RESUMO
Moraxella catarrhalis is a respiratory tract pathogen commonly causing otitis media in children and acute exacerbations in patients suffering from chronic obstructive pulmonary disease. Cartilage oligomeric matrix protein (COMP) functions as a structural component in cartilage, as well as a regulator of complement activity. Importantly, COMP is detected in resident macrophages and monocytes, alveolar fluid, and the endothelium of blood vessels in lung tissue. We show that the majority of clinical isolates of M. catarrhalis (n = 49), but not other tested bacterial pathogens, bind large amounts of COMP. COMP interacts directly with the ubiquitous surface protein A2 of M. catarrhalis. Binding of COMP correlates with survival of M. catarrhalis in human serum by inhibiting bactericidal activity of the complement membrane attack complex. Moreover, COMP inhibits phagocytic killing of M. catarrhalis by human neutrophils. We further observed that COMP reduces bacterial adhesion and uptake by human lung epithelial cells, thus protecting M. catarrhalis from intracellular killing by epithelial cells. Taken together, our findings uncover a novel mechanism that M. catarrhalis uses to evade host innate immunity.
Assuntos
Proteína de Matriz Oligomérica de Cartilagem/imunologia , Evasão da Resposta Imune/imunologia , Imunidade Inata/imunologia , Moraxella catarrhalis/imunologia , Infecções por Moraxellaceae/imunologia , Aderência Bacteriana/imunologia , Proteína de Matriz Oligomérica de Cartilagem/metabolismo , Linhagem Celular , Citometria de Fluxo , Humanos , Moraxella catarrhalis/metabolismo , Infecções por Moraxellaceae/metabolismoRESUMO
The complement, coagulation, and fibrinolytic systems are crucial for the maintenance of tissue homeostasis. To date numerous interactions and cross-talks have been identified between these cascades. In line with this, here we propose a novel, hitherto unknown interaction between the complement inhibitor C4b-binding protein (C4BP) and plasminogen of the fibrinolytic pathway. Binding of C4BP to Streptococcus pneumoniae is a known virulence mechanism of this pathogen and it was increased in the presence of plasminogen. Interestingly, the acute phase variant of C4BP lacking the ß-chain and protein S binds plasminogen much stronger than the main isoform containing the ß-chain and protein S. Indeed, the complement control protein (CCP) 8 domain of C4BP, which would otherwise be sterically hindered by the ß-chain, primarily mediates this interaction. Moreover, the lysine-binding sites in plasminogen kringle domains facilitate the C4BP-plasminogen interaction. Furthermore, C4BP readily forms complexes with plasminogen in fluid phase and such complexes are present in human serum and plasma. Importantly, whereas the presence of plasminogen did not affect the factor I cofactor activity of C4BP, the activation of plasminogen by urokinase-type plasminogen activator to active plasmin was significantly augmented in the presence of C4BP. Taken together, our data demonstrate a novel interaction between two proteins of the complement and fibrinolytic system. Most complexes might be formed during the acute phase of inflammation and have an effect on the homeostasis at the site of injury or acute inflammation.
Assuntos
Proteína de Ligação ao Complemento C4b/metabolismo , Inflamação/metabolismo , Plasminogênio/metabolismo , Streptococcus pneumoniae/metabolismo , Proteína de Ligação ao Complemento C4b/genética , Fibrinogênio/metabolismo , Fibrinolisina/metabolismo , Fibrinólise/genética , Humanos , Inflamação/patologia , Lisina/metabolismo , Plasminogênio/genética , Ativadores de Plasminogênio/metabolismo , Ligação Proteica , Mapas de Interação de Proteínas , Proteína S/metabolismo , Streptococcus pneumoniae/patogenicidade , Ressonância de Plasmônio de Superfície , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
The fight between a human host and a bacterial pathogen is highly complicated; each party tries to outshine the other in the race for survival. In humans, the innate immune system--in particular the complement system--functions as the first line of defence against invading pathogens. During the course of evolution, however, pathogens, in order to survive and perpetuate within a host, developed multiple strategies to counteract the host complement system and to colonize. One such pathogen is Streptococcus pneumoniae (pneumococcus), a gram-positive bacterial pathogen often commensal in the human respiratory tract. Depending on the host's susceptibility, pneumococci can transform into an infectious agent, disseminating within the human host and causing mild to life-threatening diseases. This transition from commensal to infectious agent is a highly complex process, and understanding of this mechanism is essential in controlling the pathogenicity of pneumococci. Using its intricate arsenal of weapons, such as surface-presenting adhesins as well as recruitment of host factor, pneumococci successfully colonize the host, a prerequisite for establishing infection. This review describes C1q, the first subunit of the classical complement pathway, and its role in pneumococcus-host interactions, whereby pneumococci exploit C1q as a molecular bridge facilitating host cellular adherence and invasion, a function not akin to the role of C1q in the defence mechanism.
