Monoclonal antibodies against accumulation-associated protein affect EPS biosynthesis and enhance bacterial accumulation of Staphylococcus epidermidis

Because there is no effective antibiotic to eradicate Staphylococcus epidermidis biofilm infections that lead to the failure of medical device implantations, the development of anti-biofilm vaccines is necessary. Biofilm formation by S. epidermidis requires accumulation-associated protein (Aap) that contains sequence repeats known as G5 domains, which are responsible for the Zn(2+)-dependent dimerization of Aap to mediate intercellular adhesion. Antibodies against Aap have been reported to inhibit biofilm accumulation. In the present study, three monoclonal antibodies (MAbs) against the Aap C-terminal single B-repeat construct followed by the 79-aa half repeat (AapBrpt1.5) were generated. MAb(18B6) inhibited biofilm formation by S. epidermidis RP62A to 60% of the maximum, while MAb(25C11) and MAb(20B9) enhanced biofilm accumulation. All three MAbs aggregated the planktonic bacteria to form visible cell clusters. Epitope mapping revealed that the epitope of MAb(18B6), which recognizes an identical area within AapBrpt constructs from S. epidermidis RP62A, was not shared by MAb(25C11) and MAb(20B9). Furthermore, all three MAbs were found to affect both Aap expression and extracellular polymeric substance (EPS, including extracellular DNA and PIA) biosynthesis in S. epidermidis and enhance the cell accumulation. These findings contribute to a better understanding of staphylococcal biofilm formation and will help to develop epitope-peptide vaccines against staphylococcal infections.     

The metalloprotease SepA governs processing of accumulation‐associated protein and shapes intercellular adhesive surface properties in Staphylococcus epidermidis

The otherwise harmless skin inhabitant Staphylococcus epidermidis is a major cause of healthcare‐associated medical device infections. The species' selective pathogenic potential depends on its production of surface adherent biofilms. The Cell wall‐anchored protein Aap promotes biofilm formation in S. epidermidis, independently from the polysaccharide intercellular adhesin PIA. Aap requires proteolytic cleavage to act as an intercellular adhesin. Whether and which staphylococcal proteases account for Aap processing is yet unknown. Here, evidence is provided that in PIA‐negative S. epidermidis 1457Δica, the metalloprotease SepA is required for Aap‐dependent S. epidermidis biofilm formation in static and dynamic biofilm models. qRT‐PCR and protease activity assays demonstrated that under standard growth conditions, sepA is repressed by the global regulator SarA. Inactivation of sarA increased SepA production, and in turn augmented biofilm formation. Genetic and biochemical analyses demonstrated that SepA‐related induction of biofilm accumulation resulted from enhanced Aap processing. Studies using recombinant proteins demonstrated that SepA is able to cleave the A domain of Aap at residue 335 and between the A and B domains at residue 601. This study identifies the mechanism behind Aap‐mediated biofilm maturation, and also demonstrates a novel role for a secreted staphylococcal protease as a requirement for the development of a biofilm.     

The giant extracellular matrix-binding protein of Staphylococcus epidermidis mediates biofilm accumulation and attachment to fibronectin

Virulence of nosocomial pathogen Staphylococcus epidermidis is essentially related to formation of adherent biofilms, assembled by bacterial attachment to an artificial surface and subsequent production of a matrix that mediates interbacterial adhesion. Growing evidence supports the idea that proteins are functionally involved in S. epidermidis biofilm accumulation. We found that in S. epidermidis 1585v overexpression of a 460 kDa truncated isoform of the extracellular matrix-binding protein (Embp) is necessary for biofilm formation. Embp is a giant fibronectin-binding protein harbouring 59 Found In Various Architectures (FIVAR) and 38 protein G-related albumin-binding (GA) domains. Studies using defined Embp-positive and -negative S.  epidermidis strains proved that Embp is sufficient and necessary for biofilm formation. Further data showed that the FIVAR domains of Embp mediate binding of S. epidermidis to solid-phase attached fibronectin, constituting the first step of biofilm formation on conditioned surfaces. The binding site in fibronectin was assigned to the fibronectin domain type III12. Embp-mediated biofilm formation also protected S. epidermidis from phagocytosis by macrophages. Thus, Embp is a multifunctional cell surface protein that mediates attachment to host extracellular matrix, biofilm accumulation and escape from phagocytosis, and therefore is well suited for promoting implant-associated infections.     

