Colonization of air interface organ cultures by S. equi strains.Following inoculation of nasal turbinate, guttural pouch, and tracheal organ culture pieces with 1 × 105 CFU wild-type S. equi 4047 or the two mutants, all three strains were recovered from all three tissues at 4 h p.i (Fig. 3A). At 24 h p.i., wild-type S. equi and Δlgt190-685 were again recovered, but ΔprtM138-213 was not detected. Wild-type bacteria were recovered in statistically significantly higher numbers at both 4 h and 24 h p.i. from nasal turbinate (3.8 ± 0.35; 3.4 ± 0.55) and guttural pouch (4.0 ± 0.60; 4.2 ± 0.70) cultures than from tracheal cultures (2.8 ± 0.25; 1.3 ± 0.80). The numbers of Δlgt190-685 cells recovered at 4 h and 24 h p.i. from turbinate (3.9 ± 0.50; 2.8 ± 0.70), guttural pouch (3.2 ± 0.45; 3.62 ± 0.80), and tracheal (2.4 ± 0.45; 1.1 ± 0.60) cultures were not significantly different from those of wild-type S. equi. However, there were significantly fewer ΔprtM138-213 cells than both wild-type S. equi and Δlgt190-685 cells recovered at 4 h and 24 h p.i. from nasal turbinate (1.8 ± 0.30; <0.7 ± 0), guttural pouch (1.4 ± 0.45; <0.7 ± 0), and tracheal (0.7 ± 0.50) cultures.

FIG. 3.
FIG. 3.

Colonization and morphometric analysis of air interface organ cultures infected with S. equi strains. (A) Recovery of viable bacteria 4 h and 24 h postinfection of nasal turbinate, guttural pouch, and tracheal air interface organ cultures with 1 x 105 CFU wild-type S. equi or the mutant Δlgt190-685 or ΔprtM138-213. The data bars show the mean viable counts (± the standard deviation [SD]) from six independent experiments. (B) Surface morphometric analysis of nasal turbinate, guttural pouch, and tracheal air interface organ cultures 24 h after infection with 5 log10 CFU wild-type S. equi or the mutant Δlgt190-685 or ΔprtM138-213. The data bars show the mean percent surface coverage by mucus (± SD) from six independent experiments.

Changes in surface epithelial morphology of air interface organ cultures exposed to S. equi strains.The surface morphology of uninfected organ culture pieces from nasal turbinate, guttural pouch, and trachea was predominantly ciliated epithelium. The guttural pouch and tracheal cultures were densely and uniformly ciliated, whereas nasal turbinate tissue exhibited a mixture of ciliated and nonciliated epithelial cells. For all three tissues after 24 h in culture, a small percentage of the total epithelial surface area was covered with mucus (Fig. 3B) and the amount of surface coverage in the uninfected control pieces was not significantly different from that at the start of the experiment. Wild-type S. equi induced a marked mucus response which resulted in a significantly greater proportion of the epithelial surface being covered by mucus in all three tissues (nasal turbinate, 86 ± 18%; guttural pouch, 95 ± 22%; trachea, 90 ± 14%). The mucus formed a dense layer that obscured the underlying ciliated epithelium (Fig. 4B). Inoculation of Δlgt190-685 also induced a mucus response at 24 h p.i. in all three tissues. The amount of mucus coverage of the epithelial surface was significantly greater (nasal turbinate, 75 ± 12%; guttural pouch, 86 ± 16%; trachea, 82 ± 20%) than that for the uninfected control pieces but was not significantly different from that of pieces infected with wild-type S. equi (Fig. 3B). Qualitatively, the mucus layer produced appeared less dense than that produced in response to wild-type S. equi (Fig. 4C). In contrast to infection with both wild-type S. equi and Δlgt190-685, inoculation with ΔprtM138-213 did not result in a significant increase in mucus production compared to that for the uninfected control pieces (Fig. 3B), with the result that the ciliated epithelial surface was not obscured by mucus (Fig. 4D).

FIG. 4.
FIG. 4.

