Role of Surface Protein SasG in Biofilm Formation by Staphylococcus aureus

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Staphylococcus aureus and Staphylococcus epidermidis form communities (called biofilms) on inserted medical devices, leading to infections that affect many millions of patients worldwide and cause substantial morbidity and mortality. As biofilms are resistant to antibiotics, device removal is often required to resolve the infection. Thus, there is a need for new therapeutic strategies and molecular data that might assist their development. Surface proteins S. aureus surface protein G (SasG) and accumulation-associated protein ( S. epidermidis ) promote biofilm formation through their “B” regions. B regions contain tandemly arrayed G5 domains interspersed with approximately 50 residue sequences (herein called E) and have been proposed to mediate intercellular accumulation through Zn 2+ -mediated homodimerization. Although E regions are predicted to be unstructured, SasG and accumulation-associated protein form extended fibrils on the bacterial surface. Here we report structures of E–G5 and G5–E–G5 from SasG and biophysical characteristics of single and multidomain fragments. E sequences fold cooperatively and form interlocking interfaces with G5 domains in a head-to-tail fashion, resulting in a contiguous, elongated, monomeric structure. E and G5 domains lack a compact hydrophobic core, and yet G5 domain and multidomain constructs have thermodynamic stabilities only slightly lower than globular proteins of similar size. Zn 2+ does not cause SasG domains to form dimers. The work reveals a paradigm for formation of fibrils on the 100-nm scale and suggests that biofilm accumulation occurs through a mechanism distinct from the “zinc zipper.” Finally, formation of two domains by each repeat (as in SasG) might reduce misfolding in proteins when the tandem arrangement of highly similar sequences is advantageous.

Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Staphylococcal biofilm-forming protein has a contiguous rod-like structure

Gruszka

Wojdyla

Bingham

et al. 2012

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“…Although the polycationic polysaccharide intercellular adhesin has long been thought to be the main component promoting intercellular adhesion (4,5), there is now a compelling body of evidence that cell wall-anchored (CWA) proteins are also involved (2,3). Several recombinant CWA proteins have been shown to form dimers in solution (6)(7)(8)(9). In some cases (i.e., Aap), crystallographic studies have provided insights into the mechanism of dimer formation (9,10).…”

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Molecular interactions and inhibition of the staphylococcal biofilm-forming protein SdrC

Feuillie

Formosa-Dague

Hays

et al. 2017

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Staphylococcus aureus forms biofilms on indwelling medical devices using a variety of cell-surface proteins. There is growing evidence that specific homophilic interactions between these proteins represent an important mechanism of cell accumulation during biofilm formation, but the underlying molecular mechanisms are still not well-understood. Here we report the direct measurement of homophilic binding forces by the serine-aspartate repeat protein SdrC and their inhibition by a peptide. Using single-cell and single-molecule force measurements, we find that SdrC is engaged in low-affinity homophilic bonds that promote cell-cell adhesion. Low-affinity intercellular adhesion may play a role in favoring biofilm dynamics. We show that SdrC also mediates strong cellular interactions with hydrophobic surfaces, which are likely to be involved in the initial attachment to biomaterials, the first stage of biofilm formation. Furthermore, we demonstrate that a peptide derived from β-neurexin is a powerful competitive inhibitor capable of efficiently blocking surface attachment, homophilic adhesion, and biofilm accumulation. Molecular modeling suggests that this blocking activity may originate from binding of the peptide to a sequence of SdrC involved in homophilic interactions. Our study opens up avenues for understanding the role of homophilic interactions in staphylococcal adhesion, and for the design of new molecules to prevent biofilm formation during infection.Staphylococcus aureus | SdrC | adhesion | biofilms | inhibition T he nosocomial pathogen Staphylococcus aureus is a major cause of superficial and invasive infections. A remarkable trait of this bacterium is its ability to form biofilms on implanted devices, thereby triggering infections that are difficult to treat with antibiotics (1, 2). Biofilm formation is initiated by attachment of the bacteria to abiotic surfaces, protein-coated materials, and host cells, and followed by cell-cell adhesion and multiplication leading to a mature biofilm (1, 2). A variety of cell-surface components are involved in intercellular interactions (2, 3). Although the polycationic polysaccharide intercellular adhesin has long been thought to be the main component promoting intercellular adhesion (4, 5), there is now a compelling body of evidence that cell wall-anchored (CWA) proteins are also involved (2, 3). Several recombinant CWA proteins have been shown to form dimers in solution (6-9). In some cases (i.e., Aap), crystallographic studies have provided insights into the mechanism of dimer formation (9, 10). Although very useful, in vitro methods provide information on purified molecules that are removed from their cellular context. Clearly, elucidating the molecular mechanisms by which CWA proteins self-associate in vivo is key to enhancing our understanding of biofilm accumulation.The increase of multidrug-resistant strains has created an urgent need for new therapeutics for bacterial infections. Biofilm inhibitors, where bacteria are prevented from forming biofilms rather than...