Biofilm formation by Staphylococcus epidermidis depends on functional RsbU, an activator of the sigB operon: differential activation mechanisms due to ethanol and salt stress

Staphylococcus epidermidis is a common pathogen in medical device-associated infections. Its major pathogenetic factor is the ability to form adherent biofilms. The polysaccharide intercellular adhesin (PIA), which is synthesized by the products of the icaADBC gene cluster, is essential for biofilm accumulation. In the present study, we characterized the gene locus inactivated by Tn917 insertions of two isogenic, icaADBC-independent, biofilm-negative mutants, M15 and M19, of the biofilm-producing bacterium S. epidermidis 1457. The insertion site was the same in both of the mutants and was located in the first gene, rsbU, of an operon highly homologous to the sigB operons of Staphylococcus aureus and Bacillus subtilis. Supplementation of Trypticase soy broth with NaCl (TSB(NaCl)) or ethanol (TSB(EtOH)), both of which are known activators of sigB, led to increased biofilm formation and PIA synthesis by S. epidermidis 1457. Insertion of Tn917 into rsbU, a positive regulator of alternative sigma factor sigma(B), led to a biofilm-negative phenotype and almost undetectable PIA production. Interestingly, in TSB(EtOH), the mutants were enabled to form a biofilm again with phenotypes similar to those of the wild type. In TSB(NaCl), the mutants still displayed a biofilm-negative phenotype. No difference in primary attachment between the mutants and the wild type was observed. Similar phenotypic changes were observed after transfer of the Tn917 insertion of mutant M15 to the independent and biofilm-producing strain S. epidermidis 8400. In 11 clinical S. epidermidis strains, a restriction fragment length polymorphism of the sigB operon was detected which was independent of the presence of the icaADBC locus and a biofilm-positive phenotype. Obviously, different mechanisms are operative in the regulation of PIA expression in stationary phase and under stress induced by salt or ethanol.     

ica and beyond: biofilm mechanisms and regulation in Staphylococcus epidermidis and Staphylococcus aureus

Recent progress in elucidating the role of the icaADBC-encoded polysaccharide intercellular adhesin (PIA) or polymeric N-acetyl-glucosamine (PNAG) in staphylococcal biofilm development has in turn contributed significantly to our understanding of the pathogenesis of device-related infections. Nevertheless, our understanding of how the ica locus and PIA/PNAG biosynthesis are regulated is far from complete and many questions remain. Moreover, beyond ica, evidence is now emerging for the existence of ica-independent biofilm mechanisms in both Staphylococcus aureus and Staphylococcus epidermidis. Teichoic acids, which are a major carbohydrate component of the S. epidermidis biofilm matrix and the major cell wall autolysin, play an important role in the primary attachment phase of biofilm development, whereas the cell surface biofilm-associated protein and accumulation-associated protein are capable of mediating intercellular accumulation. These findings raise the exciting prospect that other surface proteins, which typically function as antigenic determinants or in binding to extracellular matrix proteins, may also act as biofilm adhesins. Given the impressive array of surface proteins expressed by S. aureus and S. epidermidis, future research into their potential role in biofilm development either independent of PIA/PNAG or in cooperation with PIA/PNAG will be of particular interest.     

Staphylococcus epidermidis polysaccharide intercellular adhesin activates complement

Staphylococcus epidermidis is a frequent cause of nosocomial infections. The central virulence factor of S. epidermidis is biofilm formation. Polysaccharide intercellular adhesin (PIA) constitutes the major biofilm matrix-component. PIA and biofilm have been implicated in S. epidermidis evasion of host immune defence. We examined the effects of S. epidermidis PIA on the inflammatory response with focus on complement activation. We used a human whole-blood ex vivo model of infection and compared the effects of a PIA-positive S. epidermidis strain (SE1457) and its PIA-negative isogenic mutant (M10). The independent effect of purified PIA on complement activation was investigated. In glucose-rich media, the mutant formed a proteinacious DNA-rich biofilm, whereas SE1457 formed a thick PIA-biofilm. In biofilm growth, SE1457 induced a stronger activation of the complement system compared with M10. We verified that purified PIA was independently responsible for a strong activation of the complement system. In contrast, M10 induced higher granulocyte activation by expression of CD11b and higher secretion of cytokines. We conclude that PIA has potent pro-inflammatory properties by activating the complement system. However, in a complex balance of the immune response, the decreased activation of granulocytes and cytokines by a PIA biofilm may limit host eradication of S. epidermidis.     

