RESUMEN
During infection, the host employs nutritional immunity to restrict access to iron. Staphylococcus lugdunensis has been recognized for its ability to utilize host-derived heme to overcome iron restriction. However, the mechanism behind this process involves the release of hemoglobin from erythrocytes, and the hemolytic factors of S. lugdunensis remain poorly understood. S. lugdunensis encodes four phenol-soluble modulins (PSMs), short peptides with hemolytic activity. The peptides SLUSH A, SLUSH B, and SLUSH C are ß-type PSMs, and OrfX is an α-type PSM. Our study shows the SLUSH locus to be essential for the hemolytic phenotype of S. lugdunensis. All four peptides individually exhibited hemolytic activity against human and sheep erythrocytes, but synergism with sphingomyelinase was observed exclusively against sheep erythrocytes. Furthermore, our findings demonstrate that SLUSH is crucial for allowing the utilization of erythrocytes as the sole source of nutritional iron and confirm the transcriptional regulation of SLUSH by Agr. Additionally, our study reveals that SLUSH peptides stimulate the human immune system. Our analysis identifies SLUSH as a pivotal hemolytic factor of S. lugdunensis and demonstrates its concerted action with heme acquisition systems to overcome iron limitation in the presence of host erythrocytes.
Asunto(s)
Eritrocitos , Hemólisis , Hierro , Staphylococcus lugdunensis , Eritrocitos/metabolismo , Staphylococcus lugdunensis/metabolismo , Humanos , Hierro/metabolismo , Animales , Ovinos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Péptidos/metabolismo , Péptidos/química , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/metabolismo , Toxinas BacterianasRESUMEN
Staphylococcus lugdunensis is a species of coagulase-negative staphylococcus (CoNS) that causes serious infections in humans akin to those of S. aureus It was often misidentified as S. aureus, but this has been rectified by recent routine use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in diagnostic laboratories. It encodes a diverse array of virulence factors for adhesion, cytotoxicity, and innate immune evasion, but these are less diverse than those encoded by S. aureus It expresses an iron-regulated surface determinant (Isd) system combined with a novel energy-coupling factor (ECF) mechanism for extracting heme from hemoproteins. Small cytolytic S. lugdunensis synergistic hemolysins (SLUSH), peptides related to phenol-soluble modulins of S. aureus, act synergistically with ß-toxin to lyse erythrocytes. S. lugdunensis expresses a novel peptide antibiotic, lugdunin, that can influence the nasal and skin microbiota. Endovascular infections are initiated by bacterial adherence to fibrinogen promoted by a homologue of Staphylococcus aureus clumping factor A and to von Willebrand factor on damaged endothelium by an uncharacterized mechanism. S. lugdunensis survives within mature phagolysosomes of macrophages without growing and is released only following apoptosis. This differs fundamentally from S. aureus, which actively grows and expresses bicomponent leukotoxins that cause membrane damage and could contribute to survival in the infected host. S. lugdunensis is being investigated as a probiotic to eradicate S. aureus from the nares of carriers. However, this is contraindicated by its innate virulence. Studies to obtain a deeper understanding of S. lugdunensis colonization, virulence, and microbiome interactions are therefore warranted.
Asunto(s)
Infecciones Estafilocócicas , Staphylococcus lugdunensis , Humanos , Hierro , Staphylococcus aureus , Factores de VirulenciaRESUMEN
Staphylococcus aureus is an important bacterial pathogen that can cause a wide spectrum of diseases in humans and other animals. S. aureus expresses a variety of virulence factors that promote infection with this pathogen. These include cell-surface proteins that mediate adherence of the bacterial cells to host extracellular matrix components, such as fibronectin and fibrinogen. Here, using immunoblotting, ELISA, and surface plasmon resonance analysis, we report that the iron-regulated surface determinant B (IsdB) protein, besides being involved in heme transport, plays a novel role as a receptor for the plasma and extracellular matrix protein vitronectin (Vn). Vn-binding activity was expressed by staphylococcal strains grown under iron starvation conditions when Isd proteins are expressed. Recombinant IsdB bound Vn dose dependently and specifically. Both near-iron transporter motifs NEAT1 and NEAT2 of IsdB individually bound Vn in a saturable manner, with KD values in the range of 16-18 nm Binding of Vn to IsdB was specifically blocked by heparin and reduced at high ionic strength. Furthermore, IsdB-expressing bacterial cells bound significantly higher amounts of Vn from human plasma than did an isdB mutant. Adherence to and invasion of epithelial and endothelial cells by IsdB-expressing S. aureus cells was promoted by Vn, and an αvß3 integrin-blocking mAb or cilengitide inhibited adherence and invasion by staphylococci, suggesting that Vn acts as a bridge between IsdB and host αvß3 integrin.
