Group A Streptococcus
strains vary because of the vast number of M-protein types, and this variation is associated with varying frequency of isolation and exacerbation of disease [40
]. The M41-, M28-, M3-, and M1-type strains selected for the current study represent a significant intraspecies diversity among clinical isolates of GAS. M41 GAS was a major causative agent of superficial skin infections [42
], and strain MGAS6183, harboring the Scl1.41 protein, has been studied extensively [19
]. M28-type GAS (strain MGAS6143) has historically been associated with puerperal fever and currently is responsible for extensive human infections world-wide [45
]. M1T1 GAS, represented by strain MGAS5005, is a globally disseminated clone responsible for both pharyngitis and invasive infections [46
]. The M3-type strains of GAS cause a disproportionally large number of invasive GAS infections that are responsible for traumatic morbidity and death [49
Initial studies by Lembke et al.
that characterized biofilm formation among various M types of GAS typically included several strains of the same M type [1
]. These studies reported a significant strain-to-strain variation in ability to form biofilms within each M type. Studies that followed compared biofilm formation by defined isogenic WT and mutant strains to assess the contribution of specific GAS surface components responsible for a biofilm phenotype, including M and M-like proteins, hyaluronic acid capsule, lipoteichoic acid, and pili [12
]. In the current study, we have assessed the role and contribution of the surface protein Scl1 in the ability to support biofilm formation by GAS strains of four distinct M types.
Recent advances in molecular mega- and pathogenomics has enabled the characterization of numerous M3-type strains with a single nucleotide resolution [51
]. Interestingly, all five M3-type strains MGAS158, 274, 315, 335, and 1313 that were originally used for scl1
-gene sequencing [14
], plus an additional strain MGAS2079 (not reported) harbor the same scl1.3
allele containing a null mutation that would result in secretion of a truncated Scl1.3-protein variant. Here, we demonstrate that these GAS strains do not form biofilm on an abiotic surface. Recently, bioinformatic screening of the sequences of ~250 invasive M3-type strains isolated globally, has led to the detection of this single nucleotide polymorphism that results in disruption of Scl1.3 protein (Steve Beres and Jim Musser, personal communication). Lembke et al.
reported heterogeneous biofilm formation among four M3-type GAS strains examined over a 24, 48, and 72-h period [28
]. Biofilm was detected for one strain at a 48 h time point, on a fibrinogen-coated surface; however, it is not known whether this clinical isolate forms biofilm on abiotic surface, whether it expresses the truncated or full-length Scl1.3 protein, and whether it produces an unknown fibrinogen-binding protein, which could augment the attachment and biofilm formation. Therefore, additional studies are necessary to define the contributions of other biofilm-formation determinants in M3-type strains.
Inasmuch as, variation in biofilm formation among GAS isolates of the same M-type has been established, the molecular basis of this phenotypic variation is not known. Several GAS surface-associated and secreted components were shown to contribute to variation in biofilm [12
]. In addition, transcription regulators, such as Mga, CovR, and Srv are likely to play substantial roles in GAS biofilm formation [11
] due to their transcriptional regulation of numerous genes. Therefore, it is logical to assume that the combination of genomic/proteomic make up, allelic polymorphisms, and transcription regulation all contribute to this phenomenon. In addition, discrepancies between in vitro
data obtained with laboratory-stored strains and microcolony formation in vivo
likely exist and add yet another unknown to the complexity of GAS biofilm/microcolony formation and its role in pathogenesis. Despite this complexity, the analyses involving isogenic strains of the same genetic background provide valuable information that allows assessment of the role and contribution of a given GAS component to biofilm formation.
The M1 MGAS5005 strain was shown to form biofilm in vitro
and in experimental animals [8
], and the present study demonstrates a significant role of Scl1.1 in this process. Likewise, the MGAS6183 strain, representing M41-type isolates often associated with pyoderma, produced a more robust biofilm biomass under the same experimental conditions and Scl1.41-deficient mutant was found to be an important determinant in this process. Similarly, Scl1.28 protein significantly contributes to a robust biofilm made by the M28-type strain MGAS6143. However, a recent study reported that another surface protein, designated AspA, found in M28-type GAS significantly contributed to biofilm formation [54
]. The ΔaspA
isogenic mutant showed 60% reduction in biofilm formation. The strain MGAS6180, which they used, expresses the same Scl1.28 variant present in the MGAS6143 strain we used; our Scl1.28 mutant showed ~44% reduction in 24 h biofilm. We propose that several surface proteins contribute to biofilm formation by M28-type strains including proteins AspA and Scl1.28, and potentially, proteins F1/SfbI and F2 that are also present in these strains [22
]. This redundancy is likely responsible for the observed residual biofilms produced by the AspA- and Scl1.28-deficient mutants.
The observed heterogeneity in biofilm architecture of different GAS strains was previously observed by Lembke et al.