Assuntos
Complemento C1q/metabolismo , Evasão da Resposta Imune , Infecções Pneumocócicas/imunologia , Sistema Respiratório/imunologia , Streptococcus pneumoniae/imunologia , Animais , Aderência Bacteriana , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Infecções Pneumocócicas/transmissão , Sistema Respiratório/microbiologia , Streptococcus pneumoniae/patogenicidade , Simbiose , Fatores de VirulênciaRESUMO
The Gram-positive bacterium Streptococcus pneumoniae is a major human pathogen that causes infections ranging from acute otitis media to life-threatening invasive disease. Pneumococci have evolved several strategies to circumvent the host immune response, in particular the complement attack. The pneumococcal glycolytic enzyme phosphoglycerate kinase (PGK) is both secreted and bound to the bacterial surface and simultaneously binds plasminogen and its tissue plasminogen activator tPA. In the present study we demonstrate that PGK has an additional role in modulating the complement attack. PGK interacted with the membrane attack complex (MAC) components C5, C7, and C9, thereby blocking the assembly and membrane insertion of MAC resulting in significant inhibition of the hemolytic activity of human serum. Recombinant PGK interacted in a dose-dependent manner with these terminal pathway proteins, and the interactions were ionic in nature. In addition, PGK inhibited C9 polymerization both in the fluid phase and on the surface of sheep erythrocytes. Interestingly, PGK bound several MAC proteins simultaneously. Although C5 and C7 had partially overlapping binding sites on PGK, C9 did not compete with either one for PGK binding. Moreover, PGK significantly inhibited MAC deposition via both the classical and alternative pathway at the pneumococcal surface. Additionally, upon activation plasmin(ogen) bound to PGK cleaved the central complement protein C3b thereby further modifying the complement attack. In conclusion, our data demonstrate for the first time to our knowledge a novel pneumococcal inhibitor of the terminal complement cascade aiding complement evasion by this important pathogen.
Assuntos
Proteínas de Bactérias/metabolismo , Inativadores do Complemento/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Fosfoglicerato Quinase/metabolismo , Streptococcus pneumoniae/enzimologia , Animais , Proteínas de Bactérias/genética , Ativação do Complemento/efeitos dos fármacos , Complemento C3b/metabolismo , Complemento C5/metabolismo , Complemento C7/metabolismo , Complemento C9/metabolismo , Inativadores do Complemento/farmacologia , Complexo de Ataque à Membrana do Sistema Complemento/efeitos dos fármacos , Proteínas do Sistema Complemento/metabolismo , Relação Dose-Resposta a Droga , Eritrócitos/metabolismo , Citometria de Fluxo , Hemólise , Humanos , Fosfoglicerato Quinase/genética , Plasminogênio/metabolismo , Polimerização/efeitos dos fármacos , Ligação Proteica , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Soro/metabolismo , Ovinos , Streptococcus pneumoniae/genéticaRESUMO
The Gram-positive species Streptococcus pneumoniae is a human pathogen causing severe local and life-threatening invasive diseases associated with high mortality rates and death. We demonstrated recently that pneumococcal endopeptidase O (PepO) is a ubiquitously expressed, multifunctional plasminogen and fibronectin-binding protein facilitating host cell invasion and evasion of innate immunity. In this study, we found that PepO interacts directly with the complement C1q protein, thereby attenuating the classical complement pathway and facilitating pneumococcal complement escape. PepO binds both free C1q and C1 complex in a dose-dependent manner based on ionic interactions. Our results indicate that recombinant PepO specifically inhibits the classical pathway of complement activation in both hemolytic and complement deposition assays. This inhibition is due to direct interaction of PepO with C1q, leading to a strong activation of the classical complement pathway, and results in consumption of complement components. In addition, PepO binds the classical complement pathway inhibitor C4BP, thereby regulating downstream complement activation. Importantly, pneumococcal surface-exposed PepO-C1q interaction mediates bacterial adherence to host epithelial cells. Taken together, PepO facilitates C1q-mediated bacterial adherence, whereas its localized release consumes complement as a result of its activation following binding of C1q, thus representing an additional mechanism of human complement escape by this versatile pathogen.
