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SdrC induces staphylococcal biofilm formation through a homophilic interaction.
Serine-aspartate repeat-containing protein C

Systems used to automatically annotate proteins with high accuracy:. Select item s and click on "Add to basket" to create your own collection here entries max. Manual assertion according to rules i. Interacts with host NRXN1; this interaction mediates bacterial attachment to host cells. Manual assertion inferred from sequence similarity to i. You are using a version of browser that may not display all the features of this website.

SdrC induces staphylococcal biofilm formation through a homophilic interaction.

Either your web browser doesn't support Javascript or it is currently turned off. In the latter case, please turn on Javascript support in your web browser and reload this page. Free to read. The molecular pathogenesis of many Staphylococcus aureus infections involves growth of bacteria as biofilm.

In addition to polysaccharide intercellular adhesin PIA and extracellular DNA, surface proteins appear to mediate the transition of bacteria from planktonic growth to sessile lifestyle as well as biofilm growth, and can enable these processes even in the absence of PIA expression. However, the molecular mechanisms by which surface proteins contribute to biofilm formation are incompletely understood.

Here we demonstrate that self-association of the serine-aspartate repeat protein SdrC promotes both bacterial adherence to surfaces and biofilm formation. However, this homophilic interaction is not required for the attachment of bacteria to abiotic surfaces.

We identified the subdomain that mediates SdrC dimerization and subsequent cell-cell interactions. In addition, we determined that two adjacently located amino acid sequences within this subdomain are required for the SdrC homophilic interaction.

Comparative amino acid sequence analysis indicated that these binding sites are conserved. In summary, our study identifies SdrC as a novel molecular determinant in staphylococcal biofilm formation and describes the mechanism responsible for intercellular interactions.

Furthermore, these findings contribute to a growing body of evidence suggesting that homophilic interactions between surface proteins present on neighboring bacteria induce biofilm growth. Bacterial biofilms are communities of microorganisms growing attached to biotic or abiotic surfaces. Within the biofilm, bacteria encase themselves in a self-secreted matrix with precise micro-architectural properties that allow free circulation of nutrients, water and metabolites Costerton et al.

Besides acting as a scaffold, the matrix enables microbes to survive and persist as reservoirs, avoid desiccation or antimicrobials, and subvert host defenses. Thus, it has long been recognized that their effective therapy management is challenging. Widely known as an opportunistic pathogen, Staphylococcus aureus is a frequent cause of biofilm-related infections Otto, The transition of staphylococci from planktonic organisms to sessile growth is triggered by environmental cues or receptor availability Donlan, Overall bacterial surface hydrophobicity determines the degree of attachment to inert materials Pasqual et al.

In the case of S. Upon attachment, bacteria proliferate and synthesize a scaffolding matrix composed of intercellular polysaccharide adhesin PIA Cramton et al. Accumulating evidence suggest that CWA proteins important for biofilm growth include b iofilm a ssociated p rotein Bap Cucarella et al.

However, Bap has been found only in bovine mastitis isolates Cucarella et al. The molecular mechanism by which these proteins contribute to intercellular interaction is largely unknown, but recent studies suggest the involvement of metal-ion dependent interactions between CWA proteins present on adjacent cells Conrady et al. The initiation and development of biofilm appears to be chiefly regulated by the quorum sensing accessory gene regulator Agr system in response to cell density.

Typically, at low density staphylococci express surface proteins involved in cell attachment and biofilm accumulation Periasamy et al. When the population reaches high density, Agr activation triggers the upregulation of toxin, proteases and surfactants involved in biofilm dispersal and bacterial dissemination. Conversely, inactivation of Agr leads to an augmented biofilm phenotype, in part due to the deregulation of surface proteins involved in adherence and biofilm growth.

In agreement, recent studies have indicated that these mutants form a thicker and more resistant biofilms presumably due to perpetual expression of CWA proteins that engage newborn cells within the structure. Notably, agr spontaneous mutants are frequently isolated from biofilms Shopsin et al. An amino terminal signal sequence is followed by the ligand-binding A region subdivided into three subdomains N1, N2, and N3 , where N2 and N3 each adopt an IgG-like fold.

The carboxyl terminal section of the proteins contains the LPXTG motif required for cell wall anchoring reviewed in Foster et al. Schematic overall representation of SdrC domains. Also shown, are the enriched phage-displayed peptides aligned with the relevant SdrC sequence. Consensus sequences are highlighted in yellow. Binding was detected with an anti-MHRP antibody. The data shown is representative of three individual experiments performed in triplicate.

Using phage display, we previously identified peptides targeting the putative ligand-binding region of SdrC. As a result, SdrC promotes bacterial intercellular interactions and contributes to biofilm formation. Exploiting heterologous expression in the non-pathogenic bacterium Lactococcus lactis , we show that SdrC also triggers bacterial adhesion to abiotic surfaces.