Morphology of air interface organ cultures exposed to S. equi strains. Representative SEM micrographs of uninfected nasal turbinate organ culture pieces or pieces infected with 1 × 105 CFU wild-type S. equi or one of the two mutants after 24 h in culture. (A) Uninfected control; (B) wild-type S. equi 4047; (C) Δlgt190-685; (D) ΔprtM138-213. All images are shown at ×2,000 magnification. Scale bars, 10 μm.

Virulence of S. equi mutants in a mouse model of strangles.The virulence of the mutants in a mouse intranasal infection model of strangles was determined (9). As expected, approximately 60% of mice challenged with the parent 4047 strain lost or failed to gain weight over the 5-day study period, indicative of S. equi infection (Fig. 5A). S. equi 4047 also induced sneezing from 3 days postchallenge (Fig. 5B) and induced significant levels of disease in mice, as determined by postmortem examination 5 days postchallenge (Fig. 5C and D). Deletion of either the lgt or prtM gene significantly attenuated S. equi on intranasal challenge of mice as measured by weight gain, sneezing rate, pathological score, and the overall incidence of disease (Fig. 5A to D). Mice challenged with either Δlgt190-685 or ΔprtM138-213 generally continued to gain weight in line with the mock-challenged controls. However, 2 of 30 mice challenged with the Δlgt190-685 strain had reduced weight gain compared with that of the unchallenged controls. Two mice challenged with Δlgt190-685, including one of the mice that had failed to gain weight, also had histological disease on postmortem examination (Fig. 5D).

FIG. 5.
FIG. 5.

Challenge of mice with the Δlgt190-685 and ΔprtM138-213 deletion strains. (A) The mean percent increase in weight per mouse was calculated for each of the challenge groups. Mice succumbing to infection with wild-type S. equi (n = 30) lost or failed to gain weight in comparison to the uninfected controls (n = 10). Groups of 30 mice challenged with the Δlgt190-685 (lgt) or ΔprtM138-213 (prtM) mutant continued to gain weight during the course of the study. Error bars indicate the standard error of the mean. * indicates a statistical significance of P < 0.05 compared with results for wild type-infected ponies. (B) The mean number of sneezes in 2 min for groups of five cohoused mice was calculated for each of the challenge groups. Mice infected with the parental S. equi 4047 strain had a significantly elevated sneezing rate compared with that of the uninfected and Δlgt190-685- and ΔprtM138-213-challenged groups. Error bars indicate the standard error of the mean. * indicates a statistical significance of P < 0.05 compared with results for wild-type-infected ponies. (C) The extent of disease on histological examination of mice was quantified according to the scoring system outlined in Materials and Methods. The mean total score per mouse was calculated. Error bars indicate the standard error of the mean. * indicates a statistical significance of P < 0.05 compared with results for wild type-infected ponies. (D) The number of mice with histological signs of disease attributable to S. equi infection following postmortem examination was compared to the number without histological signs of disease by Fisher's exact test to determine if deletion of the lgt or prtM genes significantly attenuated S. equi in the mouse infection model.

Virulence of S. equi mutants in a pony challenge study.The parent strain and both mutants were assayed for virulence in Welsh Mountain ponies. The early clinical signs of strangles disease such as pyrexia, nasal discharge, and swelling of the submandibular lymph nodes were apparent from day 2 postchallenge in 3 of 4 ponies challenged with the parental strain 4047 and from day 4 in 3 of 5 ponies challenged with the Δlgt190-685 deletion mutant (Fig. 6A -C). In contrast, there were no signs of disease observed in ponies challenged with the ΔprtM138-213 deletion strain throughout the 17-day study period (Fig. 6A to C). There was a rise in mean rectal temperature from day 4 postchallenge in the ponies challenged with wild-type 4047 compared to that for those challenged with either Δlgt190-685 or ΔprtM138-213 (Fig. 6A). Moreover, pyrexia (a temperature exceeding 39.0°C) was evident in 3 of 4 of the 4047-challenged group compared with 1 of 5 of the Δlgt190-685-challenged group and 0 of 5 of the ΔprtM138-213-challenged group (Fisher's exact test, P = 0.048) (Fig. 6B). Other clinical signs were also significantly reduced in ΔprtM138-213-challenged ponies compared with those in the wild type-challenged group (Kruskal-Wallis test, P = 0.0267) (Fig. 6C). There was no significant difference in the mean clinical scores of the Δlgt190-685-challenged group compared to those for the 4047-challenged ponies (Fig. 6C). Similarly, whereas neutrophilia (>6.5 × 106 ml−1) was observed by day 17 in both the wild type- and Δlgt190-685-challenged groups, neutrophil levels in ΔprtM138-213-challenged ponies remained stable (Fig. 6D).

