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

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.

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 Δlgt_190-685 or ΔprtM_138-213.

Analysis of the presence and localization of lipoproteins.Lack of Lgt activity in the Δlgt_190-685 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 Δlgt_190-685 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, Δlgt_190-685, or ΔprtM_138-213 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, ΔprtM_138-213, or Δlgt_190-685S. 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 ΔprtM_138-213 or Δlgt_190-685, 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.

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

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 Δlgt_190-685 was confirmed by PCR and sequencing.

Radiolabeling of lipoproteins in S. equi 4047 and S. equi Δlgt_190-685.To confirm the absence of Lgt activity in the allelic-replacement mutant, S. equi 4047 and S. equi Δlgt_190-685 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.

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FIG. 1.

Labeling of lipoproteins from S. equi 4047 and the Δlgt_190-685 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 Δlgt_190-685 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 Δlgt_190-685 (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 Δlgt_190-685 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 Δlgt_190-685 (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 Δlgt_190-685 (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 Δlgt_190-685 (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 Δlgt_190-685 compared to that of the parent strain.

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FIG. 2.

Changes in the nature (A) and the activity (B) of an acid phosphatase (LppC) in the S. equi Δlgt_190-685 mutant. (A) Proteins in SDS extracts prepared from cells of the parent (4047) and mutant strain (Δlgt_190-685) 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 Δlgt_190-685. (B) Whole-cell acid phosphatase activity was determined for strain 4047 (⧫) and the Δlgt_190-685 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 Δlgt_190-685 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 (ΔprtM_138-213) 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 Δlgt_190-685 and the ΔprtM_138-213 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 Δlgt_190-685 were again recovered, but ΔprtM_138-213 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 Δlgt_190-685 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 ΔprtM_138-213 cells than both wild-type S. equi and Δlgt_190-685 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.

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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 Δlgt_190-685 or ΔprtM_138-213. 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 Δlgt_190-685 or ΔprtM_138-213. 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 Δlgt_190-685 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 Δlgt_190-685, inoculation with ΔprtM_138-213 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).

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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) Δlgt_190-685; (D) ΔprtM_138-213. 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 Δlgt_190-685 or ΔprtM_138-213 generally continued to gain weight in line with the mock-challenged controls. However, 2 of 30 mice challenged with the Δlgt_190-685 strain had reduced weight gain compared with that of the unchallenged controls. Two mice challenged with Δlgt_190-685, including one of the mice that had failed to gain weight, also had histological disease on postmortem examination (Fig. 5D).

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FIG. 5.

Challenge of mice with the Δlgt_190-685 and ΔprtM_138-213 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 Δlgt_190-685 (lgt) or ΔprtM_138-213 (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 Δlgt_190-685- and ΔprtM_138-213-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 Δlgt_190-685 deletion mutant (Fig. 6A -C). In contrast, there were no signs of disease observed in ponies challenged with the ΔprtM_138-213 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 Δlgt_190-685 or ΔprtM_138-213 (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 Δlgt_190-685-challenged group and 0 of 5 of the ΔprtM_138-213-challenged group (Fisher's exact test, P = 0.048) (Fig. 6B). Other clinical signs were also significantly reduced in ΔprtM_138-213-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 Δlgt_190-685-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 Δlgt_190-685-challenged groups, neutrophil levels in ΔprtM_138-213-challenged ponies remained stable (Fig. 6D).

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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 ΔprtM_138-213 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 ΔprtM_138-213 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 Δlgt_190-685 strain (lgt) developed neutrophilia 17 days postchallenge, and no signs of neutrophilia were observed in ponies challenged with the ΔprtM_138-213 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 Δlgt_190-685 strain, and 0 of 5 ponies (P = 0.008) challenged with the ΔprtM_138-213 strain (Fig. 7A). The mean pathological scores determined at postmortem were very similar for the 4047 and Δlgt_190-685 groups, whereas the low score obtained for the ΔprtM_138-213 group reflected low-grade pathology not indicative of strangles (Fig. 7B). S. equi was isolated from the abscesses of ponies in the 4047 and Δlgt_190-685 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 ΔprtM_138-213-challenged ponies on postmortem examination, suggesting that this strain was not able to persist in vivo for the 3-week duration of this study.

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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 Δlgt_190-685 strain had no significant signs of disease, this was not statistically significant (P = 0.44). However, ponies challenged with the ΔprtM_138-213 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.