RESUMO
Most bacterial glycoproteins identified to date are virulence factors of pathogenic bacteria, i.e. adhesins and invasins. However, the impact of protein glycosylation on the major human pathogen Staphylococcus aureus remains incompletely understood. To study protein glycosylation in staphylococci, we analyzed lysostaphin lysates of methicillin-resistant Staphylococcus aureus (MRSA) strains by SDS-PAGE and subsequent periodic acid-Schiff's staining. We detected four (>300, â¼250, â¼165, and â¼120 kDa) and two (>300 and â¼175 kDa) glycosylated surface proteins with strain COL and strain 1061, respectively. The â¼250, â¼165, and â¼175 kDa proteins were identified as plasmin-sensitive protein (Pls) by mass spectrometry. Previously, Pls has been demonstrated to be a virulence factor in a mouse septic arthritis model. The pls gene is encoded by the staphylococcal cassette chromosome (SCC)mec type I in MRSA that also encodes the methicillin resistance-conferring mecA and further genes. In a search for glycosyltransferases, we identified two open reading frames encoded downstream of pls on the SCCmec element, which we termed gtfC and gtfD. Expression and deletion analysis revealed that both gtfC and gtfD mediate glycosylation of Pls. Additionally, the recently reported glycosyltransferases SdgA and SdgB are involved in Pls glycosylation. Glycosylation occurs at serine residues in the Pls SD-repeat region and modifying carbohydrates are N-acetylhexosaminyl residues. Functional characterization revealed that Pls can confer increased biofilm formation, which seems to involve two distinct mechanisms. The first mechanism depends on glycosylation of the SD-repeat region by GtfC/GtfD and probably also involves eDNA, while the second seems to be independent of glycosylation as well as eDNA and may involve the centrally located G5 domains. Other previously known Pls properties are not related to the sugar modifications. In conclusion, Pls is a glycoprotein and Pls glycosyl residues can stimulate biofilm formation. Thus, sugar modifications may represent promising new targets for novel therapeutic or prophylactic measures against life-threatening S. aureus infections.
Assuntos
Antibacterianos/farmacologia , Fibrinolisina/metabolismo , Resistência a Meticilina , Staphylococcus aureus Resistente à Meticilina/genética , Infecções Estafilocócicas/microbiologia , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fibrinolisina/genética , Glicoproteínas , Humanos , Meticilina/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Camundongos , Fatores de VirulênciaRESUMO
The coagulase-negative species Staphylococcus lugdunensis is an emerging cause of serious and potentially life-threatening infections, such as infective endocarditis. The pathogenesis of these infections is characterized by the ability of S. lugdunensis to form biofilms on either biotic or abiotic surfaces. To elucidate the genetic basis of biofilm formation in S. lugdunensis, we performed transposon (Tn917) mutagenesis. One mutant had a significantly reduced biofilm-forming capacity and carried a Tn917 insertion within the competence gene comEB. Site-directed mutagenesis and subsequent complementation with a functional copy of comEB verified the importance of comEB in biofilm formation. In several bacterial species, natural competence stimulates DNA release via lysis-dependent or -independent mechanisms. Extracellular DNA (eDNA) has been demonstrated to be an important structural component of many bacterial biofilms. Therefore, we quantified the eDNA in the biofilms and found diminished eDNA amounts in the comEB mutant biofilm. High-resolution images and three-dimensional data obtained via confocal laser scanning microscopy (CSLM) visualized the impact of the comEB mutation on biofilm integrity. The comEB mutant did not show reduced expression of autolysin genes, decreased autolytic activities, or increased cell viability, suggesting a cell lysis-independent mechanism of DNA release. Furthermore, reduced amounts of eDNA in the comEB mutant biofilms did not result from elevated levels or activity of the S. lugdunensis thermonuclease NucI. In conclusion, we defined here, for the first time, a role for the competence gene comEB in staphylococcal biofilm formation. Our findings indicate that comEB stimulates biofilm formation via a lysis-independent mechanism of DNA release.
