Mutation of the maturase lipoprotein attenuates the virulence of Streptococcus equi to a greater extent than does loss of general lipoprotein lipidation

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 (DeltaprtM(138-213), 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 (Deltalgt(190-685)). Moreover, mucus production was significantly greater in both wild-type-infected and Deltalgt(190-685)-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 Deltalgt(190-685) mutant did still exhibit signs of disease. In contrast, only the DeltaprtM(138-213) 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 Deltalgt(190-685) 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.

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.

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.

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.

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.

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.

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.

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.