Assuntos
Células Epiteliais Alveolares/imunologia , Proteínas de Bactérias/metabolismo , Complemento C1q/metabolismo , Endopeptidases/metabolismo , Infecções Pneumocócicas/imunologia , Streptococcus pneumoniae/metabolismo , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/microbiologia , Proteínas de Bactérias/imunologia , Adesão Celular/imunologia , Linhagem Celular , Complemento C1q/imunologia , Complemento C3b/imunologia , Complemento C3b/metabolismo , Proteína de Ligação ao Complemento C4b/imunologia , Proteína de Ligação ao Complemento C4b/metabolismo , Via Clássica do Complemento/imunologia , Endopeptidases/imunologia , Hemólise/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata/imunologia , Infecções Pneumocócicas/metabolismo , Mucosa Respiratória/citologia , Mucosa Respiratória/imunologia , Streptococcus pneumoniae/imunologia , Streptococcus pneumoniae/patogenicidade , VirulênciaRESUMO
Streptococcus pneumoniae (pneumococcus) is a major human pathogen, which evolved numerous successful strategies to colonize the host. In this study, we report a novel mechanism of pneumococcal-host interaction, whereby pneumococci use a host complement protein C1q, primarily involved in the host-defense mechanism, for colonization and subsequent dissemination. Using cell-culture infection assays and confocal microscopy, we observed that pneumococcal surface-bound C1q significantly enhanced pneumococcal adherence to and invasion of host epithelial and endothelial cells. Flow cytometry demonstrated a direct, Ab-independent binding of purified C1q to various clinical isolates of pneumococci. This interaction was seemingly capsule serotype independent and mediated by the bacterial surface-exposed proteins, as pretreatment of pneumococci with pronase E but not sodium periodate significantly reduced C1q binding. Moreover, similar binding was observed using C1 complex as the source of C1q. Furthermore, our data show that C1q bound to the pneumococcal surface through the globular heads and with the host cell-surface receptor(s)/glycosaminoglycans via its N-terminal collagen-like stalk, as the presence of C1q N-terminal fragment and low m.w. heparin but not the C-terminal globular heads blocked C1q-mediated pneumococcal adherence to host cells. Taken together, we demonstrate for the first time, to our knowledge, a unique function of complement protein C1q, as a molecular bridge between pneumococci and the host, which promotes bacterial cellular adherence and invasion. Nevertheless, in some conditions, this mechanism could be also beneficial for the host as it may result in uptake and clearance of the bacteria.
Assuntos
Aderência Bacteriana , Complemento C1q/imunologia , Infecções Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Streptococcus pneumoniae/metabolismo , Streptococcus pneumoniae/patogenicidade , Linhagem Celular , Ativação do Complemento , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Interações Hospedeiro-Patógeno , Humanos , Ácido Periódico , Infecções Pneumocócicas/metabolismo , Infecções Pneumocócicas/microbiologia , Polissacarídeos Bacterianos/metabolismo , PronaseRESUMO
Streptococcus pneumoniae infections remain a major cause of morbidity and mortality worldwide. Therefore a detailed understanding and characterization of the mechanism of host cell colonization and dissemination is critical to gain control over this versatile pathogen. Here we identified a novel 72-kDa pneumococcal protein endopeptidase O (PepO), as a plasminogen- and fibronectin-binding protein. Using a collection of clinical isolates, representing different serotypes, we found PepO to be ubiquitously present both at the gene and protein level. In addition, PepO protein was secreted in a growth phase-dependent manner to the culture supernatants of the pneumococcal isolates. Recombinant PepO bound human plasminogen and fibronectin in a dose-dependent manner and plasminogen did not compete with fibronectin for binding PepO. PepO bound plasminogen via lysine residues and the interaction was influenced by ionic strength. Moreover, upon activation of PepO-bound plasminogen by urokinase-type plasminogen activator, generated plasmin cleaved complement protein C3b thus assisting in complement control. Furthermore, direct binding assays demonstrated the interaction of PepO with epithelial and endothelial cells that in turn blocked pneumococcal adherence. Moreover, a pepO-mutant strain showed impaired adherence to and invasion of host cells compared with their isogenic wild-type strains. Taken together, the results demonstrated that PepO is a ubiquitously expressed plasminogen- and fibronectin-binding protein, which plays role in pneumococcal invasion of host cells and aids in immune evasion.