Assuntos
Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Elementos de DNA Transponíveis , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Staphylococcus lugdunensis/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , DNA Bacteriano/metabolismo , Teste de Complementação Genética , Loci Gênicos , Viabilidade Microbiana , Nuclease do Micrococo/genética , Nuclease do Micrococo/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Transdução de Sinais , Staphylococcus lugdunensis/metabolismo , Staphylococcus lugdunensis/ultraestruturaRESUMO
Although it belongs to the group of coagulase-negative staphylococci, Staphylococcus lugdunensis has been known to cause aggressive courses of native and prosthetic valve infective endocarditis with high mortality similar to Staphylococcus aureus. In contrast to S. aureus, only little is known about the equipment of S. lugdunensis with virulence factors including adhesins and their role in mediating attachment to extracellular matrix and plasma proteins and host cells. In this study, we show that the multifunctional autolysin/adhesin AtlL of S. lugdunensis binds to the extracellular matrix and plasma proteins fibronectin, fibrinogen, and vitronectin as well as to human EA.hy926 endothelial cells. Furthermore, we demonstrate that AtlL also plays an important role in the internalization of S. lugdunensis by eukaryotic cells: The atlL-deficient mutant Mut17 adheres to and becomes internalized by eukaryotic cells to a lesser extent than the isogenic wild-type strain Sl253 and the complemented mutant Mut17 (pCUatlL) shows an increased internalization level in comparison to Mut17. Thus, surface localized AtlL that exhibits a broad binding spectrum also mediates the internalization of S. lugdunensis by eukaryotic cells. We therefore propose an internalization pathway for S. lugdunensis, in which AtlL plays a major role. Investigating the role of AtlL in biofilm formation of S. lugdunensis, Mut17 shows a significantly reduced ability for biofilm formation, which is restored in the complemented mutant. Thus, our data provide evidence for a significant role for AtlL in adherence and internalization processes as well as in biofilm formation of S. lugdunensis.
Assuntos
Adesinas Bacterianas/metabolismo , Aderência Bacteriana , Biofilmes/crescimento & desenvolvimento , Endocitose , Células Endoteliais/microbiologia , Staphylococcus lugdunensis/fisiologia , Fatores de Virulência/metabolismo , Adesinas Bacterianas/genética , Linhagem Celular , Fibrinogênio/metabolismo , Fibronectinas/metabolismo , Deleção de Genes , Teste de Complementação Genética , Humanos , Ligação Proteica , Staphylococcus lugdunensis/metabolismo , Fatores de Virulência/genética , Vitronectina/metabolismoRESUMO
OBJECTIVE: Staphylococcus aureus can induce platelet aggregation. The rapidity and degree of this correlates with the severity of disseminated intravascular coagulation, and depends on platelet peptidoglycans. Surface-located thiol isomerases play an important role in platelet activation. The staphylococcal extracellular adherence protein (Eap) functions as an adhesin for host plasma proteins. Therefore we tested the effect of Eap on platelets. METHODS AND RESULTS: We found a strong stimulation of the platelet-surface thiol isomerases protein disulfide isomerase and endoplasmic reticulum stress proteins 57 and 72 by Eap. Eap induced thiol isomerase-dependent glycoprotein IIb/IIIa activation, granule secretion, and platelet aggregation. Treatment of platelets with thiol blockers, bacitracin, and anti-protein disulfide isomerase antibody inhibited Eap-induced platelet activation. The effect of Eap on platelets and protein disulfide isomerase activity was completely blocked by glycosaminoglycans. Inhibition by the hydrophobic probe bis(1-anilinonaphthalene 8-sulfonate) suggested the involvement of hydrophobic sites in protein disulfide isomerase and platelet activation by Eap. CONCLUSIONS: In the present study, we found an additional and yet unknown mechanism of platelet activation by a bacterial adhesin, involving stimulation of thiol isomerases. The thiol isomerase stimulatory and prothrombotic features of a microbial secreted protein are probably not restricted to S aureus and Eap. Because many microorganisms are coated with amyloidogenic proteins, it is likely that the observed mechanism is a more general one.
Assuntos
Proteínas de Bactérias/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Isomerases de Dissulfetos de Proteínas/fisiologia , Proteínas de Ligação a RNA/farmacologia , Staphylococcus aureus/patogenicidade , Naftalenossulfonato de Anilina/farmacologia , Plaquetas/enzimologia , Ácido Ditionitrobenzoico/farmacologia , Humanos , Selectina-P/sangue , Proteoglicanas/farmacologia , Tetraspanina 30/sangueRESUMO
Invasion of host cells is an important feature of Staphylococcus aureus. The main internalization pathway involves binding of the bacteria to host cells, e.g., endothelial cells, via a fibronectin (Fn) bridge between S. aureus Fn binding proteins and α5ß1-integrin, followed by phagocytosis. The secreted extracellular adherence protein (Eap) has been shown to promote this cellular uptake pathway of not only S. aureus, but also of bacteria otherwise poorly taken up by host cells, such as Staphylococcus carnosus. The exact mechanisms are still unknown. Previously, we demonstrated that Eap induces platelet activation by stimulation of the protein disulfide isomerase (PDI), a catalyst of thiol-disulfide exchange reactions. Here, we show that Eap promotes PDI activity on the surface of endothelial cells, and that this contributes critically to Eap-driven staphylococcal invasion. PDI-stimulated ß1-integrin activation followed by increased Fn binding to host cells likely accounts for the Eap-enhanced uptake of S. aureus into non-professional phagocytes. Additionally, Eap supports the binding of S. carnosus to Fn-α5ß1 integrin, thereby allowing its uptake into endothelial cells. To our knowledge, this is the first demonstration that PDI is crucial for the uptake of bacteria into host cells. We describe a hitherto unknown function of Eap-the promotion of an enzymatic activity with subsequent enhancement of bacterial uptake-and thus broaden mechanistic insights into its importance as a driver of bacterial pathogenicity. IMPORTANCE Staphylococcus aureus can invade and persist in non-professional phagocytes, thereby escaping host defense mechanisms and antibiotic treatment. The intracellular lifestyle of S. aureus contributes to the development of infection, e.g., in infective endocarditis or chronic osteomyelitis. The extracellular adherence protein secreted by S. aureus promotes its own internalization as well as that of bacteria that are otherwise poorly taken up by host cells, such as Staphylococcus carnosus. In our study, we demonstrate that staphylococcal uptake by endothelial cells requires catalytic disulfide exchange activity by the cell-surface protein disulfide isomerase, and that this critical enzymatic function is enhanced by Eap. The therapeutic application of PDI inhibitors has previously been investigated in the context of thrombosis and hypercoagulability. Our results add another intriguing possibility: therapeutically targeting PDI, i.e., as a candidate approach to modulate the initiation and/or course of S. aureus infectious diseases.
