Insight into the molecular basis of pathogen abundance: Group A Streptococcus inhibitor of complement inhibits bacterial adherence and internalization into human cells -- Data Supplement - Supporting Methods -- Proceedings of the National Academy of Sciences

Supporting information for Hoe et al. (May 21, 2001) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.112039899.

Supporting Methods
Adherence Assays.
Trypan blue exclusion analysis indicated A549 cell viability was >90% after a 1-h incubation with all group A Streptococcus (GAS) strains tested. We did not detect any intracellular bacteria in cells infected with the wild-type or mutant strains using an immunogold--silver-staining protocol (1) that differentiates intra- and extracellular bacteria (data not shown); hence, the mean number of bacteria per A549 cell is presented as percent bacterial adherence. The mean number of bacteria per A549 cell ranged from 15 to 23 for MGAS5005sic in the absence of exogenously added Sic protein.

To determine whether polyclonal antibody to Sic decreased the inhibitory effect, the adherence assay was performed as described, except that 10 ml of affinity-purified rabbit anti-Sic antibody or affinity-purified antibody to the streptococcal cell-surface protein Spy0843 (2) was added to the A549 cells immediately before the addition of the bacteria. To determine the effect of pretreating A549 cells with purified Sic, the adherence assay was performed as described, except the host cells were incubated with Sic1.01 (10 mg) for 5 min at 37°C and washed twice with Hanks’ balanced salt solution (HBSS), and 1 ml of fresh medium was added. To determine the effect of pretreating GAS with Sic, 4 ´ 10⁸ GAS in 1 ml of F12K containing 10% FBS were incubated with 10 mg of Sic1.01 for 5 min at 37°C. The bacteria were washed twice with HBSS and suspended in 50 ml of HBSS. The A549 cells were washed once with HBSS, and 1 ml of fresh growth medium and 50 ml of the washed GAS were added to the wells. The remaining steps of the assay were performed as described.

Preparation of A549 Cell Extracts.
A549 cells were removed from culture flasks by incubation with PBS containing 1 mM EDTA for 10 min at 37°C. Cells were lysed in 50 mM Tris·HCl (pH 7.5)/1 mM CaCl₂/1 mM MgCl₂/40 mM n-octyl b-D-glucopyranoside (Sigma)/0.5 mM phenylmethylsulfonyl fluoride/10 mg/ml of aprotonin/1 mg/ml of Pepstatin A, and incubated at room temperature for 15 min. The cell lysate was centrifuged at 20,000 ´ g for 15 min to remove insoluble material, and the supernatant was collected and used immediately in affinity chromatography. Peptide Inhibition Assays.
Densitometric values for Sic in each sample were normalized to the value for the glutathione S-transferase fusion protein in that sample. Results are expressed as percent Sic binding relative to the value obtained for the no-peptide control, set at 100%. All assays were performed in triplicate. Phlebotomy.
Heparinized venous blood was obtained from healthy individuals in accordance with a protocol approved by the Institutional Review Board for Human Subjects, National Institute of Allergy and Infectious Diseases. Labeling and Opsonization of GAS.
Before incubation with human polymorphonuclear leukocytes (PMNs), GAS strains were grown to an OD at 600 nm of 0.4 and labeled with 0.75 m g/ml of FITC for 20 min at 37° C. FITC-labeled GAS were opsonized in 50% immune serum obtained from an individual with recent GAS-induced pharyngitis for 30 min at 37°C. PMN Microbicidal Activity.
GAS strains were grown to an OD at 600 nm of 0.4, washed, and opsonized with immune serum as described above. PMNs (10⁶) were combined with 10⁵ opsonized MGAS5005 or MGAS5005sic in wells of a 96-well microtiter plate on ice (final volume = 200 ml) and centrifuged to synchronize phagocytosis. After 2- to 3-hr incubation at 37°C, PMNs were permeabilized with 0.1% saponon on ice for 15–20 min. Controls consisted of opsonized bacteria incubated under identical conditions in the absence of PMNs. Samples were plated on Brain Heart Infusion agar, and colony-forming units were enumerated the following day. Percent GAS killed was calculated by using the equation (1 – (CFU_PMN+/CFU_PMN–)) ´ 100.

References:

1. Van Putten, J. P., Weel, J. F. & Grassme, H. U. (1994) Methods Enzymol. 236, 420–437.

2. Reid, S. D., Green, N. M., Buss, J. K., Lei, B. & Musser, J. M. (2001) Proc. Natl. Acad. Sci. USA 98, 7552–7557.