Assuntos
Proteínas de Bactérias/imunologia , Endopeptidases/imunologia , Fibronectinas/imunologia , Evasão da Resposta Imune/imunologia , Imunidade Inata/imunologia , Plasminogênio/imunologia , Aderência Bacteriana/genética , Aderência Bacteriana/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Complemento C3b/imunologia , Complemento C3b/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Fibrinolisina/imunologia , Fibrinolisina/metabolismo , Fibronectinas/metabolismo , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/microbiologia , Humanos , Microscopia Confocal , Mutação , Plasminogênio/metabolismo , Ligação Proteica , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/imunologia , Streptococcus pneumoniae/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/imunologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Streptococcus pyogenes AP1, a strain of the highly virulent M1 serotype, uses exclusively protein H to bind the complement inhibitor C4b-binding protein (C4BP). We found a strong correlation between the ability of AP1 and its isogenic mutants lacking protein H to inhibit opsonization with complement C3b and binding of C4BP. C4BP bound to immobilized protein H or AP1 bacteria retained its cofactor activity for degradation of (125)I-C4b. Furthermore, C4b deposited from serum onto AP1 bacterial surfaces was processed into C4c/C4d fragments, which did not occur on strains unable to bind C4BP. Recombinant C4BP mutants, which (i) lack certain CCP domains or (ii) have mutations in single aa as well as (iii) mutants with additional aa between different CCP domains were used to determine that the binding is mainly mediated by a patch of positively charged amino acid residues at the interface of domains CCP1 and CCP2. Using recombinant protein H fragments, we narrowed down the binding site to the N-terminal domain A. With a peptide microarray, we identified one single 18-amino acid-long peptide comprising residues 92-109, which specifically bound C4BP. Biacore was used to determine KD = 6 × 10(-7) M between protein H and a single subunit of C4BP. C4BP binding also correlated with elevated levels of adhesion and invasion to endothelial cells. Taken together, we identified the molecular basis of C4BP-protein H interaction and found that it is not only important for decreased opsonization but also for invasion of endothelial cells by S. pyogenes.
Assuntos
Aderência Bacteriana/fisiologia , Complemento C4b/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/patogenicidade , Fatores de Virulência/metabolismo , Sítios de Ligação , Células Cultivadas , Complemento C4b/genética , Proteína de Ligação ao Complemento C4b , Feminino , Células Endoteliais da Veia Umbilical Humana/microbiologia , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Masculino , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptococcus pyogenes/genética , Ressonância de Plasmônio de Superfície , Fatores de Virulência/genéticaRESUMO
Colonization of Streptococcus pneumoniae (pneumococci) is a prerequisite for bacterial dissemination and their capability to enter the bloodstream. Pneumococci have evolved various successful strategies to colonize the mucosal epithelial barrier of humans. A pivotal mechanism of host cell invasion implicated with invasive diseases is promoted by the interaction of pneumococcal PspC with the polymeric Ig-receptor (pIgR). However, the mechanism(s) of pneumococcal endocytosis and the intracellular route of pneumococci upon uptake by the PspC-pIgR-interaction are not known. Here, we demonstrate by using a combination of pharmacological inhibitors and genetics interference approaches the involvement of active dynamin-dependent caveolae and clathrin-coated vesicles for pneumococcal uptake via the PspC-pIgR mechanism. Depleting cholesterol from host cell membranes and disruption of lipid microdomains impaired pneumococcal internalization. Moreover, chemical inhibition of clathrin or functional inactivation of dynamin, caveolae or clathrin by RNA interference significantly affected pneumococcal internalization suggesting that clathrin-mediated endocytosis (CME) and caveolae are involved in the bacterial uptake process. Confocal fluorescence microscopy of pIgR-expressing epithelial cells infected with pneumococci or heterologous Lactococcus lactis expressing PspC demonstrated bacterial co-localization with fluorescent-tagged clathrin and early as well as recycling or late endosomal markers such as Lamp1, Rab5, Rab4, and Rab7, respectively. In conclusion these data suggest that PspC-promoted uptake is mediated by both CME and caveolae. After endocytosis pneumococci are routed via the endocytic pathway into early endosomes and are then sorted into recycling or late endosomes, which can result in pneumococcal killing in phagolysosomes or transcytosis via recycling endosomes.
Assuntos
Aderência Bacteriana , Caveolinas/metabolismo , Clatrina/metabolismo , Endocitose , Células Epiteliais/fisiologia , Receptores de Imunoglobulina Polimérica/metabolismo , Streptococcus pneumoniae/fisiologia , Animais , Proteínas de Bactérias/metabolismo , Linhagem Celular , Cães , Células Epiteliais/microbiologia , Humanos , Ligação ProteicaRESUMO
Streptococcus pneumoniae (pneumococcus) is a pathogen that causes severe local and life-threatening invasive diseases, which are associated with high mortality rates. Pneumococci have evolved several strategies to evade the host immune system, including complement to disseminate and to survive in various host niches. Thus, pneumococci bind complement inhibitors such as C4b-binding protein (C4BP) and factor H via pneumococcal surface protein C, thereby inhibiting the classical and alternative complement pathways. In this study, we identified the pneumococcal glycolytic enzyme enolase, a nonclassical cell surface and plasminogen-binding protein, as an additional pneumococcal C4BP-binding protein. Furthermore, we demonstrated that human, but not mouse, C4BP bound pneumococci. Recombinant enolase bound in a dose-dependent manner C4BP purified from plasma, and the interaction was reduced by increasing ionic strength. Enolase recruited C4BP and plasminogen, but not factor H, from human serum. Moreover, C4BP and plasminogen bound to different domains of enolase as they did not compete for the interaction with enolase. In direct binding assays with recombinant C4BP mutants lacking individual domains, two binding sites for enolase were identified on the complement control protein (CCP) domain 1/CCP2 and CCP8 of the C4BP α-chains. C4BP bound to the enolase retained its cofactor activity as determined by C4b degradation. Furthermore, in the presence of exogenously added enolase, an increased C4BP binding to and subsequently decreased C3b deposition on pneumococci was observed. Taken together, pneumococci specifically interact with human C4BP via enolase, which represents an additional mechanism of human complement control by this versatile pathogen.
