RESUMEN
Mycoplasma pneumoniae (Mp) infections cause tracheobronchitis and "walking" pneumonia, and are linked to asthma and other reactive airway diseases. As part of the infectious process, the bacterium expresses a 591-aa virulence factor with both mono-ADP ribosyltransferase (mART) and vacuolating activities known as Community-Acquired Respiratory Distress Syndrome Toxin (CARDS TX). CARDS TX binds to human surfactant protein A and annexin A2 on airway epithelial cells and is internalized, leading to a range of pathogenetic events. Here we present the structure of CARDS TX, a triangular molecule in which N-terminal mART and C-terminal tandem ß-trefoil domains associate to form an overall architecture distinct from other well-recognized ADP-ribosylating bacterial toxins. We demonstrate that CARDS TX binds phosphatidylcholine and sphingomyelin specifically over other membrane lipids, and that cell surface binding and internalization activities are housed within the C-terminal ß-trefoil domain. The results enhance our understanding of Mp pathogenicity and suggest a novel avenue for the development of therapies to treat Mp-associated asthma and other acute and chronic airway diseases.
Asunto(s)
Proteínas Bacterianas/química , Toxinas Bacterianas/química , Citotoxinas/química , Mycoplasma pneumoniae/metabolismo , Vacuolas/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , ADP Ribosa Transferasas/química , ADP Ribosa Transferasas/metabolismo , Adenosina Difosfato Ribosa/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Dominio Catalítico , Citotoxinas/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Fosfatidilcolinas/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Esfingomielinas/metabolismo , Relación Estructura-ActividadRESUMEN
Enterococcus faecium is an important cause of hospital-associated infections, including urinary tract infections (UTIs), bacteremia, and infective endocarditis. Pili have been shown to play a role in the pathogenesis of Gram-positive bacteria, including E. faecium We previously demonstrated that a nonpiliated ΔempABC::cat derivative of E. faecium TX82 was attenuated in biofilm formation and in a UTI model. Here, we studied the contributions of the individual pilus subunits EmpA, EmpB, and EmpC to pilus architecture, biofilm formation, adherence to extracellular matrix (ECM) proteins, and infection. We identified EmpA as the tip of the pili and found that deletion of empA reduced biofilm formation to the same level as deletion of the empABC operon, a phenotype that was restored by reconstituting in situ the empA gene. Deletion of empB also caused a reduction in biofilm, while EmpC was found to be dispensable. Significant reductions in adherence to fibrinogen and collagen type I were observed with deletion of empA and empB, while deletion of empC had no adherence defect. Furthermore, we showed that each deletion mutant was significantly attenuated in comparison to the isogenic parental strain, TX82, in a mixed-inoculum UTI model (P < 0.001 to 0.048), that reconstitution of empA restored virulence in the UTI model, and that deletion of empA also resulted in attenuation in an infective endocarditis model (P = 0.0088). Our results indicate that EmpA and EmpB, but not EmpC, contribute to biofilm and adherence to ECM proteins; however, all the Emp pilins are important for E. faecium to cause infection in the urinary tract.
Asunto(s)
Adhesión Bacteriana , Biopelículas/crecimiento & desarrollo , Enterococcus faecium/fisiología , Matriz Extracelular/metabolismo , Fimbrias Bacterianas/metabolismo , Infecciones por Bacterias Grampositivas/microbiología , Factores de Virulencia/metabolismo , Animales , Colágeno Tipo I/metabolismo , Modelos Animales de Enfermedad , Endocarditis Bacteriana/microbiología , Endocarditis Bacteriana/patología , Enterococcus faecium/genética , Femenino , Fibrinógeno/metabolismo , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Eliminación de Gen , Prueba de Complementación Genética , Infecciones por Bacterias Grampositivas/patología , Masculino , Ratones Endogámicos ICR , Operón , Biogénesis de Organelos , Ratas Sprague-Dawley , Infecciones Urinarias/microbiología , Infecciones Urinarias/patologíaRESUMEN
EfbA is a PavA-like fibronectin adhesin of Enterococcus faecalis previously shown to be important in experimental urinary tract infection. Here, we expressed and purified the E. faecalis OG1RF EfbA and confirmed that this protein binds with high affinity to immobilized fibronectin, collagen I, and collagen V. We constructed an efbA deletion mutant and demonstrated that its virulence was significantly attenuated (P < 0.0006) versus the wild type in a mixed inoculum rat endocarditis model. Furthermore, efbA deletion resulted in diminished ability to bind fibronectin (P < 0.0001) and reduced biofilm (P < 0.001). Reintroduction of efbA into the original chromosomal location restored virulence, adherence to fibronectin, and biofilm formation to wild-type levels. Finally, vaccination of rats with purified recombinant EfbA protein protected against OG1RF endocarditis (P = 0.008 versus control). Taken together, our results demonstrate that EfbA is an important factor involved in E. faecalis endocarditis and that rEfbA immunization is effective in preventing such infection, likely by interfering with bacterial adherence.