FIG. 6.
FIG. 6.

Effect of intranasal challenge of ponies on rectal temperature, clinical scores, and neutrophil levels. (A) The rectal temperatures of the ponies were taken daily from the day before challenge to day 17 postchallenge, and the mean temperature per pony for each challenge group is shown. (B) The numbers of ponies in each group suffering from pyrexia were compared by Fisher's exact test. Ponies were considered pyrexic when their temperatures exceeded 39°C. Only ponies challenged with the ΔprtM138-213 strain had a significantly reduced incidence of pyrexia (P = 0.048). (C) The mean clinical score for each challenge group was calculated according to the scoring system presented in Table 2. Comparison of the total clinical score per pony over the study period using the Kruskal-Wallis test indicated that only the ΔprtM138-213 deletion strain (prtM) was significantly attenuated (P = 0.0267). (D) The mean number of neutrophils per milliliter of blood was quantified for each pony. Ponies developed signs of neutrophilia (neutrophil count of > 6.5 × 106 ml−1) 6 days postchallenge with the parental 4047 strain, whereas ponies challenged with the Δlgt190-685 strain (lgt) developed neutrophilia 17 days postchallenge, and no signs of neutrophilia were observed in ponies challenged with the ΔprtM138-213 strain (prtM; P < 0.05). Error bars indicate the standard error of the mean. * indicates a statistical significance of P < 0.05 compared with results for wild-type-infected ponies.

On postmortem examination, lymph node abscesses were apparent in all 4 ponies challenged with the parental 4047 strain, 3 of 5 ponies (P = 0.44) challenged with the Δlgt190-685 strain, and 0 of 5 ponies (P = 0.008) challenged with the ΔprtM138-213 strain (Fig. 7A). The mean pathological scores determined at postmortem were very similar for the 4047 and Δlgt190-685 groups, whereas the low score obtained for the ΔprtM138-213 group reflected low-grade pathology not indicative of strangles (Fig. 7B). S. equi was isolated from the abscesses of ponies in the 4047 and Δlgt190-685 groups in high yields (in excess of 109 CFU/ml of pus), and these isolates were confirmed by PCR to have the full-length or truncated lgt gene, respectively, thus confirming the source of infection and in vivo stability of the lgt deletion. No S. equi was reisolated from any of the ΔprtM138-213-challenged ponies on postmortem examination, suggesting that this strain was not able to persist in vivo for the 3-week duration of this study.

FIG. 7.
FIG. 7.

Effect of intranasal challenge of ponies on the disease identified on postmortem examination. (A) The numbers of ponies in each group with significant pathological signs of strangles attributable to infection with S. equi on postmortem examination were compared using Fisher's exact test. Although 2 of 5 ponies challenged with the Δlgt190-685 strain had no significant signs of disease, this was not statistically significant (P = 0.44). However, ponies challenged with the ΔprtM138-213 strain did have significantly reduced disease on postmortem examination (P = 0.008). (B) The mean pathology score per pony was calculated for each of the challenge groups on postmortem examination using the scoring system outlined in Materials and Methods. Error bars indicate the standard error of the mean. * indicates a statistical significance of P < 0.05 compared with results for wild-type-infected ponies.

DISCUSSION

Comparatively little is known about the molecular basis of S. equi virulence (22). Greater understanding should identify rational candidates for the development of an effective vaccine. It is thought that prevention of strangles is likely to be the only effective mechanism for combating the disease, as the lack of vascularity associated with abscess formation prevents the delivery of effective doses of antibiotics and other drugs to the sites of infection.