Assuntos
Proteína de Ligação ao Complemento C4b/metabolismo , Inativadores do Complemento/metabolismo , Evasão da Resposta Imune , Fosfopiruvato Hidratase/metabolismo , Streptococcus pneumoniae/enzimologia , Streptococcus pneumoniae/imunologia , Proteína de Ligação ao Complemento C4b/antagonistas & inibidores , Glicólise , Humanos , Plasminogênio/metabolismo , Ligação Proteica/imunologia , Streptococcus pneumoniae/metabolismoRESUMO
OBJECTIVE: Fetus with intrauterine stress may exhibit programmed changes that can alter its metabolism and bear severe risk for diseases in adult life. The current study was designed to assess the correlation between cord blood lipid profile with the anthropometric data in neonates. MATERIALS AND METHODS: 146 newborn babies born at Dr. T M A Pai Hospital, Udupi were screened and their birth weight, length, head circumference and abdominal circumference were noted at birth. Umbilical cord blood samples were analyzed for total cholesterol, triglycerides (TG), high density lipoprotein (HDL) and low density lipoprotein (LDL). Infants were also grouped further based on gestational age (GA) and sex-adjusted birth weight percentiles into three groups i.e. Small for gestational age (SGA), Appropriate for gestational age (AGA) and Large for gestational age (LGA) for comparison of their lipid profiles. Inclusion criteria were normal fetal heart rate at birth and an APGAR score >7. Statistical significance of relation between lipid profile and anthropometry was done using ANOVA and Pearson correlation coefficient. RESULTS: Triglycerides were significantly higher in babies with higher ponderal index (PI) than those with lower PI (P = 0.011). The TG level of SGA babies were significantly higher as compared to AGA group (P = 0.001). The LDL levels in neonates with higher abdominal circumference were significantly lower than those with lower AC (P = 0.019). Mean HDL levels were higher in neonates with larger AC, but not statistically significant. Maternal BMI had no influence on neonates' lipid profile. CONCLUSION: Abnormal intrauterine milieu created by maternal changes during gestation may bear a profound impact on lipid metabolism in neonates, which may account for their differences in lipid profile and anthropometry at birth.
RESUMO
The pneumococcal surface protein C (PspC) is a major adhesin of Streptococcus pneumoniae (pneumococci) that interacts in a human-specific manner with the ectodomain of the human polymeric immunoglobulin receptor (pIgR) produced by respiratory epithelial cells. This interaction promotes bacterial colonization and bacterial internalization by initiating host signal transduction cascades. Here, we examined alterations of intracellular calcium ([Ca(2+)](i)) levels in epithelial cells during host cell infections with pneumococci via the PspC-hpIgR mechanism. The release of [Ca(2+)](i) from intracellular stores in host cells was significantly increased by wild-type pneumococci but not by PspC-deficient pneumococci. The increase in [Ca(2+)](i) was dependent on phospholipase C as pretreatment of cells with a phospholipase C-specific inhibitor U73122 abolished the increase in [Ca(2+)](i). In addition, we demonstrated the effect of [Ca(2+)](i) on pneumococcal internalization by epithelial cells. Uptake of pneumococci was significantly increased after pretreatment of epithelial cells with the cell-permeable calcium chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid-tetraacetoxymethyl ester or use of EGTA as an extracellular Ca(2+)-chelating agent. In contrast, thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+)ATPase, which increases [Ca(2+)](i) in a sustained fashion, significantly reduced pIgR-mediated pneumococcal invasion. Importantly, pneumococcal adherence to pIgR-expressing cells was not altered in the presence of inhibitors as demonstrated by immunofluorescence microscopy. In conclusion, these results demonstrate that pneumococcal infections induce mobilization of [Ca(2+)](i) from intracellular stores. This may constitute a defense response of host cells as the experimental reduction of intracellular calcium levels facilitates pneumococcal internalization by pIgR-expressing cells, whereas elevated calcium levels diminished bacterial internalization by host epithelial cells.