Asunto(s)
Adhesinas Bacterianas/inmunología , Biopelículas/crecimiento & desarrollo , Endocarditis Bacteriana/prevención & control , Enterococcus faecalis/genética , Fibronectinas/metabolismo , Infecciones por Bacterias Grampositivas/prevención & control , Adhesinas Bacterianas/administración & dosificación , Adhesinas Bacterianas/genética , Animales , Sitios de Unión , Colágeno Tipo I/inmunología , Colágeno Tipo I/metabolismo , Colágeno Tipo V/inmunología , Colágeno Tipo V/metabolismo , Endocarditis Bacteriana/inmunología , Endocarditis Bacteriana/microbiología , Endocarditis Bacteriana/patología , Enterococcus faecalis/inmunología , Enterococcus faecalis/patogenicidad , Escherichia coli/genética , Escherichia coli/metabolismo , Fibronectinas/inmunología , Expresión Génica , Prueba de Complementación Genética , Infecciones por Bacterias Grampositivas/inmunología , Infecciones por Bacterias Grampositivas/microbiología , Infecciones por Bacterias Grampositivas/patología , Inmunización , Mutación , Unión Proteica , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
The interaction between bacteria and fibronectin is believed to play an important role in the pathogenicity of clinically important Gram-positive cocci. In the present study, we identified a gene encoding a predicted fibronectin-binding protein of Enterococcus faecium (fnm), a homologue of Streptococcus pneumoniae pavA, in the genomes of E. faecium strain TX82 and all other sequenced E. faecium isolates. Full-length recombinant Fnm from strain TX82 bound to immobilized fibronectin in a concentration-dependent manner and also appeared to bind collagen type V and laminin, but not other proteins, such as transferrin, heparin, bovine serum albumin, mucin, or collagen IV. We demonstrated that the N-terminal fragment of Fnm is required for full fibronectin binding, since truncation of this region caused a 2.4-fold decrease (P < 0.05) in the adhesion of E. faecium TX82 to fibronectin. Deletion of fnm resulted in a significant reduction (P < 0.001) in the ability of the mutant, TX6128, to bind fibronectin relative to that of the wild-type strain; in situ reconstitution of fnm in the deletion mutant strain restored adherence. In addition, the Δfnm mutant was highly attenuated relative to TX82 (P ≤ 0.0001) in a mixed-inoculum rat endocarditis model. Taken together, these results demonstrate that Fnm affects the adherence of E. faecium to fibronectin and is important in the pathogenesis of experimental endocarditis.
Asunto(s)
Proteínas Bacterianas/metabolismo , Endocarditis Bacteriana/microbiología , Enterococcus faecium/patogenicidad , Genoma Bacteriano , Infecciones por Bacterias Grampositivas/microbiología , Proteínas Recombinantes/metabolismo , Secuencia de Aminoácidos , Animales , Adhesión Bacteriana , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Colágeno Tipo V/metabolismo , Endocarditis Bacteriana/metabolismo , Endocarditis Bacteriana/patología , Enterococcus faecium/genética , Fibronectinas/metabolismo , Infecciones por Bacterias Grampositivas/metabolismo , Infecciones por Bacterias Grampositivas/patología , Proteínas Inmovilizadas/metabolismo , Laminina/metabolismo , Ratones Endogámicos ICR , Datos de Secuencia Molecular , Unión Proteica , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismo , VirulenciaRESUMEN
The collagen adhesin Acm was the first virulence determinant reported to be important for the pathogenesis of Enterococcus faecium in a rat infective endocarditis model. We had previously reported that there was a slight growth delay associated with acm allelic replacement (cat) mutant strain TX6051 used in that study. Recently, we generated a nonpolar markerless acm deletion mutant and did not observe a delay in growth. We therefore performed comparative genome sequence analysis of wild-type strain TX82 and TX6051 and found a single mutation, a nonsense mutation in the ccpA gene of TX6051. After correcting this mutation, the growth defect of TX6051 was abolished, implicating a role for CcpA in the growth of E. faecium. To confirm this, we created a ccpA deletion mutant of TX82, which also exhibited a slight delay in growth. Furthermore, the ccpA deletion mutant was attenuated (P = 0.0024) in a mixed-inoculum (TX82 plus TX82 ΔccpA) rat endocarditis model and also in an in vitro competitive growth assay; a ccpA-complemented strain showed neither reduced growth nor reduced virulence. We also found attenuation in the endocarditis model with the new acm deletion mutant although not as great as that previously observed with TX6051 carrying the ccpA mutation. Taken together, our data confirm the role of Acm in the pathogenesis of endocarditis. We also show that CcpA affects the growth of E. faecium, that an intact ccpA gene is important for full virulence, and that a ccpA mutation was partly responsible for the highly attenuated phenotype of TX6051.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Enterococcus faecium/genética , Enterococcus faecium/metabolismo , Virulencia/genética , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Secuencia de Aminoácidos , Animales , Adhesión Bacteriana/genética , Biopelículas/crecimiento & desarrollo , Endocarditis Bacteriana , Datos de Secuencia Molecular , Ratas , Eliminación de Secuencia/genéticaRESUMEN
