Bacterial strains and growth
GAS strain 5448 (serotype M1T1, ndoS-positive) and GAS strain NZ131 (serotype M49, ndoS-negative) are well-characterized and were selected for use in this study [30, 31]. Escherichia coli MC1061 was used as cloning tool . The streptococcal and E. coli strains were propagated on Todd-Hewitt agar (THA). For selection, erythromycin (erm) was used at 5 μg/mL (5448), 2 μg/mL (NZ131) and 500 μg/mL (MC1061). GAS and its isogenic mutant were grown in Todd-Hewitt broth (THB (Difco, Detroit, MI)) at 37°C without shaking. For in vitro and in vivo experiments, fresh overnight cultures were diluted 1:10 in THB and grown to mid logarithmic phase (OD600 = 0.4) and resuspended in PBS, or in mid-log supernatants for neutrophil assays with NZ131. For analysis of streptococcal supernatants, strains were grown in C-medium (0.5% (w/v) Proteose Peptone no. 2 (Difco), 1.5% (w/v) yeast extract, 10 mM K2HPO4, 0.4 mM MgSO4, 17 mM NaCl pH 7.5) to maximize EndoS expression.
EndoS is encoded by the gene ndoS. A precise, in-frame allelic replacement of ndoS with chloramphenicol transferase, cat, was created in M1T1 GAS strain 5448 by a method previously described  and was denoted 5448ΔndoS. Briefly, a 798 bp fragment upstream, and 987 bp fragment downstream of ndoS was amplified using polymerase chain reaction, PCR, using primers ndoS-up-F-XbaI (GCATCTAGAGCTTGTCGGTCTTGGGGTAGC), ndoS-up-R (GGTGGTATATCCAGTGATTTTTTTCTCCATTTGGACACTCCTTATTTTTGGTACTAAGT C) and ndoS-dn-F (TACTGCGATGAGTGGCAGGGCGGGGCGTAAACAAAGTAACTTTCTTAGATAGCAACATT CAG), ndoS-dn-R-BamHI (GCGGATCCGTTCTTGCGCCATGACACCTCC) respectively. The primers adjacent to ndoS contained 30 bp overhang of the cat gene corresponding to the 5' and 3' ends of cat, respectively. The upstream and downstream fragments were combined with the 650 bp cat gene in a fusion PCR using primers ndoS-up-F-XbaI and ndoS-dn-R-BamHI. This triple fragment was digested using restriction enzymes XbaI and BamHI and ligated using T4 ligase into the temperature sensitive vector pHY304, bearing erythromycin resistance, to generate the knockout plasmid pHY-ndoS-KO. pHY-ndoS-KO was transformed into GAS 5448 by electroporation and transformants were grown at the permissive temperature of 30°C with erythromycin. Transformants were then grown at the non-permissive temperature of 37°C with erythromycin present to select for homologous recombination and integration of the plasmid into the genome. Single crossovers were confirmed by PCR analysis. Relaxation of the plasmid was carried out at 30°C with no antibiotic selection to allow the plasmid to reform, outside the chromosome. Growing the bacteria at 37°C without antibiotic pressure resulted in loss of the plasmid. Finally, screening for erythromycin sensitive colonies was used to identify double crossover events and allelic replacement mutants were confirmed by PCR. In frame allelic replacement ndoS mutant, 5448ΔndoS, was confirmed by multiple PCR reactions showing the insertion of the cat gene and absence of the ndoS gene in the genome. Heterologous expression of EndoS in M49 GAS strain NZ131 was established by transformation with the EndoS expression plasmid pNdoS. ndoS was amplified from the M1 genome using primers ndoS-F-EcoRI (GCGAATTCATGGATAAACATTTGTTGGTAAAAAGAAC) and ndoS-R-BamHI (GCGGATCCTTATTTTTTTAGCAGCTGCCTTTTCTC), digested with EcoRI and BamHI prior to T4-ligation into the expression vector pDCerm, denoted pNdoS. As a control, GAS strain NZ131 was transformed with the empty vector pDCerm to generate NZ131[empty vector].
Supernatants from stationary phase (16 h) GAS strains 5448, 5448ΔndoS, NZ131[empty vector] and NZ131[pNdoS] were precipitated with 5% final concentration of trichloroacetic acid and separated on a 10% SDS-PAGE gel and blotted onto a methanol activated PVDF membrane. The membrane was blocked in 5% skimmed milk (Difco) for 1 h and washed 3 × 10 minutes in phosphate buffered saline, PBS (137 mM NaCl, 2.7 M KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4). The membrane was then incubated with polyclonal rabbit antiserum against rEndoS at 1:2000 dilution in 0.5% skimmed milk and incubated for 1 h at 37°C. The membrane was washed as before and incubated with goat anti-rabbit IgG conjugated with Horse radish peroxidase (Bio-Rad), at 1:5,000 in 0.5% skimmed milk for 1 h at 37°C. After washing, the membrane was developed using Supersignal West Pico Chemiluminescent (Thermo Scientific, Rockford, IL) and analyzed on a Chemidoc XRS (Bio-Rad, Hercules, CA).