Assuntos
Proteínas de Bactérias/metabolismo , Infecções Pneumocócicas/metabolismo , Receptores de Imunoglobulina Polimérica/metabolismo , Streptococcus pneumoniae/metabolismo , Animais , Proteínas de Bactérias/genética , Cálcio/metabolismo , Linhagem Celular , Quelantes/farmacologia , Ácido Egtázico/farmacologia , Estrenos/farmacologia , Humanos , Camundongos , Inibidores de Fosfodiesterase/farmacologia , Infecções Pneumocócicas/genética , Pirrolidinonas/farmacologia , Receptores de Imunoglobulina Polimérica/genética , Streptococcus pneumoniae/genética , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismoRESUMO
Streptococcus pneumoniae, a human pathogen, recruits complement regulator factor H to its bacterial cell surface. The bacterial PspC protein binds Factor H via short consensus repeats (SCR) 8-11 and SCR19-20. In this study, we define how bacterially bound Factor H promotes pneumococcal adherence to and uptake by epithelial cells or human polymorphonuclear leukocytes (PMNs) via a two-step process. First, pneumococcal adherence to epithelial cells was significantly reduced by heparin and dermatan sulfate. However, none of the glycosaminoglycans affected binding of Factor H to pneumococci. Adherence of pneumococci to human epithelial cells was inhibited by monoclonal antibodies recognizing SCR19-20 of Factor H suggesting that the C-terminal glycosaminoglycan-binding region of Factor H mediates the contact between pneumococci and human cells. Blocking of the integrin CR3 receptor, i.e. CD11b and CD18, of PMNs or CR3-expressing epithelial cells reduced significantly the interaction of pneumococci with both cell types. Similarly, an additional CR3 ligand, Pra1, derived from Candida albicans, blocked the interaction of pneumococci with PMNs. Strikingly, Pra1 inhibited also pneumococcal uptake by lung epithelial cells but not adherence. In addition, invasion of Factor H-coated pneumococci required the dynamics of host-cell actin microfilaments and was affected by inhibitors of protein-tyrosine kinases and phosphatidylinositol 3-kinase. In conclusion, pneumococcal entry into host cells via Factor H is based on a two-step mechanism. The first and initial contact of Factor H-coated pneumococci is mediated by glycosaminoglycans expressed on the surface of human cells, and the second step, pneumococcal uptake, is integrin-mediated and depends on host signaling molecules such as phosphatidylinositol 3-kinase.
Assuntos
Fator H do Complemento/metabolismo , Streptococcus pneumoniae/metabolismo , Actinas/metabolismo , Animais , Aderência Bacteriana , Transporte Biológico , Células CHO , Linhagem Celular , Fator H do Complemento/química , Sequência Consenso , Cricetinae , Cricetulus , Citoesqueleto/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Glicosaminoglicanos/metabolismo , Heparina/química , Heparina/metabolismo , Humanos , Antígeno de Macrófago 1/metabolismo , Neutrófilos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Sequências Repetitivas de Aminoácidos , Transdução de Sinais , Solubilidade , Streptococcus pneumoniae/fisiologiaRESUMO
Streptococcus pneumoniae are commensals of the human nasopharynx with the capacity to invade mucosal respiratory cells. PspC, a pneumococcal surface protein, interacts with the human polymeric immunoglobulin receptor (pIgR) to promote bacterial adherence to and invasion into epithelial cells. Internalization of pneumococci requires the coordinated action of actin cytoskeleton rearrangements and the retrograde machinery of pIgR. Here, we demonstrate the involvement of Src protein-tyrosine kinases (PTKs), focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) but not p38 mitogen-activated protein kinases (MAPK) in pneumococcal invasion via pIgR. Pharmacological inhibitors of PTKs and MAPKs and genetic interference with Src PTK and FAK functions caused a significant reduction of pIgR-mediated pneumococcal invasion but did not influence bacterial adhesion to host cells. Furthermore, pneumococcal ingestion by host cells induces activation of ERK1/2 and JNK. In agreement with activated JNK, its target molecule and DNA-binding protein c-Jun was phosphorylated. We also show that functionally active Src PTK is essential for activation of ERK1/2 upon pneumococcal infections. In conclusion, these data illustrate the importance of a coordinated signaling between Src PTKs, ERK1/2, and JNK during PspC-pIgR-mediated uptake of pneumococci by host epithelial cells.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Receptores de Imunoglobulina Polimérica/metabolismo , Transdução de Sinais/fisiologia , Streptococcus pneumoniae/metabolismo , Quinases da Família src/metabolismo , Animais , Aderência Bacteriana/fisiologia , Proteínas de Bactérias/metabolismo , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Ativação Enzimática , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Flavonoides/farmacologia , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Genisteína/farmacologia , Interações Hospedeiro-Patógeno , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Microscopia Confocal , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Receptores de Imunoglobulina Polimérica/genética , Streptococcus pneumoniae/fisiologia , Transfecção , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/genéticaRESUMO