Bacterial lipoproteins are attractive as potential vaccine candidates, since they may be exposed on the bacterial surface and thus may be potentially accessible to host immune molecules (28, 51). Furthermore, a wide variety of functions has been attributed to bacterial lipoproteins, at least some of which are likely to be crucial to bacterial colonization and survival within the host (46-48). Thus, immune responses directed at such proteins may be highly opsonic and may also block the activity of essential proteins. In the present study, we investigated the contribution of lipoproteins to S. equi physiology and virulence. To this end, we characterized the consequences of both disrupting the lipoprotein biosynthetic pathway per se and inactivating a specific lipoprotein, PrtM.

In order to construct a lipoprotein-deficient mutant of S. equi 4047, the wild-type lgt gene was replaced by a mutant allele that contained a 495-bp internal deletion that removed the central portion of Lgt, including the highly conserved sequence motif that defines the Lgt family of enzymes. An allelic-replacement strategy was chosen so as to minimize the potential for polar effects due to the mutation. The successful construction of an Lgt-deficient mutant (S. equi Δlgt190-685) was confirmed by PCR and by palmitate radiolabeling, which clearly showed that the mutant strain lacked labeled lipoproteins (at least 10 of which could be seen in the parent strain). That we could generate a viable, Lgt-deficient mutant indicates that the absence of Lgt in S. equi is not lethal, a finding also observed for other gram-positive organisms such as B. subtilis (12, 29, 39), S. pneumoniae (34), and S. aureus (45).

The failure to transfer a lipid moiety to preprolipoproteins due to Lgt inactivation would likely result in either the accumulation of signal peptide-anchored preprolipoproteins in the cell membrane or the release of the lipoprotein derivatives into the culture medium, due to either shedding or signal peptide processing at “cryptic” signal peptidase I processing sites (“shaving”). In order to determine which of these possibilities did indeed occur for individual lipoproteins in the S. equi Δlgt190-685 strain, the localization of a proven lipoprotein was investigated by Western blot analysis. While it was clear that there were reductions in the levels of LppC acid phosphatase in cell extracts of the S. equi Δlgt190-685 mutant, there was no corresponding increase in the amount of this protein in culture supernatants. It appears, therefore, that the failure of this protein to become lipid modified may result in its degradation, either before or during secretion, although it remains possible that expression of this protein is altered in the S. equi Δlgt190-685 mutant.

Despite the demonstration, by palmitate labeling, that the S. equi Δlgt190-685 mutant contained no detectable lipoproteins, a protein cross-reacting with the anti-LppC antibody was present in cell extracts of the S. equi Δlgt190-685 mutant, albeit at significantly lower levels than in the parent strain. The cross-reacting band also appeared to have a molecular weight very similar to that of the wild-type (mature) lipoprotein. The most likely explanation for this observation is that the LppC protein seen in S. equi Δlgt190-685 is not lipidated but that the LppC preprolipoprotein is proteolytically modified, yielding a product similar in size to the mature acid phosphatase in the parent strain. Comparable “mature-like lipoproteins” have been reported previously for B. subtilis, L. monocytogenes, and S. suis mutants lacking the Lsp signal peptidase II (13, 37, 53). Most significantly, differential processing of lipoprotein precursors has been observed in an lsp mutant strain of M. tuberculosis: whereas the mutant accumulated both prolipoprotein and a “mature-like” form of a 19-kDa lipoprotein, only the prolipoprotein forms of a 27-kDa and the MPT83 lipoproteins accumulated (40).

Although the phenotype of S. equi Δlgt190-685 did not correspond with that originally predicted (i.e., preprolipoprotein accumulation), convincing autoradiographic assays, Western blotting analysis, acid phosphatase enzyme assays, and immunolocalization data all confirmed a significant defect in lipoprotein processing in this strain.

We intended to investigate the virulence of the S. equi Δlgt190-685 mutant strain in a variety of in vitro and in vivo models of colonization and disease and also decided to generate a mutant that was deficient in a single specific lipoprotein. For the purpose of this study we chose the putative maturase lipoprotein PrtM, one of four lipoproteins released in large amounts by an Lgt mutant strain of S. aureus (45).