Assuntos
Adesinas Bacterianas , Infecções Estafilocócicas , Humanos , Adesinas Bacterianas/metabolismo , Proteínas de Bactérias/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Células Endoteliais/metabolismo , Staphylococcus aureus/metabolismo , Integrinas/metabolismoRESUMO
The role of the pore-forming Staphylococcus aureus toxin Panton-Valentine leukocidin (PVL) in severe necrotizing diseases is debated due to conflicting data from epidemiological studies of community-associated methicillin-resistant S. aureus (CA-MRSA) infections and various murine disease-models. In this study, we used neutrophils isolated from different species to evaluate the cytotoxic effect of PVL in comparison to other staphylococcal cytolytic components. Furthermore, to study the impact of PVL we expressed it heterologously in a non-virulent staphylococcal species and examined pvl-positive and pvl-negative clinical isolates as well as the strain USA300 and its pvl-negative mutant. We demonstrate that PVL induces rapid activation and cell death in human and rabbit neutrophils, but not in murine or simian cells. By contrast, the phenol-soluble modulins (PSMs), a newly identified group of cytolytic staphylococcal components, lack species-specificity. In general, after phagocytosis of bacteria different pvl-positive and pvl-negative staphylococcal strains, expressing a variety of other virulence factors (such as surface proteins), induced cell death in neutrophils, which is most likely associated with the physiological clearing function of these cells. However, the release of PVL by staphylococcal strains caused rapid and premature cell death, which is different from the physiological (and programmed) cell death of neutrophils following phagocytosis and degradation of virulent bacteria. Taken together, our results question the value of infection-models in mice and non-human primates to elucidate the impact of PVL. Our data clearly demonstrate that PVL acts differentially on neutrophils of various species and suggests that PVL has an important cytotoxic role in human neutrophils, which has major implications for the pathogenesis of CA-MRSA infections.
Assuntos
Toxinas Bacterianas/efeitos adversos , Exotoxinas/efeitos adversos , Leucocidinas/efeitos adversos , Staphylococcus aureus Resistente à Meticilina/metabolismo , Neutrófilos/microbiologia , Animais , Humanos , Macaca fascicularis , Camundongos , Necrose , Neutrófilos/metabolismo , Neutrófilos/patologia , Coelhos , Especificidade da EspécieRESUMO
Neutrophil extracellular traps (NETs) are webs of DNA covered with antimicrobial molecules that constitute a newly described killing mechanism in innate immune defense. Previous publications reported that NETs take up to 3-4 h to form via an oxidant-dependent event that requires lytic death of neutrophils. In this study, we describe neutrophils responding uniquely to Staphylococcus aureus via a novel process of NET formation that did not require neutrophil lysis or even breach of the plasma membrane. The multilobular nucleus rapidly became rounded and condensed. During this process, we observed the separation of the inner and outer nuclear membranes and budding of vesicles, and the separated membranes and vesicles were filled with nuclear DNA. The vesicles were extruded intact into the extracellular space where they ruptured, and the chromatin was released. This entire process occurred via a unique, very rapid (5-60 min), oxidant-independent mechanism. Mitochondrial DNA constituted very little if any of these NETs. They did have a limited amount of proteolytic activity and were able to kill S. aureus. With time, the nuclear envelope ruptured, and DNA filled the cytoplasm presumably for later lytic NET production, but this was distinct from the vesicular release mechanism. Panton-Valentine leukocidin, autolysin, and a lipase were identified in supernatants with NET-inducing activity, but Panton-Valentine leukocidin was the dominant NET inducer. We describe a new mechanism of NET release that is very rapid and contributes to trapping and killing of S. aureus.