Iron oxide impregnated tamarind hull carbon (IOITHC) was developed for use as an adsorbent for the removal of As(V) from water. Tamarind hull was used as the source of carbonaceous material, which was first treated with ferric chloride and ammonium hydroxide solutions with successive calcination at 873-974 K in a muffle furnace for 1 h to prepare an arsenic adsorbent. The B.E.T surface area of IOITHC was found to be 304.6 m(2) g(-1) and the average iron content in the adsorbent was found to be 7 wt%. The point of zero charge (pH(zpc)) of IOITHC was found to be 6.9. As(V) and arsenic (as total) adsorption on IOITHC were investigated in batch mode using As(V) spiked distilled water and real contaminated groundwater (CGW). The effects of speed of agitation, adsorbent particle size, temperature, pH of the solution, and concentration of the adsorbate on the adsorption process were investigated. The maximum adsorption capacity of about 1.2 mg g(-1) As(V) was achieved. The removal of As(V) on IOITHC was compared with the untreated tamarind hull carbon as well as with the activated commercial carbon and IOITHC was found to be better adsorbent. Arsenic adsorption from arsenic contaminated groundwater (CGW) on IOITHC in batch mode indicates that 98% removal was achieved for adsorbent loading of 3.0 g L(-1) with initial arsenic concentration of 264 microg L(-1). Desorption study of arsenic from As(V)-loaded IOITHC was performed using aqueous solution in the pH range of 3 to 12.
Assuntos
Arsênio/isolamento & purificação , Compostos Férricos/química , Tamarindus , Poluentes Químicos da Água/isolamento & purificação , Carbono/química , Carvão Vegetal/químicaRESUMO
BibA, a group B streptococcus (GBS) surface protein, has been shown to protect the pathogen from phagocytic killing by sequestering a complement inhibitor: C4b-binding protein (C4BP). Here, the X-ray crystallographic structure of a GBS BibA fragment (BibA126-398) and a low-resolution small-angle X-ray scattering (SAXS) structure of the full-length N-terminal domain (BibA34-400) are described. The BibA126-398 fragment crystal structure displayed a novel and predominantly helical structure. The tertiary arrangement of helices forms four antiparallel three-helix-bundle-motif repeats, with one long helix from a bundle extending into the next. Multiple mutations on recombinant BibA34-400 delayed the degradation of the protein, and circular dichroism spectroscopy of BibA34-400 suggested a similar secondary-structure composition to that observed in the crystallized BibA126-398 fragment. A model was generated for the 92 N-terminal residues (BibA34-125) using structural similarity prediction programs, and a BibA34-400 model was generated by combining the coordinates of BibA34-126 and BibA126-398. The X-ray structure of BibA126-398 and the model of BibA34-400 fitted well into the calculated SAXS envelope. One possible binding site for the BibA N-terminal domain was localized to the N-terminal CCP (complement-control protein) domains of the C4BP α-chain, as indicated by the decreased binding of BibA to a ΔCCP1 C4BP α-chain mutant. In summary, it is suggested that the GBS surface protein BibA, which consists of three antiparallel α-helical-bundle motifs, is unique and belongs to a new class of Gram-positive surface adhesins.
Assuntos
Adesinas Bacterianas/química , Streptococcus agalactiae/metabolismo , Sítios de Ligação , Proteína de Ligação ao Complemento C4b/química , Cristalografia por Raios X , Conformação Proteica em alfa-HéliceRESUMO
Helicobacter pylori is an important human pathogen and a common cause of peptic ulcers and gastric cancer. Despite H. pylori provoking strong innate and adaptive immune responses, the bacterium is able to successfully establish long-term infections. Vitronectin (Vn), a component of both the extracellular matrix and plasma, is involved in many physiological processes, including regulation of the complement system. The aim of this study was to define a receptor in H. pylori that binds Vn and determine the significance of the interaction for virulence. Surprisingly, by using proteomics, we found that the hydrogen peroxide-neutralizing enzyme catalase KatA is a major Vn-binding protein. Deletion of the katA gene in three different strains resulted in impaired binding of Vn. Recombinant KatA was generated and shown to bind with high affinity to a region between heparin-binding domain 2 and 3 of Vn that differs from previously characterised bacterial binding sites on the molecule. In terms of function, KatA protected H. pylori from complement-mediated killing in a Vn-dependent manner. Taken together, the virulence factor KatA is a Vn-binding protein that moonlights on the surface of H. pylori to promote bacterial evasion of host innate immunity.
