Adaptation of the group A Streptococcus adhesin Scl1 to bind fibronectin type III repeats within wound‐associated extracellular matrix: implications for cancer therapy

Summary The human‐adapted pathogen group A Streptococcus (GAS) utilizes wounds as portals of entry into host tissue, wherein surface adhesins interact with the extracellular matrix, enabling bacterial colonization. The streptococcal collagen‐like protein 1 (Scl1) is a major adhesin of GAS that selectively binds to two fibronectin type III (FnIII) repeats within cellular fibronectin, specifically the alternatively spliced extra domains A and B, and the FnIII repeats within tenascin‐C. Binding to FnIII repeats was mediated through conserved structural determinants present within the Scl1 globular domain and facilitated GAS adherence and biofilm formation. Isoforms of cellular fibronectin that contain extra domains A and B, as well as tenascin‐C, are present for several days in the wound extracellular matrix. Scl1‐FnIII binding is therefore an example of GAS adaptation to the host's wound environment. Similarly, cellular fibronectin isoforms and tenascin‐C are present in the tumor microenvironment. Consistent with this, FnIII repeats mediate GAS attachment to and enhancement of biofilm formation on matrices deposited by cancer‐associated fibroblasts and osteosarcoma cells. These data collectively support the premise for utilization of the Scl1‐FnIII interaction as a novel method of anti‐neoplastic targeting in the tumor microenvironment.


Table S1
Sequences of Scl-V chimeras. Inserted loops are underlined and in bold. Sequence identities and root mean square deviations from Scl2.3 structure are given in the last two columns.

Figure S1
Fig. S1. Specificity of anti-ECM antibodies and background controls.
A. Antibodies against TnC (BC-24), EDA containing (IST-9) or EDB containing (C6) isoforms of cFn, as well as anti-fibronectin monoclonal antibody (IST-4), were analyzed by western immunoblotting of commercial preparations of cFn. Molecular weights, in kDa, are shown for PageRuler™ Plus protein ladder.
B. Immunofluorescent control images. 1 µg of BSA, 2 µg of plasma fibronectin (Sigma), or CAF-deposited matrices, were prepared on glass coverslips and incubated with primary mAbs, outlined in Fig. S1A. Secondary Ab conjugated with Alexa Fluor® 568 was used for detection. A representative image of BSA background (using anti-EDB antibody, C6) is shown in upper left panel, secondary only background on CAF-deposited ECM on upper right panel, and plasma fibronectin coatings in the bottom two rows. Images were taken using Nikon A1-R confocal microscope with 60x objective; representative images are shown from 2 independent experiments, imaging 10 arbitrary fields per coverslip.
A. Assessment of biofilm formation on rEDB-coated surfaces. M1 and M41 WT and their Δscl1 isogenic mutants were compared. Biofilm formation was evaluated spectrophotometrically following crystal violet staining. Graphic bars indicate the mean OD600nm normalized against BSA controls. Statistical analysis was calculated using Student's two-tailed t-test from three independent experiments (N=3±SD); ***P≤0.001.
B. Microscopy imaging of GAS biofilms formed on CAF-derived ECM. GFP-expressing M41 WT and Δscl1 mutant strains were grown for 24 h on CAF-ECM matrix deposited on glass coverslips. Twodimensional orthogonal views of GAS biofilms are representative of Z stacks from 10 fields within a single experiment. Average vertical thickness is indicated in micrometers below two-dimensional orthogonal views, taken from 10 arbitrary fields per experiment.

Figure S3
Fig. S3. Scl1-mediated GAS attachment to and biofilm formation on ECM deposited by bone osteosarcoma cells. WT GAS strains M1 and M41, and their isogenic Δscl1 mutants were compared for attachment and biofilm formation on the ECM produced by osteosarcoma Saos-2 cells.
A. Representative image of Ponceau S staining of ECM network deposited by Saos-2 cells.
B. Characterization of the ECM deposited by Saos-2 cells by ELISA. The presence of total Fn, EDA/cFn, EDB/cFn, and TnC was assessed with specific anti-ECM mAbs and secondary HRP-conjugated antibody.
Graph bars indicate the mean OD415nm from three independent experiments, each in triplicate wells (N=3±SD). Dashed line indicate threshold OD415nm +2SD values recorded for BSA control wells.
C. GAS attachment on Saos-2-derived ECM. Isogenic GFP-GAS strains were inoculated onto Saos-2derived matrices, allowed to attach for 1 h, and imaged using fluorescent confocal microscope with 100x objective. Top, representative images of attached GAS were taken in 20 fields. Bottom, quantification of GAS attachment with WT binding set as a 100%. Bacteria were counted in 20 fields and the average was calculated. Statistical analysis was calculated using Student's two-tailed t-test from two independent experiments, each performed in duplicate wells (N=2±SD); **P≤0.01, ***P≤0.001. Statistical significance evaluates the difference between adherence to Saos-2-derived matrices by the WT and their respective isogenic Δscl1 mutants. Each symbol represents one imaged-field. D. GAS biofilm formation on Saos-2-derived ECM. Isogenic GAS strains were inoculated onto Saos-2derived matrices and grown for 24 hours. Bacterial biomass was evaluated spectrophotometrically following crystal violet staining. Graphic bars indicate the mean OD600nm normalized against BSA controls. Statistical analysis was calculated using Student's two-tailed t-test from three independent experiments (N=3±SD); ***P≤0.001.