] and was also documented in the current study using FESEM. In addition, here we report the differences in GAS-cell surface morphology and within cell-to-cell junctions in biofilms formed by M1- and M41-type strains. The structural and genetic determination of these differences is not known since M41 genome has not been sequenced, but may be associated with the presence of additional surface proteins, such as the F2 protein [55
] encoded by prtf2
gene found in this strain [22
]. Even more striking was an observed difference in the amount of the extracellular material associated with each strain, referred to as BAEM (bacteria-associated extracellular matrix). It has been shown that extracellular matrix, also called glycocalyx, is produced by biofilm-forming bacteria. DNA, lipids, proteins [33
], polysaccharides and dead cell debris [56
] were identified in this matrix and for gram-positive bacteria, teichoic acids have also been detected [57
]. The exopolysaccharide component of the glycocalyx is detected using carbohydrate-binding lectins, such as concanavalin A (ConA) [10
]. Both FESEM analysis and ConA staining detected more BAEM associated with M1 biofilm compared to M41, which produced larger biofilm. These observations suggest that GAS biofilm is stabilized differently by different strains and that higher BAEM production does not necessarily pre-determine larger biofilm mass. Consequently, a combination of biofilm features rather than biofilm size alone may be more relevant to pathogenicity of a given GAS strain.
Diminished adherence and biofilm formation could be associated with changes in cell surface hydrophobicity [59
] of the scl1
mutants. Indeed, the lack of Scl1 resulted in both decreased hydrophobicity and the ability to form biofilm, albeit in a somewhat disproportionate manner. A decrease in the hydrophobicity index by only ~8%, as compared to the wild type-strain, was measured for the M41Δscl1
mutant and this modest decrease was accompanied by a rather large reduction in biofilm formation capacity after 24 h by 30%. Greater decrease in cell-surface hydrophobicity was measured for the M1Δscl1
(~21%) and M28Δscl1
(~22%) mutants, which was accompanied by a significant loss in biofilm formation after 24 h by both isogenic strains by ~55% and ~41% (P
≤ 0.001 for each comparison), respectively. In addition, heterologous expression of Scl.41 in L. lactis
increased hydrophobicity index of this organism to ~137% of the WT level, which was accompanied by significant increase in its ability to form biofilm. Similar observations have been reported for the M and M-like protein mutants that typically, but not always, exhibit concurrent loss of both biological features [12
]. For example, isogenic ΔMrp49 mutant had a non-significant drop in hydrophobicity (~2%) but significantly lower biofilm formation after 48 h by ~30%, whereas ΔEmm1 mutant lost ~78% hydrophobicity and ~44% biofilm formation capacity. In summary: (i) here we report that the Scl1 adhesin is also a hydrophobin with varying contribution to the overall surface hydrophobicity among GAS strains representing different M types and (ii) Scl1-associated surface hydrophobicity is likely to contribute to Scl1-mediated biofilm formation.
To test whether Scl1 alone could support biofilm formation, we used a heterologous L. lactis
strain, which provides an expression system for membrane-bound proteins of gram- positive bacteria with LPXTG cell-wall anchoring motifs [39
], including the group A streptococcal M6 protein [38
]. In a recent study by Maddocks et al.
] it was shown that heterologous expression of AspA GAS surface protein was able to induce a biofilm phenotype in L. lactis
MG1363. We were also able to achieve a gain-of-function derivative of the L. lactis
WT MG1363 strain, (MG1363::pSL230), displaying an altered phenotype associated with biofilm formation, as compared to wild-type parental and vector-only controls. These data support our current model that Scl1 protein is an important determinant of GAS biofilm formation.
As shown by crystal violet staining and CLSM, biofilm formation by the Scl1-negative mutants was compromised during the initial stage of adherence, as well as microcolony stabilization and maturation. Consequently, their capacity for biofilm formation as compared to the respective WT controls was greatly reduced. This comparison identifies for the first time that the Scl1 protein contributes significantly to biofilm assembly and stability. Based on these observations, as well as previous work by us and others, we propose the following model of Scl1 contribution to GAS tissue microcolony formation (Figure ). First, the Scl1 hydrophobin (current study) initiates bacterial adhesion to animate surfaces within the host [59
]. Next, the Scl1 adhesin anchors the outside edge of growing microcolony in tissue by direct binding to tissue extracellular matrix components, cellular fibronectin and laminin [19
]. Microcolony development is stabilized by Scl1-Scl1 scaffolding resulting from Scl1's capacity to form head-to-head dimers [64
] between molecules located on adjacent chains. This model will be tested experimentally in future studies.
Figure 6 Scl1-mediated model of GAS biofilm (not to scale). Scl1 hydrophobin (current study) initiates bacterial adhesion to animate surfaces  within the host (blue field). Scl1 adhesin anchors the growing microcolony by direct binding to tissue extracellular (more ...)