We identified Mpn133 as a Ca(2+)-dependent cytotoxic nuclease of Mycoplasma pneumoniae. Flow cytometry analysis and immunofluorescence studies revealed the binding and internalization of recombinant Mpn133 (rMpn133) in human airway A549 cells. Amino acid sequence comparisons of Mpn133 with other mycoplasma nucleases demonstrated the presence of a unique glutamic acid-, lysine- and serine-rich region (EKS region; amino acids 72-110). Deletion of this EKS peptide (rMpn133(Δ72-110)) abrogated its binding and internalization but not its nuclease activity. The function of the EKS region in host cell trafficking and nuclear localization was reinforced by the successful delivery of EKS-conjugated mCherry protein into A549 cells. rMpn133, but not rMpn133(Δ72-110), induced apoptosis-like death in A549 cells. This observation suggested a unique role of Mpn133 as an important contributor to M. pneumoniae-associated life cycle events and as a virulence factor in host-associated cytopathologies. In addition, the distinct property of the EKS peptide in delivery of proteins, like mCherry, into target cells opens new avenues to the establishment of novel concepts of drug delivery and therapy.
Asunto(s)
Apoptosis , Esterasas/genética , Esterasas/metabolismo , Mycoplasma pneumoniae/enzimología , Mycoplasma pneumoniae/patogenicidad , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Secuencia de Aminoácidos , Línea Celular , Células Epiteliales/fisiología , Humanos , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Enterococcus faecium, a major cause of nosocomial infections, is often isolated from conditions where biofilm is considered to be important in the establishment of infections. We investigated biofilm formation among E. faecium isolates from diverse sources and found that the occurrence and amount of biofilm formation were significantly greater in clinical isolates than fecal isolates from community volunteers. We also found that the presence of the empfm (E. faecium pilus) operon was associated with the amount of biofilm formation. Furthermore, we analyzed the possible association between the distribution of 16 putative virulence genes and the occurrence of biofilm production. Even though the prevalence of these virulence genes was significantly higher in clinical isolates, we did not observe any correlation with the occurrence of biofilm formation.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Enterococcus faecium/fisiología , Infecciones por Bacterias Grampositivas/microbiología , Factores de Virulencia/genética , Adhesión Bacteriana/genética , Proteínas Bacterianas/genética , Infección Hospitalaria , Enterococcus faecium/genética , Enterococcus faecium/aislamiento & purificación , Enterococcus faecium/patogenicidad , Heces/microbiología , Fimbrias Bacterianas/genética , Humanos , VirulenciaRESUMEN
UNLABELLED: Mycoplasma pneumoniae synthesizes a novel human surfactant protein A (SP-A)-binding cytotoxin, designated community-acquired respiratory distress syndrome (CARDS) toxin, that exhibits ADP-ribosylating and vacuolating activities in mammalian cells and is directly linked to a range of acute and chronic airway diseases, including asthma. In our attempt to detect additional CARDS toxin-binding proteins, we subjected the membrane fraction of human A549 airway cells to affinity chromatography using recombinant CARDS toxin as bait. A 36-kDa A549 cell membrane protein bound to CARDS toxin and was identified by time of flight (TOF) mass spectroscopy as annexin A2 (AnxA2) and verified by immunoblotting with anti-AnxA2 monoclonal antibody. Dose-dependent binding of CARDS toxin to recombinant AnxA2 reinforced the specificity of the interaction, and further studies revealed that the carboxy terminus of CARDS toxin mediated binding to AnxA2. In addition, pretreatment of viable A549 cells with anti-AnxA2 monoclonal antibody or AnxA2 small interfering RNA (siRNA) reduced toxin binding and internalization. Immunofluorescence