Role for the A Domain of Unprocessed Accumulation-Associated Protein (Aap) in the Attachment Phase of the Staphylococcus epidermidis Biofilm Phenotype

The polysaccharide intercellular adhesin or the cell wall-anchored accumulation-associated protein (Aap) mediates cellular accumulation during Staphylococcus epidermidis biofilm maturation. Mutation of sortase, which anchors up to 11 proteins (including Aap) to the cell wall, blocked biofilm development by the cerebrospinal fluid isolate {"type":"entrez-protein","attrs":{"text":"CSF41498","term_id":"904025233","term_text":"CSF41498"}}CSF41498. Aap was implicated in this phenotype when Western blots and two-dimensional (2D) electrophoresis revealed increased levels of the protein in culture supernatants. Unexpectedly, reduced levels of primary attachment were associated with impaired biofilm formation by {"type":"entrez-protein","attrs":{"text":"CSF41498","term_id":"904025233","term_text":"CSF41498"}}CSF41498 srtA and aap mutants. In contrast to previous studies, which implicated Aap proteolytic cleavage and, specifically, the Aap B domains in biofilm accumulation, the {"type":"entrez-protein","attrs":{"text":"CSF41498","term_id":"904025233","term_text":"CSF41498"}}CSF41498 Aap protein was unprocessed. Furthermore, aap appeared to play a less important role in the biofilm phenotype of S. epidermidis 1457, in which the Aap protein is processed. Anti-Aap A-domain IgG inhibited primary attachment and biofilm formation in strain {"type":"entrez-protein","attrs":{"text":"CSF41498","term_id":"904025233","term_text":"CSF41498"}}CSF41498 but not in strain 1457. The nucleotide sequences of the aap gene A-domain region and cleavage site in strains {"type":"entrez-protein","attrs":{"text":"CSF41498","term_id":"904025233","term_text":"CSF41498"}}CSF41498 and 1457 were identical, implicating altered protease activity in the differential Aap processing results in the two strains. These data reveal a new role for the A domain of unprocessed Aap in the attachment phase of biofilm formation and suggest that extracellular protease activity can influence whether Aap contributes to the attachment or accumulation phases of the S. epidermidis biofilm phenotype.     

Staphylococcal biofilm exopolysaccharide protects against Caenorhabditis elegans immune defenses

Staphylococcus epidermidis and Staphylococcus aureus are leading causes of hospital-acquired infections that have become increasingly difficult to treat due to the prevalence of antibiotic resistance in these organisms. The ability of staphylococci to produce biofilm is an important virulence mechanism that allows bacteria both to adhere to living and artificial surfaces and to resist host immune factors and antibiotics. Here, we show that the icaADBC locus, which synthesizes the biofilm-associated polysaccharide intercellular adhesin (PIA) in staphylococci, is required for the formation of a lethal S. epidermidis infection in the intestine of the model nematode Caenorhabditis elegans. Susceptibility to S. epidermidis infection is influenced by mutation of the C. elegans PMK-1 p38 mitogen-activated protein (MAP) kinase or DAF-2 insulin-signaling pathways. Loss of PIA production abrogates nematocidal activity and leads to reduced bacterial accumulation in the C. elegans intestine, while overexpression of the icaADBC locus in S. aureus augments virulence towards nematodes. PIA-producing S. epidermidis has a significant survival advantage over ica-deficient S. epidermidis within the intestinal tract of wild-type C. elegans, but not in immunocompromised nematodes harboring a loss-of-function mutation in the p38 MAP kinase pathway gene sek-1. Moreover, sek-1 and pmk-1 mutants are equally sensitive to wild-type and icaADBC-deficient S. epidermidis. These results suggest that biofilm exopolysaccharide enhances virulence by playing an immunoprotective role during colonization of the C. elegans intestine. These studies demonstrate that C. elegans can serve as a simple animal model for studying host-pathogen interactions involving staphylococcal biofilm exopolysaccharide and suggest that the protective activity of biofilm matrix represents an ancient conserved function for resisting predation.     