Supernatants from GAS strains 5448, 5448ΔndoS, NZ131[empty vector] and NZ131[pNdoS] at stationary phase (16 h) was incubated with 1 μg murine IgG (mIgG) for 2 h at 37°C at static conditions. As a positive control, IgG was incubated with 1 μg rEndoS. The glycan hydrolyzing activity was analyzed with SDS-PAGE and lectin blot using biotinylated Lens culinaris agglutinin (LCA) (Vector Laboratories, Burlingame, CA). LCA lectin recognizes the α-1,3 mannose residue found on the N-linked glycan on IgG. Briefly, the supernatants and mIgG were separated on 10% SDS-PAGE gels, onestained with Coomassie blue and the other blotted onto Immobilon PVDF membranes (Millipore, Bedford, MA). The membrane was blocked in lectin buffer (10 mM HEPES, 0.15 M NaCl, 0,1% Tween 20, 0.01 mM MnCl2, 0.1 mM CaCl2, pH = 7.5) for 1 h. 10 μg LCA in lectin buffer was incubated with the membrane for 1 h at RT. The membrane was then washed for 3 × 10 min in lectin buffer and incubated with 2 μg streptavidin linked HRP (Vector Laboratories) for 1 h. After washing as above the blot was developed using Supersignal West Pico Chemiluminescent (Thermo Scientific) as described for Western blots.
Neutrophil killing assay
Neutrophils were purified from healthy donors using PolyMorphPrep-kit (Axis-Shield, Oslo, Norway) and RBCs lysed with sterile H20 as previously described . Neutrophils were seeded at 2 × 105 cells/well in 96-well microtiter plates in RPMI.
Plasma was obtained from healthy volunteers as previously described . All neutrophil and plasma donors exhibited high serum titer (>1:20,000) against serotype M1 and M49 GAS (Additional file 1 Table S1). GAS strains were grown as described and opsonized for 1 h at 37°C in 80% plasma, with or without pretreatment using recombinant EndoS (rEndoS) under rotating conditions. For pretreatment, 1 mL of plasma was incubated with 50 μg of rEndoS or PBS (control) at 37°C for 2 h with rotation. The bacteria were then diluted to the desired concentration in RPMI with a final concentration of 2% plasma and added to the neutrophils at a multiplicity of infection (MOI) of 10 bacteria per cell. Control wells contained GAS in RPMI and 2% plasma without neutrophils. The plate was centrifuged at 500 × g for 10 min and incubated for 30 min at 37°C with 5% CO2 before being serially diluted in sterile H2O and triplicate wells were plated on Todd-Hewitt agar (THA) plates for enumeration. Percent survival of the bacteria was calculated relative to control wells. Data from three separate experiments were normalized to 5448 or NZ131[empty vector] and combined.
Monocyte killing assay
The human monocytic cell line U937 was seeded at 5 × 105 cells/well in RPMI supplemented with 10% fetal bovine serum (FBS) in 24-well plates. GAS was grown and pre-opsonized in human plasma with or without rEndoS treatment, as described above. Bacteria were grown as described above and added to the U937 cells at MOI = 10 and incubated at 37°C with 5% CO2. Samples were collected at 1, 2, 3 and 4 h when monocytes were lysed with 0.025% Triton X-100 (MP Biomedicals, Aurora, OH) and triturated vigorously. Surviving bacteria from triplicate wells were plated on THA for enumeration. Percentage of surviving bacteria was calculated relative to the initial innoculum. Data from at least three separate experiments were normalized to 5448 or NZ131[empty vector] and combined.
Determination of donor serum titers
Blood from healthy human donors was collected in glass venous blood collection tubes with no additives (BD Biosciences, San Jose, CA) and clotted at room temperature for 15 min. Blood was centrifuged at 3,200 × g for 10 min at 4°C. The serum fraction was collected and stored at -80°C.
GAS strains NZ131 (serotype M49) and 5448 (serotype M1) were grown to mid-log phase in THB. Bacteria were resuspended in PBS and heat-killed at 95°C for 10 min. Heat-killed bacteria were mixed with a final concentration of 0.1 M NaHCO3 pH 9.6 and 106 bacteria per well were coated to 96-well high-bind ELISA plates (Costar, Cambridge, MA) at 4°C overnight. Plates were washed with PBS + 0.05% Tween (PBS-T) and blocked with 4% BSA + 10% FBS in PBS-T for 1 h at 37°C. Serum samples were diluted in blocking solution and incubated for 2 h at 37°C. Plates were washed with PBS-T and incubated with 1:5000 dilution of HRP-conjugated goat anti-human IgG antibody (Promega, Madison, WI) for 1 h at room temperature. Plates were washed five times with PBS-T and incubated with TMB substrate reagent (BD OptEIA TMB Substrate Reagent Set, BD Biosciences) at room temperature for 30 min. The reaction was stopped with an equal volume of 0.2 N sulfuric acid, and the plate was read at 450 nm. End point titer was determined as the dilution giving signal above a calculation cutoff determined using a mouse serum negative control and the calculation method described in .
In vivo mouse model
To evaluate the contribution of EndoS to GAS virulence in vivo, we utilized a murine model of systemic infection. GAS strains were grown as described and resuspended in PBS with 5% mucin for an inoculum of 2 × 107 cfu for WT M1T1 strain 5448 and isogenic mutant 5448ΔndoS, and 5 × 108 cfu for NZ131[empty vector] and NZ131[pNdoS]. 8-10 week old female CD-1 mice (n = 6 for 5448, n = 10 for NZ131) were infected intraperitoneally with GAS strains and mortality was monitored daily for 10 days.
Cfu enumeration in neutrophil and monocyte killing assays were statistically analyzed by unpaired Student's t-test. Differences were considered significant if P < 0.05. The in vivo results were evaluated with log-rank (Mantel-Cox) test for comparison of survival curves. Differences in survival were considered significant if P < 0.05. All statistical analysis was performed using GraphPad Prism v.5 (GraphPad Software).
Permission to collect human blood under informed consent was approved by the UCSD Human Research Protections Program. All animal use and procedures were approved by the UCSD Institutional Animal Care and Use Committee.