Air interface respiratory organ cultures of nasal turbinate, guttural pouch, and tracheal tissues were used to compare the abilities of wild-type S. equi, the Δlgt190-685 mutant strain, and a PrtM-deficient mutant strain (ΔprtM138-213) to colonize tissues derived from a variety of anatomical sites within the equine upper respiratory tract (URT) and to assess the response of URT tissues to infection. Previous studies have shown that culturing respiratory tract tissues at an air interface provides a more physiological infection environment for bacterial pathogens than submerged culture systems (31, 59). Viable and contamination-free tissues from all three regions were successfully maintained for the duration of the experiment. Colonization was assessed by measuring the numbers of bacteria adherent to tissues at 4 h and 24 h p.i. Wild-type S. equi cells were present in higher numbers on nasal turbinate and guttural pouch tissues than on tracheal tissues, suggesting less-efficient colonization of the trachea. The numbers of cells of the Δlgt190-685 mutant strain adherent to organ culture pieces were not significantly different from those of wild-type S. equi, suggesting that this mutant was capable of colonizing nasal turbinate, guttural pouch, and tracheal tissues as efficiently as wild-type bacteria. In contrast, however, cells of the ΔprtM138-213 mutant were present in significantly reduced numbers compared to those for wild-type S. equi in all three tissue sites at both time points measured. These data suggest that the ΔprtM138-213 mutant strain has impaired ability to colonize the equine URT.

The response of the equine URT to infection with wild-type S. equi and the two mutants was assessed by SEM morphometric analysis of the epithelial surface. Uninfected cultures from all three anatomical regions had a small surface area covered by mucus. Wild-type S. equi induced a marked mucus response that obscured the epithelial surface. It was not possible to assess the underlying epithelium by SEM analysis, although light microscopy suggested that this was intact, and furthermore, organ culture pieces infected with the wild-type bacteria continued to clear beads despite the production of mucus (data not shown). This extensive mucus response has been noted for other bacterial pathogens, using air interface organ cultures of human (59) and canine (1) origin. The mutant Δlgt190-685 strain also induced a marked mucus response that was not different from that of wild-type S. equi. Although the amount of mucus produced in these experiments was not quantifiable, the depth of the mucus layer induced by Δlgt190-685 appeared thinner than that produced by wild-type infection because the epithelial surface could be discerned beneath. In contrast, ΔprtM138-213 did not induce a mucus response, and organ culture pieces infected with this mutant were indistinguishable from the uninfected control pieces in this regard.

Taken together, the organ culture colonization and tissue response data suggest that colonization of the URT (and/or possibly production of soluble factors by the bacteria) is required to induce a mucus response. Wild-type S. equi and Δlgt190-685 colonized the epithelium to a similar extent and induced a similar mucus response, whereas ΔprtM138-213 was less able to colonize, was cleared from all tissue sites within 24 h p.i., and did not induce a mucus response. Since colonization is the first step in pathogenesis, these in vitro data suggest that ΔprtM138-213 is likely to have reduced virulence in the natural host. However, note that persistence and abscess formation in the horse would require evasion of the immune response, which was not assessed in the air interface organ culture models.

In the mouse S. equi model (9), challenge with the parental S. equi 4047 strain induced disease in 57% (17 of 30) of mice during the 5-day study period, as determined by changes in weight gain, rate of sneezing, and histopathological analysis. The deletion of the prtM gene significantly attenuated S. equi in the mouse model of S. equi infection (P < 0.001). None of the mice challenged with ΔprtM138-213 showed signs of disease (either reduced weight gain or sneezing) throughout the study period, and no disease was detected histologically. The Δlgt190-685 strain was also significantly attenuated in the mouse S. equi infection model (P < 0.001). However, 3 of 30 mice challenged with Δlgt190-685 had histological signs of disease and/or reduced weight gain compared to that of the unchallenged controls, indicating that this strain is not completely avirulent in mice.