葡萄糖类似物甲基葡萄糖对表皮葡萄球菌生物被膜形成的影响及调节机制的研究

生物被膜(Biofilm)是条件致病菌表皮葡萄球菌(Staphylococcusepidermidis)的主要致病因素,生物被膜的形成依赖多糖PIA的合成,PIA合成与细菌糖代谢相关。通过研究葡萄糖类似物甲基葡萄糖(MethylDglucoside,MG)对生物被膜的形成及相关基因表达的影响,考察生物被膜形成的调控机制并寻找抑制生物被膜形成的方法。甲基葡萄糖能抑制97337株生物被膜的形成,而且不同浓度的甲基葡萄糖对生物膜作用不同。甲基葡萄糖对97337株生物被膜形成的早期的粘附有较强的抑制作用;不同浓度的甲基葡萄糖处理后对ica和AtlE基因的mRNA表达水平影响不大,但能诱导agr基因的表达,这与甲基葡萄糖处理不同时间后的结果一致;而且甲基葡萄糖处理后97337的表面相关蛋白的组成明显改变。甲基葡萄糖对生物膜的抑制并不直接由于它对生长的抑制,它对细菌生长和生物被膜形成的抑制与其在细菌糖代谢中的竞争性相关;甲基葡萄糖能通过调控agr基因的表达改变细菌表面从而抑制97337的早期粘附和生物被膜的形成,但没有通过调控icaADBC、icaR的表达抑制生物膜的形成,可能与其对合成PIA相关糖基转移酶的竞争性抑制相关。     

Modulation of poly-N-acetylglucosamine accumulation within mature Staphylococcus epidermidis biofilms grown in excess glucose

PNAG is a major component of Staphylococcus epidermidis biofilms involved in intercellular adhesion as well as in the interaction of the biofilm with components of the host immune response. Synthesis of PNAG has been found to be regulated by several environmental factors. In the present study, the effect of glucose metabolism-dependent culture medium acidification in PNAG accumulation was evaluated. Established S. epidermidis biofilms were allowed to grow in excess glucose with or without maintained pH conditions. PNAG accumulation in these biofilms was determined by flow cytometry and fluorescence microscopy using wheat germ agglutinin as a fluorescent probe. Biofilms grown in maintained pH conditions presented significantly higher amounts of this polymer as well as higher icaA expression than biofilms grown in acidic pH conditions. Moreover, PNAG accumulation in biofilms grown in non-maintained pH conditions occurred in association with cell death. Overall, we show that glucose metabolism by decreasing the culture pH affects biofilm physiology in respect to PNAG production and cell death. The reported in vitro modulation of PNAG accumulation within S. epidermidis biofilms further highlights the role of environment on determining the biofilm physiological state.     

五倍子水煎剂对表皮葡萄球菌生物膜抑制的研究

通过五倍子水煎剂对表皮葡萄球菌MIC测定和生物膜形成干预的研究,为表皮葡萄球菌引起感染提供新的治疗途径。用微量肉汤稀释法分别测定五倍子水煎剂对表皮葡萄球菌的MIC;刚果红及刚果红红霉素、五倍子水煎剂琼脂平板测定表皮葡萄球菌PIA生成与抑制;五倍子水煎剂、红霉素干预表皮葡萄球菌生物膜形成,于光镜和电镜下观察其生物膜形态。134株表皮葡萄球菌五倍子水煎剂的MIC50为0.488 mg/mL,MIC90为0.977 mg/mL。134株表皮葡萄球菌中有50株为PIA阳性,PIA阳性的50株菌全部产生生物膜,红霉素对表皮葡萄球菌生物膜形成有抑制,而五倍子水煎剂则无。表皮葡萄球菌PIA的相互作用在其生物膜的生成中起主要作用;五倍子水煎剂对表皮葡萄球菌生长有明显的抑制但对生物膜形成无干预作用。     