Asunto(s)
Proteínas de Transporte de Catión/química , Staphylococcus aureus/química , Vitronectina/química , Proteínas de Transporte de Catión/metabolismo , Humanos , Integrina alfaVbeta3/química , Integrina alfaVbeta3/metabolismo , Unión Proteica , Staphylococcus aureus/metabolismo , Vitronectina/metabolismoRESUMEN
[This corrects the article DOI: 10.1371/journal.pgen.1006246.].
RESUMEN
Staphylococcus aureus is a Gram-positive bacterium that can cause both superficial and deep-seated infections. Histones released by neutrophils kill bacteria by binding to the bacterial cell surface and causing membrane damage. We postulated that cell wall-anchored proteins protect S. aureus from the bactericidal effects of histones by binding to and sequestering histones away from the cell envelope. Here, we focused on S. aureus strain LAC and by using an array of biochemical assays, including surface plasmon resonance and ELISA, discovered that fibronectin-binding protein B (FnBPB) is the main histone receptor. FnBPB bound all types of histones, but histone H3 displayed the highest affinity and bactericidal activity and was therefore investigated further. H3 bound specifically to the A domain of recombinant FnBPB with a KD of 86 nm, â¼20-fold lower than that for fibrinogen. Binding apparently occurred by the same mechanism by which FnBPB binds to fibrinogen, because FnBPB variants defective in fibrinogen binding also did not bind H3. An FnBPB-deletion mutant of S. aureus LAC bound less H3 and was more susceptible to its bactericidal activity and to neutrophil extracellular traps, whereas an FnBPB-overexpressing mutant bound more H3 and was more resistant than the WT. FnBPB bound simultaneously to H3 and plasminogen, which after activation by tissue plasminogen activator cleaved the bound histone. We conclude that FnBPB provides a dual immune-evasion function that captures histones and prevents them from reaching the bacterial membrane and simultaneously binds plasminogen, thereby promoting its conversion to plasmin to destroy the bound histone.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Antiinfecciosos/farmacología , Histonas/farmacología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/metabolismo , Antiinfecciosos/metabolismo , Pared Celular/efectos de los fármacos , Pared Celular/metabolismo , Histonas/metabolismo , Concentración Osmolar , Plasminógeno/metabolismo , Unión Proteica , Staphylococcus aureus/citologíaRESUMEN
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 ß-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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Biopelículas , Proteínas del Tejido Nervioso/química , Péptidos/farmacología , Staphylococcus aureus/fisiología , Adhesión Bacteriana , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Sitios de Unión , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Péptidos/química , Unión Proteica , Análisis de la Célula IndividualRESUMEN
Staphylococcus aureus surface protein SasG promotes cell-cell adhesion during the accumulation phase of biofilm formation, but the molecular basis of this interaction remains poorly understood. Here, we unravel the mechanical properties of SasG on the surface of living bacteria, that is, in its native cellular environment. Nanoscale multiparametric imaging of living bacteria reveals that Zn(2+) strongly increases cell wall rigidity and activates the adhesive function of SasG. Single-cell force measurements show that SasG mediates cell-cell adhesion via specific Zn(2+)-dependent homophilic bonds between ß-sheet-rich G5-E domains on neighboring cells. The force required to unfold individual domains is remarkably strong, up to â¼500 pN, thus explaining how SasG can withstand physiological shear forces. We also observe that SasG forms homophilic bonds with the structurally related accumulation-associated protein of Staphylococcus epidermidis, suggesting the possibility of multispecies biofilms during host colonization and infection. Collectively, our findings support a model in which zinc plays a dual role in activating cell-cell adhesion: adsorption of zinc ions to the bacterial cell surface increases cell wall cohesion and favors the projection of elongated SasG proteins away from the cell surface, thereby enabling zinc-dependent homophilic bonds between opposing cells. This work demonstrates an unexpected relationship between mechanics and adhesion in a staphylococcal surface protein, which may represent a general mechanism among bacterial pathogens for activating cell association.
