Figure 3

Field emission scanning electron microscopy of GAS biofilms. 24-h biofilms of the M1- and M41-type GAS MLN4924 price strains were grown on glass cover slips and analyzed by FESEM. (a-b) Architecture of GAS microcolonies shown at low magnification. (c-d) Cell surface morphology and cell-to-cell junctions observed at higher magnification. Enlargements of cell-to-cell junctions are shown below. GAS biofilms differ in production of bacterial-associated extracellular matrix The production of BAEM has been shown to be an integral component in the structural integrity of a biofilm, imparting protection from dehydration, host immune attack, and antibiotic sensitivity [30, 31]. GAS cells encased in a glycocalyx were first identified by Akiyama et al. this website GS-1101 supplier in skin biopsies obtained from impetigo patients. We therefore compared the production of BAEM within biofilms employing GFP-expressing GAS strains of the M1 and M41 type (Figure 4). Cells

were grown to form biofilms on glass cover slips for 24 h and stained with TRITC-concanavalin A (ConA), a fluorescently-labeled lectin that binds to the extracellular polysaccharides in biofilms [32]. Fluorescent microscopy was performed to compare matrix production (red staining) by GAS strains (green). Visual screening of both biofilms indicated that the M41-type strain formed a more dispersed extracellular matrix as compared to the M1 strain, which had a dense, more closely associated matrix. In addition, averages of at least 10 fields of ConA stained matrix by CLSM support our FESEM observations that more BAEM is deposited within the biofilm by the M1 GAS cells as compared to M41 GAS. This is in agreement with the report from Akiyama et al that showed a substantial FITC-ConA stained matrix associated with T1-type GAS microcolonies in vivo and in vitro [10]. Figure 4 Production of bacterial-associated extracellular matrix. GFP-expressing wild type (WT) M41- and M1-type GAS strains were

grown on glass cover slips for 24 h and stained with TRITC-conjugated concanavalin A (ConA). Confocal laser scanning microscopic (CLSM) images were separated to represent green GFP-expressing GAS cells (left images) and red ConA-TRITC staining (right images) for detection Reverse transcriptase of extracellular matrix associated with each strain. Images are from one representative experiment. Scl1 protein significantly contributes to biofilm formation by GAS Variations in GAS pathogenicity and capacity to form biofilm are driven by specific proteins and components present on the cell surface or are secreted by the organism. It has been shown that deletion of the M and M-like surface proteins or capsule, as well as increased expression of the secreted SpeB protease decreases biofilm formation dramatically for some strains of GAS [12, 33, 34].