Assuntos
Toxinas Bacterianas/imunologia , Cromatina/imunologia , DNA Mitocondrial/imunologia , Exotoxinas/imunologia , Imunidade Inata/imunologia , Leucocidinas/imunologia , Neutrófilos/imunologia , Staphylococcus aureus/imunologia , Citoplasma/imunologia , Humanos , OxirreduçãoRESUMO
The coagulase-negative staphylococcal (CoNS) species Staphylococcus lugdunensis is unique in causing serious infections in humans that resemble those of Staphylococcus aureus rather than those of other CoNS species. The colonization and invasion of host tissue presupposes the presence of adherence factors, but only a few proteins mediating adhesion of S. lugdunensis to biotic surfaces are known yet. Here, we report on the functionality of the S. lugdunensis enolase (SlEno), which performs two distinct roles, first, as the metabolic enzyme of the glycolysis, and second, as an adherence factor to the extracellular matrix (ECM) of cells. Phylogenetic analyses of the SlEno confirmed their high conservation to enolases of other species and revealed a closer relationship to Staphylococcus epidermidis than to S. aureus. Using matrix-assisted laser desorption/ionization time of flight mass spectrometry and Western blot experiments, we identified SlEno to be located in the cytoplasm as well as on the cell surface of S. lugdunensis. Recombinantly generated and surface-associated SlEno showed the usual enolase activity by catalyzing the conversion of 2-phosphoglycerate to phosphoenolpyruvate but, in addition, also displayed strong binding to immobilized laminin, fibronectin, fibrinogen, and collagen type IV in a dose-dependent manner. We also showed a strong binding of SlEno to plasminogen (Plg) and observed a tissue plasminogen activator (tPA)-dependent conversion of Plg to plasmin (Pln) whereby the Plg activation significantly increased in the presence of SlEno. This interaction might be dependent on lysines of the SlEno protein as binding to Plg was inhibited by ε-aminocaproic acid. Furthermore, the enhanced activation of the Plg/Pln system by SlEno enabled S. lugdunensis to migrate through a fibrin matrix. This migration was about 10-fold higher than without exogenously added SlEno. Finally, we observed a significantly higher clearance of S. lugdunensis by freshly prepared granulocytes and in the presence of anti-SlEno antibodies. In conclusion, these data demonstrate for the first time a moonlighting function of the S. lugdunensis enolase, which is an underrated virulence factor for colonization and invasion of tissues. Hence, SlEno might be a potential vaccine candidate to prevent severe infections caused by this pathogen.
RESUMO
Repeated cases of low pathogenic influenza A/H9N2 virus (IAV/H9N2) have been reported in commercial chickens since its emergence in 1998 in Pakistan. However, recently increased mortality and severe respiratory complications under field conditions have been noticed, suggesting concomitant influenza infections with respiratory viral and/or bacterial pathogens. Therefore, the present study aimed to investigate the presence of IAV/H9N2 coinfecting with multiple viral and bacterial pathogens in broiler chicken flocks. We surveyed 60 broiler flocks with respiratory signs from March through July 2019 in Punjab, Pakistan. Suspected flocks were screened for the presence of IAV using a lateral-flow device. Tracheal, cloacal, and bone marrow samples were collected and further tested for seven viral agents (chicken anemia; Newcastle disease; infectious bronchitis; infectious laryngeotracheitis [ILT]; and IAV subtypes H9, H7, and H5) and three bacterial agents (Mycoplasma gallisepticum; Mycoplasma synovae; Ornithobacterium rhinotracheale [ORT]) using PCR assays. Upon initial screening for IAV, 35/60 (58.3%) flocks tested positive. The coinfection of IAV/H9N2 with other pathogens was detected in 25 (71.4%) flocks and only IAV/H9N2 was detected in 10 (28.6%) flocks out of total positive IAV flocks (n = 35). IAV subtypes H5 and H7, ILT, and ORT were not detected throughout the study period. The detection rate of double, triple, and quadruple combinations of coinfections with IAV/H9N2 were 37% (13 flocks), 26% (9 flocks), 9% (3 flocks), respectively. Higher average mortality (28.5%) was found in broiler chicken flocks coinfected with viral and/or bacterial pathogens than in flocks where only H9 low pathogenic IAV/H9N2 was detected (20.8%). In conclusion, higher circulation of IAV/H9N2 with other viral and bacterial pathogens may contribute to higher production and economic losses at the farm level.