Asunto(s)
Proteínas Bacterianas/metabolismo , Biopelículas/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Staphylococcus aureus/fisiología , Zinc/farmacología , Adhesión Bacteriana/efectos de los fármacos , Proteínas Bacterianas/química , Fenómenos Biomecánicos/efectos de los fármacos , Fenómenos Biofísicos/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Pared Celular/efectos de los fármacos , Pared Celular/metabolismo , Proteínas de la Membrana/química , Microscopía de Fuerza Atómica , Modelos Biológicos , Estructura Terciaria de Proteína , Staphylococcus aureus/efectos de los fármacosRESUMEN
Staphylococcus lugdunensis is a coagulase negative bacterial pathogen that is particularly associated with severe cases of infectious endocarditis. Unique amongst the coagulase-negative staphylococci, S. lugdunensis harbors an iron regulated surface determinant locus (isd). This locus facilitates the acquisition of heme as a source of nutrient iron during infection and allows iron limitation caused by "nutritional immunity" to be overcome. The isd locus is duplicated in S. lugdunensis HKU09-01 and we show here that the duplication is intrinsically unstable and undergoes accordion-like amplification and segregation leading to extensive isd copy number variation. Amplification of the locus increased the level of expression of Isd proteins and improved binding of hemoglobin to the cell surface of S. lugdunensis. Furthermore, Isd overexpression provided an advantage when strains were competing for a limited amount of hemoglobin as the sole source of iron. Gene duplications and amplifications (GDA) are events of fundamental importance for bacterial evolution and are frequently associated with antibiotic resistance in many species. As such, GDAs are regarded as evolutionary adaptions to novel selective pressures in hostile environments pointing towards a special importance of isd for S. lugdunensis. For the first time we show an example of a GDA that involves a virulence factor of a Gram-positive pathogen and link the GDA directly to a competitive advantage when the bacteria were struggling with selective pressures mimicking "nutritional immunity".
Asunto(s)
Endocarditis Bacteriana/microbiología , Hierro/metabolismo , Infecciones Estafilocócicas/metabolismo , Staphylococcus lugdunensis/metabolismo , Variaciones en el Número de Copia de ADN/genética , Endocarditis Bacteriana/genética , Duplicación de Gen , Sitios Genéticos/genética , Hemo/genética , Hemo/metabolismo , Hemoglobinas/genética , Hemoglobinas/metabolismo , Humanos , Proteínas Reguladoras del Hierro/genética , Proteínas Reguladoras del Hierro/metabolismo , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/patología , Staphylococcus lugdunensis/patogenicidad , Propiedades de SuperficieRESUMEN
Staphylococcus aureus persistently colonizes about 20 % of the population and is intermittently associated with the remainder. The organism can cause superficial skin infections and life-threatening invasive diseases. The surface of the bacterial cell displays a variety of proteins that are covalently anchored to peptidoglycan. They perform many functions including adhesion to host cells and tissues, invasion of non-phagocytic cells, and evasion of innate immune responses. The proteins have been categorized into distinct classes based on structural and functional analysis. Many surface proteins are multifunctional. Cell wall-anchored proteins perform essential functions supporting survival and proliferation during the commensal state and during invasive infections. The ability of cell wall-anchored proteins to bind to desquamated epithelial cells is important during colonization, and the binding to fibrinogen is of particular significance in pathogenesis.
Asunto(s)
Staphylococcus aureus , Adhesión Bacteriana , Proteínas Bacterianas , Pared Celular , Proteínas de la MembranaRESUMEN
Staphylococcus aureus is a commensal bacterium that has the ability to cause superficial and deep-seated infections. Like several other invasive pathogens, S. aureus can capture plasminogen from the human host where it can be converted to plasmin by host plasminogen activators or by endogenously expressed staphylokinase. This study demonstrates that sortase-anchored cell wall-associated proteins are responsible for capturing the bulk of bound plasminogen. Two cell wall-associated proteins, the fibrinogen- and fibronectin-binding proteins A and B, were found to bind plasminogen, and one of them, FnBPB, was studied in detail. Plasminogen captured on the surface of S. aureus- or Lactococcus lactis-expressing FnBPB could be activated to the potent serine protease plasmin by staphylokinase and tissue plasminogen activator. Plasminogen bound to recombinant FnBPB with a KD of 0.532 µm as determined by surface plasmon resonance. Plasminogen binding did not to occur by the same mechanism through which FnBPB binds to fibrinogen. Indeed, FnBPB could bind both ligands simultaneously indicating that their binding sites do not overlap. The N3 subdomain of FnBPB contains the full plasminogen-binding site, and this includes, at least in part, two conserved patches of surface-located lysine residues that were recognized by kringle 4 of the host protein.