analysis of CARDS toxin-treated A549 cells demonstrated the colocalization of CARDS toxin with cell surface-associated AnxA2 upon initial binding and with intracellular AnxA2 following toxin internalization. HepG2 cells, which express low levels of AnxA2, were transfected with a plasmid expressing AnxA2 protein, resulting in enhanced binding of CARDS toxin and increased vacuolization. In addition, NCI-H441 cells, which express both AnxA2 and SP-A, upon AnxA2 siRNA transfection, showed decreased binding and subsequent vacuolization. These results indicate that CARDS toxin recognizes AnxA2 as a functional receptor, leading to CARDS toxin-induced changes in mammalian cells. IMPORTANCE: Host cell susceptibility to bacterial toxins is usually determined by the presence and abundance of appropriate receptors, which provides a molecular basis for toxin target cell specificities. To perform its ADP-ribosylating and vacuolating activities, community-acquired respiratory distress syndrome (CARDS) toxin must bind to host cell surfaces via receptor-mediated events in order to be internalized and trafficked effectively. Earlier, we reported the binding of CARDS toxin to surfactant protein A (SP-A), and here we show how CARDS toxin uses an alternative receptor to execute its pathogenic properties. CARDS toxin binds selectively to annexin A2 (AnxA2), which exists both on the cell surface and intracellularly. Since AnxA2 regulates membrane dynamics at early stages of endocytosis and trafficking, it serves as a distinct receptor for CARDS toxin binding and internalization and enhances CARDS toxin-induced vacuolization in mammalian cells.
Asunto(s)
Anexina A2/metabolismo , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Mycoplasma pneumoniae/metabolismo , Animales , Anexina A2/genética , Anexina A2/inmunología , Anticuerpos Monoclonales , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Línea Celular Tumoral , Endocitosis , Técnica del Anticuerpo Fluorescente , Células Hep G2 , Humanos , Unión Proteica , ARN Interferente Pequeño/genética , Síndrome de Dificultad Respiratoria , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Vacuolas/ultraestructuraRESUMEN
UNLABELLED: The inflammasome is a major regulator of inflammation through its activation of procaspase-1, which cleaves prointerleukin-1ß (pro-IL-1ß) into its mature form. IL-1ß is a critical proinflammatory cytokine that dictates the severity of inflammation associated with a wide spectrum of inflammatory diseases. NLRP3 is a key component of the inflammasome complex, and multiple signals and stimuli trigger formation of the NLRP3 inflammasome complex. In the current study, we uncovered a yet unknown mechanism of NLRP3 inflammasome activation by a pathogen-derived factor. We show that the unique bacterial ADP-ribosylating and vacuolating toxin produced by Mycoplasma pneumoniae and designated community-acquired respiratory distress syndrome (CARDS) toxin activates the NLRP3 inflammasome by colocalizing with the NLRP3 inflammasome and catalyzing the ADP-ribosylation of NLRP3. Mutant full-length CARDS toxin lacking ADP-ribosyltransferase (ADPRT) activity and truncated CARDS toxins unable to bind to macrophages and be internalized failed to activate the NLRP3 inflammasome. These studies demonstrate that CARDS toxin-mediated ADP-ribosylation constitutes an important posttranslational modification of NLRP3, that ADPRT activity of CARDS toxin is essential for NLRP3 inflammasome activation, and that posttranslational ADPRT-mediated modification of the inflammasome is a newly discovered mechanism for inflammasome activation with subsequent release of IL-1ß and associated pathologies. IMPORTANCE: Inflammation is a fundamental innate immune response to environmental factors, including infections. The inflammasome represents a multiprotein complex that regulates inflammation via its ability to activate specific proinflammatory cytokines, resulting in an effective host protective response. However, excessive release of proinflammatory cytokines can occur following infection that skews the host response to "hyperinflammation" with exaggerated tissue damage. Mycoplasma pneumoniae, a common bacterial airway pathogen, possesses a unique protein toxin with ADP-ribosyltransferase and vacuolating properties capable of reproducing the robust inflammation and cytopathology associated with mycoplasma infection. Here, we show that the toxin uniquely activates the NLRP3 inflammasome by colocalizing with and ADP-ribosylating NLRP3, possibly leading to "hyperinflammation" and thus uncovering a novel target for therapeutic intervention.