Nota de investigación- Tasa de coinfecciones virales y bacterianas múltiples en parvadas de pollos de engorde infectadas con virus influenza A/H9N2. Se han reportado varios casos del virus de influenza A de baja patogenicidad H9N2 (IAV/H9N2) en pollos comerciales desde su aparición en 1998 en Pakistán. Sin embargo, recientemente se ha observado un aumento de la mortalidad y complicaciones respiratorias graves en condiciones de campo, lo que sugiere infecciones concomitantes de influenza con patógenos respiratorios virales y/o bacterianos. Por lo tanto, el presente estudio tuvo como objetivo investigar la presencia del virus de influenza aviar H9N2 coinfectando con múltiples patógenos virales y bacterianos en parvadas de pollos de engorde. Se evaluaron 60 parvadas de pollos de engorde con signos respiratorios desde marzo hasta julio del año 2019 en Punjab, Pakistán. Las parvadas sospechosas fueron analizadas para detectar la presencia del virus de influenza aviar utilizando un dispositivo de flujo lateral. Se recolectaron muestras traqueales, cloacales y de médula ósea y se analizaron para detectar siete agentes virales (anemia infecciosa aviar, enfermedad de Newcastle, bronquitis infecciosa, laringeotraqueítis infecciosa [ILT] y subtipos H9, H7 y H5 de influenza aviar) y tres agentes bacterianos (Mycoplasma gallisepticum ; Mycoplasma sinovae; Ornithobacterium rhinotracheale [ORT]) utilizando ensayos de PCR. Tras la detección inicial del virus de la influenza aviar, 35/60 (58.3 %) parvadas resultaron positivas. La coinfección del virus de la influenza H9N2 con otros patógenos se detectó en 25 (71.4 %) parvadas y el virus de influenza aviar H9N2 fue detectado solo en 10 (28.6 %) parvadas del total de parvadas positivas (n = 35). Los subtipos H5 y H7 del virus de influenza, ILT y ORT no se detectaron durante el período de estudio. La tasa de detección de combinaciones dobles, triples y cuádruples de coinfecciones con el virus de influenza H9N2 fue del 37 % (13 parvadas), del 26% (9 parvadas), del 9 % (3 parvadas), respectivamente. Se encontró una mortalidad promedio más alta (28.5 %) en lotes de pollos de engorde coinfectados con patógenos virales y/o bacterianos que en lotes donde solo se detectó al virus de influenza H9 de baja patogenicidad (20.8%). En conclusión, una mayor circulación del virus de influenza aviar H9N2 con otros patógenos virales y bacterianos puede contribuir a mayores pérdidas en la producción y económicas a nivel de granja.
Assuntos
Coinfecção , Vírus da Influenza A Subtipo H9N2 , Influenza Aviária , Influenza Humana , Doenças das Aves Domésticas , Animais , Galinhas , Coinfecção/epidemiologia , Coinfecção/veterinária , Humanos , Doenças das Aves Domésticas/microbiologiaRESUMO
Staphylococcus aureus is a human pathogen that secretes proteins that contribute to bacterial colonization. Here we describe the extracellular adherence protein (Eap) as a novel anti-inflammatory factor that inhibits host leukocyte recruitment. Due to its direct interactions with the host adhesive proteins intercellular adhesion molecule 1 (ICAM-1), fibrinogen or vitronectin, Eap disrupted beta(2)-integrin and urokinase receptor mediated leukocyte adhesion in vitro. Whereas Eap-expressing S. aureus induced a 2 3-fold lower neutrophil recruitment in bacterial peritonitis in mice as compared with an Eap-negative strain, isolated Eap prevented beta(2)-integrin-dependent neutrophil recruitment in a mouse model of acute thioglycollate-induced peritonitis. Thus, the specific interactions with ICAM-1 and extracellular matrix proteins render Eap a potent anti-inflammatory factor, which may serve as a new therapeutic substance to block leukocyte extravasation in patients with hyperinflammatory pathologies.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Aderência Bacteriana , Proteínas de Bactérias/farmacologia , Leucócitos/fisiologia , Staphylococcus aureus/fisiologia , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Endotélio Vascular/microbiologia , Endotélio Vascular/fisiologia , Fibronectinas/fisiologia , Humanos , Molécula 1 de Adesão Intercelular/fisiologia , Leucócitos/microbiologia , Antígeno de Macrófago 1/fisiologia , Camundongos , Camundongos Endogâmicos , Peritonite/microbiologia , Peritonite/prevenção & controle , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/prevenção & controle , Células U937 , Veias UmbilicaisRESUMO
BACKGROUND: Staphylococcus aureus is an important human pathogen of endovascular diseases, which can take a chronic course with a high relapse rate despite antimicrobial treatment. Thus far, persistent and antibiotic-refractory infections have been largely associated with a subpopulation of S. aureus, the small-colony variants (SCVs). METHODS: In this work, we used endothelial cells to investigate infection with the highly virulent wild-type isolate (6850), 2 stable isogenic SCV phenotypes (hemB mutant IIb13 and JB1), and the complemented mutant. RESULTS: All strains were highly invasive in endothelial cells but largely differed in host response induction. Microarray analysis showed that wild-type phenotypes up-regulated a large number of endothelial genes (including genes involved in innate immunity), whereas the SCVs did not cause these dramatic changes. The inflammatory response and cytotoxicity were strongest shortly after infection and largely decreased within the following days, which was accompanied by a fast elimination of intracellular wild-type bacteria. By contrast, SCVs survived within endothelial cells at high numbers. CONCLUSION: S. aureus intracellular persistence via the development of an adapted subpopulation of SCVs most likely represents an important strategy of S. aureus to hide within the host cells, which could be a reservoir for chronic infections.
