MATERIALS AND METHODS

Materials
Purified horse IgG1 and a mixture of horse IgG1 and IgG4 were kindly provided by Dr. Bettina Wagner at Cornell University. S. equi-specific mouse sera were obtained from adult female outbred CD-1 Swiss mice (Charles River Laboratories, Wilmington, Mass.) 21 days after they were inoculated subcutaneously with 1 x 107 cfu S. equi strain SEM1. Convalescent sera from 3 horses suffering from strangles were obtained 30 days after diagnosis. GAS M1 Mac was prepared as previously described [5].

Bacterial Strains and Growth
Six of 10 S. equi strains used were kindly provided by Dr. James Musser at Methodist Hospital, Houston, Texas, and these strains were isolated more than 20 years ago from horses with strangles in the Eastern U.S. (4 strains), Brazil (1 strain), and Finland (1 strain). The other 4 strains were isolated in 2003 from horses with strangles in Livingston (designated strain SEM1) [10], Pony, Great Fall, and Norris in Montana. GAS strain MGAS5005 (serotype M1) has been described [11]. Escherichia coli Novablue and BL21(DE3) (Novagen, Madison, Wis.) were used for gene cloning and protein expression, respectively. S. equi and GAS strains were routinely grown in Todd-Hewitt broth (Difco Laboratories, Detroit, Mich.) supplemented with 0.2% yeast extract (THY) in 5% CO2 at 37°C. THY agar and tryptose agar with 5% sheep blood (Becton Dickinson, Cockeysville, Md.) were used as the solid media.

Gene Cloning and Mutagenesis
The gene fragment encoding mature SeMac was cloned from SEM1 with primers 5’-ACCATGGACGA TTACCAAAGGAATGCTAC-3’ and 5’-CGAATTCT TAGCTCAGTTTCTGCCATATG-3’. The protein made from this cloned fragment lacks the presumed secretion signal sequence (amino acids 1-34). The PCR product was digested with NcoI and EcoRI and ligated into pET-21d (Novagen) to yield recombinant plasmid pSEMAC. The cloned gene was sequenced and had identical DNA sequence with the corresponding open reading frame of S. equi genome database [12]. Amino acid replacement of Cys102 and His272 or Asp294 of SeMac with serine and alanine, respectively, was achieved by site-directed mutagenesis using the Quick-Change Mutagenesis kit (Stratagene, La Jolla, Calif.). The entire mutated gene was sequenced to confirm the mutations and rule out spurious mutations.

Expression and Purification of Recombinant SeMac
Recombinant SeMac was purified from E. coli BL21 (DE3) containing plasmid pSEMAC. Bacteria were grown to optical density at 600 nm of 0.5 in 6 liters of Luria-Bertani broth supplemented with 100 mg of ampicillin per liter at 37°C, and SeMac expression was then induced with 0.5 mM IPTG 6 h. Solutions used in purification were buffered with 10 mM Tris-HCl (pH 8.0). Cell paste was sonicated for 15 min at 4°C in 60 ml of the buffer, and centrifuged at 15,000 g for 10 min. The supernatant obtained was loaded onto a DEAE-Sepharose column (2.5 by 10 cm). The column was washed with 50 ml of Tris-HCl, and SeMac was eluted with 200 ml of 50 mM NaCl. SeMac was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and peak fractions were pooled. Ammonium sulfate was added to the pool to a concentration of 1.2 M, and the resulting solution was applied to a phenyl-Sepharose column (1.5 x 10 cm). The column was washed with 50 ml of 1.2 M (NH4)2SO4 and eluted with a 100-ml linear gradient of 1.2 to 0.7 M (NH4)2SO4. Fractions containing the protein were pooled. The pooled protein was precipitated with (NH4)2SO4 at 70% of saturation, centrifuged, dialyzed against 3 liters of 10 mM Tris-HCl buffer (pH 8.0) overnight, and loaded onto a DEAE-Sepharose column (1.5 by 10 cm). The protein was eluted with a 100-ml linear gradient of 2 to 8 mM NaCl and pooled. The pooled protein was concentrated by (NH4)2SO4 precipitation and dialyzed against Tris-HCl as described above.

DNA Sequencing
Genomic DNA was isolated with a FastDNA SPIN Kit (Qbiogene, Calsdad, Calif.) according to manufacturer’s protocol. A DNA fragment containing S. equi mac was amplified by PCR using the genomic DNA samples and primers 5’-GCATCTCTACTATCTCATCAC-3’ and 5’-ACAGGCACATTAATGTTTAAC-3’. Sequences of the PCR products were obtained from both DNA strands with an Applied Biosystem 310 automated sequencer (Applied BioSystems, Inc., Foster City, Calif.).

Anti-SeMac antisera
Four female outbred CD-1 Swiss mice (4-week-old) (Charles River Laboratories) were immunized subcutaneously with 50 µg of recombinant SeMac suspended in 200 µl of saline emulsified in 44 µl of monophosphoryl lipid A-synthetic trehalose dicorynomycolate adjuvant (Corixa, Hamilton, Mont.). The animals were anesthetized by isoflurane inhalation prior to immunization and blood collection. Blood was collected prior to active immunization to prepare control sera. Mice were boosted at weeks 2 and 4 with 50 µg of protein mixed with the adjuvant. Anti-SeMac antiserum was prepared from blood obtained 1 week after the second booster.

Phagocytosis Assay
Phagocytosis assay was performed as described previously [13, 14]. Briefly, S. equi SEM1 and GAS bacteria from exponential growth phase in THY were washed with phosphate-buffered saline (PBS) and labeled with 0.75 μg/ml FITC in PBS at 37°C for 20 min. The labeled bacteria were washed and resuspended at 1 x 109 cfu/ml in PBS. Ten μl of the labeled bacteria were mixed with 100 μl of non-immune heparinized horse or human blood with or without 100 μg/ml SeMac and incubated with gentle shaking at 37°C for 5, 10, or 20 min. The samples were immediately processed using an Immunolyse Kit (Beckman Coulter) according to the manufacturer’s protocol and analyzed by flow cytometry. The percentage of PMNs with fluorescent bacteria was used as a measure of phagocytosis efficiency.

Other Assays
To assess IgG endopeptidase activity of SeMac, human, mouse or horse IgG (20 μg) was incubated with E. coli lysate containing SeMac proteins or 1 μg purified SeMac or GAS M1 Mac in PBS at 37° for 90 min, and the reaction mixture was analyzed by SDS-PAGE. Western immunoblot analysis, which was performed as described previously [15], was used to assess in vitro SeMac production and the presence of SeMac-specific antibody in the horse and mouse convalescent sera. Culture supernatant proteins of S. equi, which were used to assess the in vitro SeMac production, were prepared by the method of Lei et al. [16].