Investigation of the in vivo virulence of the Δlgt190-685 and ΔprtM138-213 deletion strains was determined in the natural host via intranasal challenge of Welsh Mountain ponies. Ponies challenged with the parental S. equi 4047 strain developed obvious signs of strangles during the 17-day study period, as determined by pyrexia (3 of 4 ponies), clinical observations (4 of 4 ponies), and postmortem examination (4 of 4 ponies). The ΔprtM138-213 strain was significantly attenuated for each of these parameters and did not induce signs of pyrexia (0 of 5 ponies; P = 0.048), significant clinical signs of disease (0 of 5 ponies; P = 0.027), or obvious signs of disease on postmortem examination (0 of 5 ponies; P = 0.008) during the same 17-day study period. The presence of lymph node swelling in 1 of 5 ponies and follicular hyperplasia of the guttural pouch in all 5 ponies challenged with ΔprtM138-213 may be indicative of an immune response directed against this strain and suggests that, although not detected at 17-days postchallenge, the ΔprtM138-213 strain may persist for a short time in vivo. The Δlgt190-685 strain generated early clinical signs of strangles in 3 of 5 ponies challenged. S. equi isolates from the lesions in these ponies all contained the deleted version of the lgt gene, indicating that the strain had not reverted in vivo and thus that the presence of functional Lgt is not an absolute requirement for virulence in the natural host. However, there were no signs of strangles in 2 of the 5 ponies challenged with Δlgt190-685, and there was an overall reduction in the degree of pyrexia in this cohort. This suggests that while this deletion mutant is not statistically significantly attenuated in the pony, there was a trend toward reduced incidence of disease compared with that for the parental 4047 strain, consistent with our findings for the other models. The stronger attenuation of the Δlgt190-685 strain in the mouse model compared with that in ponies may reflect differences in either the nature of bacterial-host interactions between the two species or differences in host responses to infection. Note, for example, that the wild-type strain causes disease in 100% of infected ponies but only 60% of infected mice. Our data demonstrate that, ultimately, it is important to evaluate virulence attenuation in the natural host.

The increased attenuation observed upon deletion of the prtM gene compared with that upon deletion of the lgt gene suggests that lack of lipidation of PrtM does not completely eliminate its functional activity. In this context, it is important to note that while deletion of lgt is not, in itself, lethal in B. subtilis (12, 29, 39) the PrsA lipoprotein is essential: reduction of the cellular levels of PrsA below a critical threshold of ca. 200 molecules per cell results in cellular lysis (56). Thus, it is likely that some residual functional activity may be retained by preprolipoproteins in Lgt mutants, perhaps during transient membrane association prior to shedding, shaving, or proteolytic degradation. Similar conclusions have previously been drawn based on the absence of a significant growth defect in an Lgt mutant of S. aureus (45) and an Lsp mutant of Lactococcus lactis (55). In B. subtilis the in vivo function of PrsA apparently derives from the interaction of the central parvulin-like domain with the flanking N- and C-terminal domains (57). However, a PrsA deletion mutant (PrsAN+C), which is comparable to the ΔprtM138-213 mutant described herein, was unable to restore growth of PrsA-depleted cells, although it did exhibit weak activity in an AmyQ secretion assay (57). Our studies suggest that deletion of the central domain of S. equi PrtM is sufficient to abrogate its function in vivo, thereby attenuating this strain. Some maturases, such as the plasmid-encoded PrtM of L. lactis, have clearly-defined substrates (19, 20), whereas the role of the chromosomally encoded PpmA maturase has not yet been defined (16). Similarly, PrsA of B. subtilis may have a more pleiotropic role in protein secretion (57), and the Bacillus anthracis genome encodes three functional PrsA homologues that may have distinct but overlapping substrate specificities (58). Further analyses of the molecular consequences of the deletion of prtM in S. equi are now required in order to identify those virulence factors reliant on its activity and which are essential to pathogenicity in the horse.

ACKNOWLEDGMENTS

This work was supported by the Wellcome Trust (project grant 056042) and the Horseracing Betting Levy Board (project grant VET/PRJ/696).

We are grateful to Horst Malke (Institute for Molecular Biology, Jena University, Germany) for providing the anti-LppC antibody used in this study. We thank Neil Chanter (Intervet UK Ltd.) for his contribution to the early stages of this project and acknowledge the invaluable help and expertise of Debs Flack and Jason Tearle (both of Animal Health Trust, Newmarket, United Kingdom).

FOOTNOTES

    • Received 17 July 2006.
    • Returned for modification 30 August 2006.
    • Accepted 21 September 2006.

  • ↵▿ Published ahead of print on 2 October 2006.

  • Editor: J. N. Weiser

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