Assuntos
Citoplasma/microbiologia , Células Endoteliais/microbiologia , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/patogenicidade , Células Cultivadas , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Among coagulase-negative staphylococci (CoNS), Staphylococcus lugdunensis has a special position as causative agent of aggressive courses of infectious endocarditis (IE) more reminiscent of IEs caused by Staphylococcus aureus than those by CoNS. To initiate colonization and invasion, bacterial cell surface proteins are required; however, only little is known about adhesion of S. lugdunensis to biotic surfaces. Cell surface proteins containing the LPXTG anchor motif are covalently attached to the cell wall by sortases. Here, we report the functionality of Staphylococcus lugdunensis sortase A (SrtA) to link LPXTG substrates to the cell wall. To determine the role of SrtA dependent surface proteins in biofilm formation and binding eukaryotic cells, we generated SrtA-deficient mutants (ΔsrtA). These mutants formed a smaller amount of biofilm and bound less to immobilized fibronectin, fibrinogen, and vitronectin. Furthermore, SrtA absence affected the gene expression of two different adhesins on transcription level. Surprisingly, we found no decreased adherence and invasion in human cell lines, probably caused by the upregulation of further adhesins in ΔsrtA mutant strains. In conclusion, the functionality of S. lugdunensis SrtA in anchoring LPXTG substrates to the cell wall let us define it as the pathogen's housekeeping sortase.
RESUMO
Staphylococcus aureus reacts to changing environmental conditions such as heat, pH, and chemicals through global regulators such as the sae (S. aureus exoprotein expression) two-component signaling system. Subinhibitory concentrations of some antibiotics were shown to increase virulence factor expression. Here, we investigated the S. aureus stress response to sublethal concentrations of a commonly used biocide (Perform), by real-time quantitative PCR (qRT-PCR), promoter activity assay, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and a flow cytometric invasion assay. Perform, acting through the production of reactive oxygen species, generally downregulated expression of extracellular proteins in strains 6850, COL, ISP479C but upregulated these proteins in strain Newman. Upregulated proteins were sae dependent. The Perform component SDS, but not paraquat (another oxygen donor), mimicked the biocide effect. Eap (extracellular adherence protein) was most prominently augmented. Upregulation of eap and sae was confirmed by qRT-PCR. Promoter activity of sae P1 was increased by Perform and SDS. Both substances enhanced cellular invasiveness, by 2.5-fold and 3.2-fold, respectively. Increased invasiveness was dependent on Eap and the sae system, whereas agr, sarA, sigB, and fibronectin-binding proteins had no major effect in strain Newman. This unique response pattern was due to a point mutation in SaeS (the sensor histidine kinase), as demonstrated by allele swapping. Newman saePQRS(ISP479C) behaved like ISP479C, whereas saePQRS(Newman) rendered ISP479C equally responsive as Newman. Taken together, the findings indicate that a point mutation in SaeS of strain Newman was responsible for increased expression of Eap upon exposure to sublethal Perform and SDS concentrations, leading to increased Eap-dependent cellular invasiveness. This may be important for understanding the regulation of virulence in S. aureus.