Assuntos
Proteínas de Bactérias/metabolismo , Catalase/metabolismo , Proteínas do Sistema Complemento/metabolismo , Helicobacter pylori/enzimologia , Fatores de Virulência/metabolismo , Vitronectina/metabolismo , Proteínas de Bactérias/genética , Catalase/genética , Deleção de Genes , Helicobacter pylori/imunologia , Evasão da Resposta Imune , Imunidade Inata , Ligação Proteica , Virulência , Fatores de Virulência/química , Vitronectina/químicaRESUMO
OBJECTIVE: Present work focuses on the use of mimosa seed gum to develop a drug delivery system making combined use of floating and pulsatile principles, for the chrono-prevention of nocturnal acid breakthrough. METHODOLOGY: The desired aim was achieved by fabricating a floating delivery system bearing time - lagged coating of Mimosa pudica seed polymer for the programmed release of Famotidine. Response Surface Methodology was the statistical tool that was employed for experiment designing, mathematical model generation and optimization study. A 3(2) full factorial design was used in designing the experiment.% weight ratio of mimosa gum to hydroxy propyl methyl cellulose in the coating combination and the coating weight were the independent variables, whereas the lag time and the cumulative % drug release in 360 minutes were the observed responses. KEY FINDINGS: Results revealed that both the coating composition and the coating weight significantly affected the release of drug from the dosage form. CONCLUSION: The optimized formulation prepared according to the computer generated software, Design-Expert(®) deciphered response which were in close proximity with the experimental responses, thus confirming the robustness as well as accuracy of the predicted model for the utilization of natural polymer like mimosa seed gum for the chronotherapeutic treatment of nocturnal acid breakthrough.
Assuntos
Antiácidos/química , Sistemas de Liberação de Medicamentos , Famotidina/química , Mimosa , Gomas Vegetais/química , Antiácidos/administração & dosagem , Química Farmacêutica , Preparações de Ação Retardada/administração & dosagem , Preparações de Ação Retardada/química , Famotidina/administração & dosagem , Gomas Vegetais/administração & dosagem , SementesRESUMO
"Blind race ends in a pit". A similar scenario is observed with the use of conventional dosage forms for different pathological conditions. Of late various disease states had regularly been reported to bear direct concurrence to the body's secretions that bear a constant rotationary cycle. Moreover the pharmacokinetic as well as pharmacodynamic responsiveness of various drugs had been reported to bear constant swings with the changing hours of the day. Thus, usage of the conventional zero order dosage form for every disease state or every active moiety developed, will only leave the researchers as well as the consumers in doldrums. Chronomodulated dosage forms are a silver lining in these overshadowed clouds. They are the dosage forms that spearhead the innovative researches because of their pre-programmed and pre-regulated pulsed release of the drug, at desired sites. Drug release pattern from these dosage forms considerably mimics the circadian timing of the body's secretion which are held responsible for the symptoms of the pathological irregularities arising in one's body. Thus, in a way these dosage forms are a shield against the inducers of the disease symptoms. Current review enlists the pathological states for which the chronomodulated delivery systems can prove to be a miraculous cure. This review emphasizes to summarize the patents granted as well as the novel researches undertaken by various researchers to upgrade the previously existing dosage form scenario. Moreover, this work is an attempt to summarize the various proprietary techniques and marketed formulations, thus trying to help the researchers to fabricate a better and novel dosage form from previously existing ones.
Assuntos
Cronofarmacoterapia , Sistemas de Liberação de Medicamentos , Preparações Farmacêuticas/administração & dosagem , Formas de Dosagem , Desenho de Fármacos , Humanos , Patentes como Assunto , Fatores de TempoRESUMO
OBJECTIVE: Present work focuses on the use of tamarind gum to develop a drug delivery system making combined use of floating and pulsatile principles, for the chrono-prevention of nocturnal acid breakthrough. METHOD: The desired aim was achieved by fabricating a floating delivery system bearing time - lagged coating of Tamarindus indica seed polymer for the programmed release of Famotidine. Response Surface METHODology was the statistical tool that was employed for experiment designing, mathematical model generation and optimization study. A 32 full factorial design was used in designing the experiment. % weight ratio of tamarind gum to ethyl cellulose in the coating combination and the coating weight were the independent variables, whereas the lag time for drug release and the cumulative % drug release in 330 minutes were the observed responses. KEY FINDINGS: Results revealed that both the coating composition and the coating weight significantly affected the release of drug from the dosage form. CONCLUSION: The optimized formulation prepared according to the computer generated software, Design-Expert® deciphered response which were in close proximity with the experimental responses, thus confirming the robustness and accuracy of the predicted model for the utilization of natural polymer like tamarind gum for the chronotherapeutic treatment of nocturnal acid breakthrough.