Asunto(s)
Adhesinas Bacterianas/química , Proteínas Bacterianas/química , Plasminógeno/química , Staphylococcus aureus/química , Adhesinas Bacterianas/metabolismo , Proteínas Bacterianas/metabolismo , Humanos , Plasminógeno/metabolismo , Unión Proteica , Dominios Proteicos , Staphylococcus aureus/metabolismoRESUMEN
Staphylococcus aureus skin infection is a frequent and recurrent problem in children with the common inflammatory skin disease atopic dermatitis (AD). S. aureus colonizes the skin of the majority of children with AD and exacerbates the disease. The first step during colonization and infection is bacterial adhesion to the cornified envelope of corneocytes in the outer layer, the stratum corneum. Corneocytes from AD skin are structurally different from corneocytes from normal healthy skin. The objective of this study was to identify bacterial proteins that promote the adherence of S. aureus to AD corneocytes. S. aureus strains from clonal complexes 1 and 8 were more frequently isolated from infected AD skin than from the nasal cavity of healthy children. AD strains had increased ClfB ligand binding activity compared to normal nasal carriage strains. Adherence of single S. aureus bacteria to corneocytes from AD patients ex vivo was studied using atomic force microscopy. Bacteria expressing ClfB recognized ligands distributed over the entire corneocyte surface. The ability of an isogenic ClfB-deficient mutant to adhere to AD corneocytes compared to that of its parent clonal complex 1 clinical strain was greatly reduced. ClfB from clonal complex 1 strains had a slightly higher binding affinity for its ligand than ClfB from strains from other clonal complexes. Our results provide new insights into the first step in the establishment of S. aureus colonization in AD patients. ClfB is a key adhesion molecule for the interaction of S. aureus with AD corneocytes and represents a target for intervention.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Dermatitis Atópica/microbiología , Células Epiteliales/microbiología , Infecciones Cutáneas Estafilocócicas/microbiología , Staphylococcus aureus/metabolismo , Adhesinas Bacterianas/genética , Adhesión Bacteriana , Preescolar , Femenino , Proteínas Filagrina , Humanos , Masculino , Cavidad Nasal/microbiología , Eliminación de Secuencia , Piel/citología , Piel/microbiología , Staphylococcus aureus/genéticaRESUMEN
Mechanisms of protective immunity to Staphylococcus aureus infection in humans remain elusive. While the importance of cellular immunity has been shown in mice, T cell responses in humans have not been characterised. Using a murine model of recurrent S. aureus peritonitis, we demonstrated that prior exposure to S. aureus enhanced IFNγ responses upon subsequent infection, while adoptive transfer of S. aureus antigen-specific Th1 cells was protective in naïve mice. Translating these findings, we found that S. aureus antigen-specific Th1 cells were also significantly expanded during human S. aureus bloodstream infection (BSI). These Th1 cells were CD45RO+, indicative of a memory phenotype. Thus, exposure to S. aureus induces memory Th1 cells in mice and humans, identifying Th1 cells as potential S. aureus vaccine targets. Consequently, we developed a model vaccine comprising staphylococcal clumping factor A, which we demonstrate to be an effective human T cell antigen, combined with the Th1-driving adjuvant CpG. This novel Th1-inducing vaccine conferred significant protection during S. aureus infection in mice. This study notably advances our understanding of S. aureus cellular immunity, and demonstrates for the first time that a correlate of S. aureus protective immunity identified in mice may be relevant in humans.