Assuntos
Antibacterianos/farmacologia , Mutação Puntual/genética , Proteínas Quinases/genética , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Proteínas de Bactérias , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/genética , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Dodecilsulfato de Sódio/farmacologia , Espectrometria de Massas em TandemRESUMO
The extracellular adherence protein (Eap) is a multifunctional Staphylococcus aureus protein and broad-spectrum adhesin for several host matrix and plasma proteins. We investigated the interactions of full-length Eap and five recombinant tandem repeat domains with host proteins by use of surface plasmon resonance (BIAcore) and ligand overlay assays. In addition, agglutination and host cell interaction, namely, adherence, invasion, and stimulation of proliferation, were determined. With plasmon resonance, the interaction of full-length Eap isoforms (from strains Newman and Wood 46) with fibrinogen, fibronectin, vitronectin, and thrombospondin-1 was found to be specific but with different affinities for the ligands tested. In the ligand overlay assay, the interactions of five single tandem repeat domains (D1 to D5) of Eap-7 (from strain CI-7) with fibronectin, fibrinogen, vitronectin, thrombospondin-1, and collagen I differed substantially. Most prominently, D3 bound most strongly to fibronectin and fibrinogen. Full-length Eap, but none of the single tandem repeat domains, agglutinated S. aureus and enhanced adherence to and invasion of host cells by S. aureus. Constructs D3-4 and D1-3 (in cis) increased adherence and invasiveness compared to what was seen for single Eap tandem repeat domains. By contrast, single Eap tandem repeat domains and full-length Eap similarly modulated the proliferation of peripheral blood mononuclear cells (PBMCs): low concentrations stimulated, whereas high concentrations inhibited, proliferation. Taken together, the data indicate that Eap tandem repeat domains appear to have distinct characteristics for the binding of soluble ligands, despite a high degree of sequence similarity. In addition, more than one Eap tandem repeat domain is required for S. aureus agglutination, adherence, and cellular invasion but not for the stimulation of PBMC proliferation.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Ligação a RNA/genética , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Sequências de Repetição em Tandem , Aglutinação/genética , Aderência Bacteriana/genética , Western Blotting , Proliferação de Células , Eletroforese em Gel de Poliacrilamida , Células Endoteliais/microbiologia , Fibroblastos/microbiologia , Humanos , Ligação Proteica/genética , Isoformas de Proteínas/genética , Proteínas Recombinantes/genética , Infecções Estafilocócicas/genética , Staphylococcus aureus/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
The cell surface-associated extracellular adherence protein (Eap) mediates adherence of Staphylococcus aureus to host extracellular matrix components and inhibits inflammation, wound healing, and angiogenesis. A well-characterized collection of S. aureus and non-S. aureus staphylococcal isolates (n = 813) was tested for the presence of the Eap-encoding gene (eap) by PCR to investigate the use of the eap gene as a specific diagnostic tool for identification of S. aureus. Whereas all 597 S. aureus isolates were eap positive, this gene was not detectable in 216 non-S. aureus staphylococcal isolates comprising 47 different species and subspecies of coagulase-negative staphylococci and non-S. aureus coagulase-positive or coagulase-variable staphylococci. Furthermore, non-S. aureus isolates did not express Eap homologs, as verified on the transcriptional and protein levels. Based on these data, the sensitivity and specificity of the newly developed PCR targeting the eap gene were both 100%. Thus, the unique occurrence of Eap in S. aureus offers a promising tool particularly suitable for molecular diagnostics of this pathogen.
Assuntos
Proteínas de Bactérias/genética , Reação em Cadeia da Polimerase/métodos , Proteínas de Ligação a RNA/genética , Infecções Estafilocócicas/diagnóstico , Staphylococcus aureus/isolamento & purificação , Proteínas de Bactérias/análise , Northern Blotting , Western Blotting , DNA Bacteriano/genética , Eletroforese em Gel de Poliacrilamida , Humanos , Proteínas de Ligação a RNA/análise , Sensibilidade e Especificidade , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/química , Staphylococcus aureus/genéticaRESUMO
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RESUMO
Globalization and migration promote the spread of Panton-Valentine leukocidin (PVL)-positive Staphylococcus aureus strains. The toxin PVL is linked to the development of thrombosis in association with osteomyelitis. The mechanisms by which PVL drives thrombosis development are however still unknown. We demonstrate that PVL-damaged neutrophils activate platelets via neutrophil secretion products, such as α-defensins and the myeloperoxidase product HOCl, as well as the formation of HOCl-modified proteins. Neutrophil damage by PVL is blocked by anti-PVL-antibodies, explaining why especially young osteomyelitis patients with a low antibody titre against PVL suffer from thrombotic complications. Platelet activation in the presence of PVL-damaged neutrophils is prevented by α-defensin inhibitors and by glutathione and resveratrol, which are both inhibitors of HOCl-modified protein-induced platelet activation. Remarkably, intravenously infused glutathione also prevents activation of human platelets in an ex vivo assay. We here describe a new mechanism of PVL-neutrophil-platelet interactions, which might be extrapolated to other toxins that act on neutrophils. Our observations may make us think about new approaches to treat and/or prevent thrombotic complications in the course of infections with PVL-producing S. aureus strains.