“…Disorder can also play other roles: it facilitates posttranslational modification and may promote allostery (4,5). SasG (Staphylococcus aureus surface protein G) is a cell wall-attached protein from S. aureus that promotes intercellular adhesion during the accumulation phase of biofilm formation via its C-terminal repetitive region (6)(7)(8). We previously showed that this part of SasG contains alternating E and G5 domains (Fig.…”

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Disorder drives cooperative folding in a multidomain protein

Gruszka

Mendonca

Paci

et al. 2016

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Many human proteins contain intrinsically disordered regions, and disorder in these proteins can be fundamental to their function-for example, facilitating transient but specific binding, promoting allostery, or allowing efficient posttranslational modification. SasG, a multidomain protein implicated in host colonization and biofilm formation in Staphylococcus aureus, provides another example of how disorder can play an important role. Approximately one-half of the domains in the extracellular repetitive region of SasG are intrinsically unfolded in isolation, but these E domains fold in the context of their neighboring folded G5 domains. We have previously shown that the intrinsic disorder of the E domains mediates long-range cooperativity between nonneighboring G5 domains, allowing SasG to form a long, rod-like, mechanically strong structure. Here, we show that the disorder of the E domains coupled with the remarkable stability of the interdomain interface result in cooperative folding kinetics across long distances. Formation of a small structural nucleus at one end of the molecule results in rapid structure formation over a distance of 10 nm, which is likely to be important for the maintenance of the structural integrity of SasG. Moreover, if this normal folding nucleus is disrupted by mutation, the interdomain interface is sufficiently stable to drive the folding of adjacent E and G5 domains along a parallel folding pathway, thus maintaining cooperative folding.IDP | protein folding | parallel pathways | protein engineering | cooperativity I t has been suggested that as much as 20% of the proteome may be intrinsically disordered (1), mainly manifested as intrinsically disordered regions within multidomain proteins, although a few proteins are apparently entirely disordered. Some proteins function as a consequence of disorder: for example, disordered PEVK regions of titin act as an entropic spring (2), whereas in the nuclear pore complex, disordered nucleoporins provide a thick selective barrier controlling nuclear import (3). Disorder can also play other roles: it facilitates posttranslational modification and may promote allostery (4, 5). SasG (Staphylococcus aureus surface protein G) is a cell wall-attached protein from S. aureus that promotes intercellular adhesion during the accumulation phase of biofilm formation via its C-terminal repetitive region (6-8). We previously showed that this part of SasG contains alternating E and G5 domains (Fig. 1A) and that E folds when it is N-terminal of a G5 domain. The disorder of E domains in isolation is essential for formation of a long, stiff, mechanically strong, rod-like structure (9) capable of projecting the N-terminal A domain, which is involved in host colonization (6).Here, we combine biophysical measurements, protein engineering, and simulation to show that the disorder in the E domains of SasG also promotes cooperative folding and unfolding pathways. We find that SasG domains have a highly polarized transition-state (TS) structure, where formation of a sm...