Asunto(s)
Memoria Inmunológica , Infecciones Estafilocócicas/inmunología , Infecciones Cutáneas Estafilocócicas/microbiología , Staphylococcus aureus/inmunología , Células TH1/inmunología , Adyuvantes Inmunológicos/farmacología , Traslado Adoptivo , Adulto , Anciano , Animales , Antígenos/inmunología , Femenino , Humanos , Interleucina-17/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Infecciones Cutáneas Estafilocócicas/inmunología , Células TH1/efectos de los fármacosRESUMEN
Staphylococcus aureus biofilms, a leading cause of persistent infections, are highly resistant to immune defenses and antimicrobial therapies. In the present study, we investigated the contribution of fibrin and staphylokinase (Sak) to biofilm formation. In both clinical S. aureus isolates and laboratory strains, high Sak-producing strains formed less biofilm than strains that lacked Sak, suggesting that Sak prevents biofilm formation. In addition, Sak induced detachment of mature biofilms. This effect depended on plasminogen activation by Sak. Host-derived fibrin, the main substrate cleaved by Sak-activated plasminogen, was a major component of biofilm matrix, and dissolution of this fibrin scaffold greatly increased susceptibility of biofilms to antibiotics and neutrophil phagocytosis. Sak also attenuated biofilm-associated catheter infections in mouse models. In conclusion, our results reveal a novel role for Sak-induced plasminogen activation that prevents S. aureus biofilm formation and induces detachment of existing biofilms through proteolytic cleavage of biofilm matrix components.
Asunto(s)
Biopelículas/efectos de los fármacos , Metaloendopeptidasas/metabolismo , Staphylococcus aureus/metabolismo , Animales , Antibacterianos/farmacología , Farmacorresistencia Bacteriana/efectos de los fármacos , Femenino , Fibrina/metabolismo , Masculino , Metaloendopeptidasas/farmacología , Ratones , Ratones Endogámicos C57BL , Plasminógeno/metabolismo , Staphylococcus aureus/efectos de los fármacosRESUMEN
In this study, we investigated the cell wall-anchored fibronectin-binding proteins SpsD and SpsL from the canine commensal and pathogen Staphylococcus pseudintermedius for their role in promoting bacterial invasion of canine progenitor epidermal keratinocytes (CPEK). Invasion was examined by the gentamicin protection assay and fluorescence microscopy. An ΔspsD ΔspsL mutant of strain ED99 had a dramatically reduced capacity to invade CPEK monolayers, while no difference in the invasion level was observed with single mutants. Lactococcus lactis transformed with plasmids expressing SpsD and SpsL promoted invasion, showing that both proteins are important. Soluble fibronectin was required for invasion, and an RGD-containing peptide or antibodies recognizing the integrin α5ß1 markedly reduced invasion, suggesting an important role for the integrin in this process. Src kinase inhibitors effectively blocked internalization, suggesting a functional role for the kinase in invasion. In order to identify the minimal fibronectin-binding region of SpsD and SpsL involved in the internalization process, recombinant fragments of both proteins were produced. The SpsD520-846 and SpsL538-823 regions harboring the major fibronectin-binding sites inhibited S. pseudintermedius internalization. Finally, the effects of staphylococcal invasion on the integrity of different cell lines were examined. Because SpsD and SpsL are critical factors for adhesion and invasion, blocking these processes could provide a strategy for future approaches to treating infections.
Asunto(s)
Proteínas Bacterianas/metabolismo , Enfermedades de los Perros/microbiología , Células Epiteliales/microbiología , Fibronectinas/metabolismo , Infecciones Estafilocócicas/veterinaria , Staphylococcus/metabolismo , Animales , Proteínas Bacterianas/genética , Línea Celular , Pared Celular/genética , Pared Celular/metabolismo , Enfermedades de los Perros/metabolismo , Perros , Unión Proteica , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/microbiología , Staphylococcus/genética , Staphylococcus/patogenicidad , VirulenciaRESUMEN
The capacity for intracellular survival within phagocytes is likely a critical factor facilitating the dissemination of Staphylococcus aureus in the host. To date, the majority of work on S. aureus-phagocyte interactions has focused on neutrophils and, to a lesser extent, macrophages, yet we understand little about the role played by dendritic cells (DCs) in the direct killing of this bacterium. Using bone marrow-derived DCs (BMDCs), we demonstrate for the first time that DCs can effectively kill S. aureus but that certain strains of S. aureus have the capacity to evade DC (and macrophage) killing by manipulation of autophagic pathways. Strains with high levels of Agr activity were capable of causing autophagosome accumulation, were not killed by BMDCs, and subsequently escaped from the phagocyte, exerting significant cytotoxic effects. Conversely, strains that exhibited low levels of Agr activity failed to accumulate autophagosomes and were killed by BMDCs. Inhibition of the autophagic pathway by treatment with 3-methyladenine restored the bactericidal effects of BMDCs. Using an in vivo model of systemic infection, we demonstrated that the ability of S. aureus strains to evade phagocytic cell killing and to survive temporarily within phagocytes correlated with persistence in the periphery and that this effect is critically Agr dependent. Taken together, our data suggest that strains of S. aureus exhibiting high levels of Agr activity are capable of blocking autophagic flux, leading to the accumulation of autophagosomes. Within these autophagosomes, the bacteria are protected from phagocytic killing, thus providing an intracellular survival niche within professional phagocytes, which ultimately facilitates dissemination.