Assuntos
Toxinas Bacterianas/farmacologia , Plaquetas/imunologia , Exotoxinas/farmacologia , Leucocidinas/farmacologia , Neutrófilos/imunologia , Osteomielite/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/patogenicidade , Plaquetas/efeitos dos fármacos , Humanos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Osteomielite/imunologia , Osteomielite/patologia , Infecções Estafilocócicas/epidemiologia , Staphylococcus aureus/efeitos dos fármacosRESUMO
The extracellular adherence protein (Eap), a broad-spectrum adhesin secreted by Staphylococcus aureus, was previously shown to curb acute inflammatory responses, presumably through its binding to endothelial cell (EC) ICAM-1. Examining the effect of Eap on endothelial function in more detail, we here show that, in addition, Eap functions as a potent angiostatic agent. Concomitant treatment of EC with purified Eap resulted in the complete blockage of the mitogenic and sprouting responses elicited by vascular endothelial growth factor (VEGF)165 or basic fibroblast growth factor (bFGF). Moreover, the induction of tissue factor and decay-accelerating factor were repressed by Eap, as determined by qRT-polymerase chain reaction (qRT-PCR), with a corresponding reduction in Egr-1 protein up-regulation seen. This angiostatic activity was accompanied by a corresponding inhibition in ERK1/2 phosphorylation, while activation of p38 was not affected. Inhibition occurred downstream of tyrosine kinase receptor activation, as comparable effects were seen on TPA-induced ERK1/2 phosphorylation. Similar to previously described angiostatic agents like angiopoietin-1 or the 16-kDa prolactin fragment, Eap blockage of the Ras/Raf/MEK/ERK cascade was localized by pull-down assay at the level of Ras activation. Eap's combined anti-inflammatory and antiangiogenic properties render this bacterial protein not only an important virulence factor during S. aureus infection but open new perspectives for therapeutic applications in pathological neovascularization.
Assuntos
Proteínas de Bactérias/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Proteínas de Ligação a RNA/farmacologia , Proteínas ras/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Fatores de Crescimento de Fibroblastos/metabolismo , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neovascularização Fisiológica/fisiologia , Fosfolipase C gama/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas ras/metabolismoRESUMO
The extracellular matrix protein Emp of Staphylococcus aureus is a secreted adhesin that mediates interactions between the bacterial surface and extracellular host structures. However, its structure and role in staphylococcal pathogenesis remain unknown. Using multidisciplinary approaches, including circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy, transmission electron (TEM) and immunogold transmission electron microscopy, functional ELISA assays and in silico techniques, we characterized the Emp protein. We demonstrated that Emp and its truncated forms bind to suprastructures in human skin, cartilage or bone, among which binding activity seems to be higher for skin compounds. The binding domain is located in the C-terminal part of the protein. CD spectroscopy revealed high contents of ß-sheets (39.58%) and natively disordered structures (41.2%), and TEM suggested a fibrous structure consisting of Emp polymers. The N-terminus seems to be essential for polymerization. Due to the uncommonly high histidine content, we suggest that Emp represents a novel type of histidine-rich protein sharing structural similarities to leucine-rich repeats proteins as predicted by the I-TASSER algorithm. These new findings suggest a role of Emp in infections of deeper tissue and open new possibilities for the development of novel therapeutic strategies.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Osso e Ossos/metabolismo , Cartilagem/metabolismo , Escherichia coli , Matriz Extracelular/ultraestrutura , Proteínas da Matriz Extracelular/química , Humanos , Cinética , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Proteínas Recombinantes , Alinhamento de Sequência , Pele/metabolismo , Staphylococcus aureusRESUMO
Besides a number of cell wall-anchored adhesins, the majority of Staphylococcus aureus strains produce anchorless, cell wall-associated proteins, such as Eap (extracellular adherence protein). Eap contains four to six tandem repeat (EAP)-domains. Eap mediates diverse biological functions, including adherence and immunomodulation, thus contributing to S. aureus pathogenesis. Eap binding to host macromolecules is unusually promiscuous and includes matrix or matricellular proteins as well as plasma proteins. The structural basis of this promiscuity is poorly understood. Here, we show that in spite of the preferential location of the binding epitopes within triple helical regions in some collagens there is a striking specificity of Eap binding to different collagen types. Collagen I, but not collagen II, is a binding substrate in monomolecular form. However, collagen I is virtually unrecognized by Eap when incorporated into banded fibrils. By contrast, microfibrils containing collagen VI as well as basement membrane-associated networks containing collagen IV, or aggregates containing fibronectin bound Eap as effectively as the monomeric proteins. Therefore, Eap-binding to extracellular matrix ligands is promiscuous at the molecular level but not indiscriminate with respect to supramolecular structures containing the same macromolecules. In addition, Eap bound to banded fibrils after their partial disintegration by matrix-degrading proteinases, including matrix metalloproteinase 1. Therefore, adherence to matrix suprastructures by S. aureus can be supported by inflammatory reactions.