However, bacterial attachment to uncoated inert surfaces is independent of self-association. Moreover, our results indicate that the SdrC contribution to biofilm formation is dependent on the strain background. We found that these sequences are located adjacent to each other positions — and — in the C-terminal region of the N2 subdomain of SdrC itself Fig.

Considering that the peptide sequences identified by phage display are found in the bait protein, we reasoned that SdrC interacts with itself. We compared the binding of SdrC subdomains to each other using a solid-phase binding assay. Recombinant polypeptides containing the N2 subdomain showed significant levels of binding to N2 subdomain-containing proteins relative to N1 and B segments Fig. To establish binding specificity, either N2 or N2N3 recombinant proteins were immobilized on microtiter plates and probed with increasing concentrations of biotin-labeled subdomains.

We found that N2-containing proteins bound in a dose-dependent and saturable manner to both proteins. The apparent dissociation constant corresponding to the concentration needed for half maximum binding ranged between 0. To further assess the binding specificity, we tested the ability of consensus peptides to inhibit N2-mediated SdrC self-association.

Of importance, the binding was completely inhibited only when both phage clones displaying consensus sequences were combined, suggesting that the identified binding sites act cooperatively Fig.

An insertless phage had no effect on the N2 subdomain interaction with its partner. The interaction was detected with avidin-HRP. B and C. Dose-dependent and saturable binding of N2 red and N2N3 blue subdomains to one another.

Increasing concentrations of biotin-labeled subdomains were incubated with either immobilized B unlabeled N2 or C N2N3. D and E. Immobilized recombinant SdrC N2N3 protein was first allowed to interact with phage or antibodies and then incubated with biotin-labeled recombinant SdrC N2 protein. Insertless phage and anti-ClfB antibodies were used as negative controls.

N2 subdomain-mediated dimer formation demonstrated by gel permeation chromatography. Gel phase distribution coefficients K AV were calculated from the respective elution volumes Ve and represented as a function of molecular mass. The relative molecular masses calculated from the linear regression based on column calibration with globular proteins revealed a 40 KDa dimeric species and an 18 KDa form indicative of a monomeric state for rSdrC N2 Fig.

In contrast, rSdrC N3 eluted from the column as a single peak with a relative molecular mass of 19 KDa, which is similar to the calculated theoretical mass of the monomer Fig. These results indicate that the N2 subdomain mediates SdrC self-association.

An earlier study investigating biofilm formation by S. Since surface molecule self-association may lead to intercellular interactions, we hypothesized that SdrC may be involved in biofilm formation. We compared the biofilm growth of the parent strain S.

A mutation in sdrC significantly inhibited biofilm formation, whereas an sdrD single mutant had no detectable effect Fig. Complete elimination of biofilm was not detected because these strains likely express additional factors involved in biofilm accumulation.

To clarify this, we assessed the biofilm formed by S. We found that SdrC expression restored biofilm formation Fig. To determine whether additional staphylococcal factors are necessary for SdrC activity, we exploited the apathogenic bacterium L.

Biofilm accumulation by lactococci constitutively expressing CWA proteins known to promote this type of growth was significant relative to the parent strain carrying the empty vector pKS Furthermore, a strong correlation between inducible surface protein expression and biofilm formation was observed for both SdrC and ClfB Fig. A and B.

C and D. Biofilm formation in M17 medium containing 0. Heterologous protein expression was induced with increasing concentrations of nisin. Static biofilm formation was measured by staining with 0. Representative images of wells containing the CV extracted from previously stained biofilm are shown above the graphs. The data shown is representative of six individual experiments.

Representative images of colony swarming motility are shown above the graphs. Previous work showed that MSCRAMMs, such as FnBPA and ClfB, involved in cell-cell aggregation and biofilm formation antagonize colony spreading motility on soft agar consistent with the hypothesis that these proteins promote intercellular adherence Tsompanidou et al.

Similarly, SdrC also reduced the efficiency of spreading of lactococci on wet surfaces, whereas SdrE had no effect Fig. These data demonstrate that SdrC contributes to staphylococcal intercellular interactions and subsequent biofilm formation in vitro.

To further investigate the mechanism of SdrC self-association, we determined the effect of anti-SdrC N2N3 antibodies on biofilm formation. SasG-driven lactococcal biofilm was not affected by the antiserum, whereas biofilm formation by L.

Next, we compared the abilities of different recombinant protein segments to inhibit biofilm formation. The rSdrC N3 protein had no effect on L. Despite repeated attempts, we were unable to determine a concentration at which biofilm was completely eradicated by the addition of recombinant SdrC fragments.

We hypothesized that the residual biofilm is due to an SdrC-dependent bacterial adherence to plastic. Thus, we assessed the ability of L. We detected complete inhibition of bacterial attachment to plastic in the presence of antibodies, whereas rSdrC N2N3 had no significant effect Fig.

These results suggest that SdrC promotes both bacterial adherence to plastic and biofilm growth. However, SdrC self-association is important only for biofilm accumulation. Inhibition of L. After one hour, unbound bacteria were removed by washing with PBS.

Bound bacteria were stained with 0.

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