Asunto(s)
Autofagia/fisiología , Proteínas Bacterianas/metabolismo , Células Dendríticas/microbiología , Infecciones Estafilocócicas/inmunología , Transactivadores/metabolismo , Animales , Bacteriemia/metabolismo , Bacteriemia/microbiología , Western Blotting , Células de la Médula Ósea/microbiología , Células Cultivadas , Modelos Animales de Enfermedad , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/inmunologíaRESUMEN
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 neighbouring bacteria induce biofilm growth.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Biopelículas , Staphylococcus aureus/fisiología , Secuencia de Aminoácidos , Adhesión Bacteriana , Proteínas Bacterianas/genética , Dimerización , Humanos , Datos de Secuencia Molecular , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/química , Staphylococcus aureus/genéticaRESUMEN
SdrG is a cell surface adhesin from Staphylococcus epidermidis which binds to the blood plasma protein fibrinogen (Fg). Ligand binding follows a 'dock, lock and latch' model involving dynamic conformational changes of the adhesin that result in a greatly stabilized adhesin-ligand complex. To date, the force and dynamics of this multistep interaction are poorly understood. Here we use atomic force microscopy (AFM) to unravel the binding strength and cell surface localization of SdrG at molecular resolution. Single-cell force spectroscopy shows that SdrG mediates time-dependent attachment to Fg-coated surfaces. Single-molecule force spectroscopy with Fg-coated AFM tips demonstrates that the adhesin forms nanoscale domains on the cell surface, which we believe contribute to strengthen cell adhesion. Notably, we find that the rupture force of single SdrG-Fg bonds is very large, â¼ 2 nN, equivalent to the strength of a covalent bond, and shows a low dissociation rate, suggesting that the bond is very stable. The strong binding force, slow dissociation and clustering of SdrG provide a molecular foundation for the ability of S. epidermidis to colonize implanted biomaterials and to withstand physiological shear forces.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Fibrinógeno/metabolismo , Staphylococcus epidermidis/metabolismo , Adhesión Bacteriana/fisiología , Ligandos , Microscopía de Fuerza Atómica , Unión Proteica , Análisis de la Célula IndividualRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/química , Biopelículas , Staphylococcus aureus/metabolismo , Staphylococcus epidermidis/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Dimerización , Datos de Secuencia Molecular , Pliegue de Proteína , TermodinámicaRESUMEN
Despite showing promise in preclinical models, anti-Staphylococcus aureus vaccines have failed in clinical trials. To date, approaches have focused on neutralizing/opsonizing antibodies; however, vaccines exclusively inducing cellular immunity have not been studied to formally test whether a cellular-only response can protect against infection. We demonstrate that nasal vaccination with targeted nanoparticles loaded with Staphylococcus aureus antigen protects against acute systemic S. aureus infection in the absence of any antigen-specific antibodies. These findings can help inform future developments in staphylococcal vaccine development and studies into the requirements for protective immunity against S. aureus.
Asunto(s)
Infecciones Estafilocócicas/prevención & control , Vacunas Estafilocócicas/administración & dosificación , Staphylococcus aureus/inmunología , Administración Intranasal , Animales , Anticuerpos Neutralizantes/sangre , Carga Bacteriana/inmunología , Femenino , Inmunidad Celular/efectos de los fármacos , Inmunidad Celular/inmunología , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Nanopartículas/administración & dosificación , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiología , Vacunas Estafilocócicas/química , Vacunas Estafilocócicas/inmunologíaRESUMEN
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 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 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 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 CSF41498 but not in strain 1457. The nucleotide sequences of the aap gene A-domain region and cleavage site in strains 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.