Solution Structural Studies of Pre-amyloid Oligomer States of the Biofilm Protein Aap

Staphylococcus epidermidis is a commensal bacterium on human skin that is also the leading cause of medical device-related infections. The accumulation-associated protein (Aap) from S. epidermidis is a critical factor for infection via its ability to mediate biofilm formation. The B-repeat superdomain of Aap is composed of 5 to 17 Zn²⁺-binding B-repeats, which undergo rapid, reversible assembly to form dimer and tetramer species. The tetramer can then undergo a conformational change and nucleate highly stable functional amyloid fibrils. In this study, multiple techniques including analytical ultracentrifugation (AUC) and small-angle X-ray scattering (SAXS) are used to probe a panel of B-repeat mutant constructs that assemble to distinct oligomeric states to define the structural characteristics of B-repeat dimer and tetramer species. The B-repeat region from Aap forms an extremely elongated conformation that presents several challenges for standard SAXS analyses. Specialized approaches, such as cross-sectional analyses, allowed for in-depth interpretation of data, while explicit-solvent calculations via WAXSiS allowed for accurate evaluation of atomistic models. The resulting models suggest mechanisms by which Aap functional amyloid fibrils form, illuminating an important contributing factor to recurrent staphylococcal infections.     

An 18 kDa Scaffold Protein Is Critical for Staphylococcus epidermidis Biofilm Formation

Virulence of the nosocomial pathogen Staphylococcus epidermidis is crucially linked to formation of adherent biofilms on artificial surfaces. Biofilm assembly is significantly fostered by production of a bacteria derived extracellular matrix. However, the matrix composition, spatial organization, and relevance of specific molecular interactions for integration of bacterial cells into the multilayered biofilm community are not fully understood. Here we report on the function of novel 18 kDa Small basic protein (Sbp) that was isolated from S. epidermidis biofilm matrix preparations by an affinity chromatographic approach. Sbp accumulates within the biofilm matrix, being preferentially deposited at the biofilm–substratum interface. Analysis of Sbp-negative S. epidermidis mutants demonstrated the importance of Sbp for sustained colonization of abiotic surfaces, but also epithelial cells. In addition, Sbp promotes assembly of S. epidermidis cell aggregates and establishment of multilayered biofilms by influencing polysaccharide intercellular-adhesin (PIA) and accumulation associated protein (Aap) mediated intercellular aggregation. While inactivation of Sbp indirectly resulted in reduced PIA-synthesis and biofilm formation, Sbp serves as an essential ligand during Aap domain-B mediated biofilm accumulation. Our data support the conclusion that Sbp serves as an S. epidermidis biofilm scaffold protein that significantly contributes to key steps of surface colonization. Sbp-negative S. epidermidis mutants showed no attenuated virulence in a mouse catheter infection model. Nevertheless, the high prevalence of sbp in commensal and invasive S. epidermidis populations suggests that Sbp plays a significant role as a co-factor during both multi-factorial commensal colonization and infection of artificial surfaces.     

In vitro activity of allicin against Staphylococcus epidermidis and influence of subinhibitory concentrations on biofilm formation

AIMS: The aim of this study is to determine the in vitro activity of allicin against Staphylococcus epidermidis and to evaluate the influence of allicin on biofilm formation. METHODS AND RESULTS: In vitro activity of allicin (diallyl thiosulphinate) against 38 strains of S. epidermidis was investigated. The activity of allicin was similar against S. epidermidis methicillin susceptible and methicillin resistant strains [minimum inhibitory concentration (MIC)90=8 mg l(-1)]. In general, subinhibitory concentrations (sub-MIC) of allicin diminished biofilm formation in the five strains analysed. CONCLUSION: The results confirm the antibacterial effect of allicin. Sub-MICs of allicin also diminished the biofilm formations by S. epidermidis. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study shows that allicin is active in vitro against S. epidermidis and that sub-MICs of allicin may play a role in the prevention of adherence of this bacteria to medical devices.