RESUMO
Group A Streptococcus (GAS) causes a wide range of human infections, ranging from simple pharyngitis to life-threatening necrotizing fasciitis and toxic shock syndrome. A globally disseminated clone of M1T1 GAS has been associated with an increase in severe, invasive GAS infections in recent decades. The secreted GAS pore-forming toxin streptolysin O (SLO), which induces eukaryotic cell lysis in a cholesterol-dependent manner, is highly upregulated in the GAS M1T1 clone during bloodstream dissemination. SLO is known to promote GAS resistance to phagocytic clearance by neutrophils, a critical first element of host defense against invasive bacterial infection. Here, we examine the role of SLO in modulating specific neutrophil functions during their early interaction with GAS. We find that SLO at subcytotoxic concentrations and early time points is necessary and sufficient to suppress neutrophil oxidative burst, in a manner reversed by free cholesterol and anti-SLO blocking antibodies. In addition, SLO at subcytotoxic concentrations blocked neutrophil degranulation, interleukin-8 secretion and responsiveness, and elaboration of DNA-based neutrophil extracellular traps, cumulatively supporting a key role for SLO in GAS resistance to immediate neutrophil killing. A non-toxic SLO derivate elicits protective immunity against lethal GAS challenge in a murine infection model. We conclude that SLO exerts a novel cytotoxic-independent function at early stages of invasive infections (<30 min), contributing to GAS escape from neutrophil clearance.
RESUMO
The role of sirtuin-1 (SIRT1) in innate immunity, and in particular the influence of SIRT1 on antimicrobial defense against infection, has yet to be reported but is important to define since SIRT1 inhibitors are being investigated as therapeutic agents in the treatment of cancer, Huntington's disease, and autoimmune diseases. Given the therapeutic potential of SIRT1 suppression, we sought to characterize the role of SIRT1 in host defense. Utilizing both pharmacologic methods and a genetic knockout, we demonstrate that SIRT1 expression has little influence on macrophage and neutrophil antimicrobial functions. Myeloid SIRT1 expression does not change mortality in gram-negative toxin-induced shock or gram-positive bacteremia, suggesting that therapeutic suppression of SIRT1 may be done safely without suppression of myeloid cell-specific immune responses to severe bacterial infections.
Assuntos
Endotoxemia/imunologia , Regulação da Expressão Gênica/imunologia , Infecções por Bactérias Gram-Positivas/imunologia , Células Mieloides/imunologia , Sirtuína 1/imunologia , Animais , Bacteriemia/genética , Bacteriemia/imunologia , Bacteriemia/metabolismo , Bacteriemia/patologia , Endotoxemia/genética , Endotoxemia/metabolismo , Endotoxemia/patologia , Regulação da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Infecções por Bactérias Gram-Positivas/genética , Infecções por Bactérias Gram-Positivas/metabolismo , Infecções por Bactérias Gram-Positivas/patologia , Células HL-60 , Humanos , Camundongos , Células Mieloides/metabolismo , Células Mieloides/patologia , Sirtuína 1/biossíntese , Sirtuína 1/genéticaRESUMO
Group A Streptococcus (GAS) is a human-specific bacterial pathogen responsible for serious morbidity and mortality worldwide. The hyaluronic acid (HA) capsule of GAS is a major virulence factor, contributing to bloodstream survival through resistance to neutrophil and antimicrobial peptide killing and to in vivo pathogenicity. Capsule biosynthesis has been exclusively attributed to the ubiquitous hasABC hyaluronan synthase operon, which is highly conserved across GAS serotypes. Previous reports indicate that hasA, encoding hyaluronan synthase, and hasB, encoding UDP-glucose 6-dehydrogenase, are essential for capsule production in GAS. Here, we report that precise allelic exchange mutagenesis of hasB in GAS strain 5448, a representative of the globally disseminated M1T1 serotype, did not abolish HA capsule synthesis. In silico whole-genome screening identified a putative HasB paralog, designated HasB2, with 45% amino acid identity to HasB at a distant location in the GAS chromosome. In vitro enzymatic assays demonstrated that recombinant HasB2 is a functional UDP-glucose 6-dehydrogenase enzyme. Mutagenesis of hasB2 alone slightly decreased capsule abundance; however, a ΔhasB ΔhasB2 double mutant became completely acapsular. We conclude that HasB is not essential for M1T1 GAS capsule biogenesis due to the presence of a newly identified HasB paralog, HasB2, which most likely resulted from gene duplication. The identification of redundant UDP-glucose 6-dehydrogenases underscores the importance of HA capsule expression for M1T1 GAS pathogenicity and survival in the human host.
Assuntos
Cápsulas Bacterianas/metabolismo , Proteínas de Bactérias/metabolismo , Óperon/genética , Streptococcus pyogenes/metabolismo , Uridina Difosfato Glucose Desidrogenase/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica , Genoma Bacteriano , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Insercional , Filogenia , Conformação Proteica , Streptococcus pyogenes/genética , Uridina Difosfato Glucose Desidrogenase/genéticaRESUMO
Detection of microbial constituents by membrane associated and cytoplasmic pattern recognition receptors is the essence of innate immunity, leading to activation of protective host responses. However, it is still unclear how immune cells specifically respond to pathogenic bacteria. Using virulent and nonvirulent strains of Bacillus anthracis, we have shown that secretion of ATP by infected macrophages and the sequential activation of the P2X7 purinergic receptor and nucleotide binding oligomerization domain (NOD)-like receptors are critical for IL-1-dependent host protection from virulent B. anthracis. Importantly, lethal toxin produced by virulent B. anthracis blocked activation of protein kinases, p38 MAPK and AKT, resulting in opening of a connexin ATP release channel and induction of macrophage death. Prevention of cell death or ATP release through constitutive p38 or AKT activation interfered with inflammasome activation and IL-1ß production, thereby compromising antimicrobial immunity.
Assuntos
Antraz/imunologia , Antígenos de Bactérias/metabolismo , Bacillus anthracis/imunologia , Toxinas Bacterianas/metabolismo , Inflamassomos/metabolismo , Macrófagos Peritoneais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Antraz/microbiologia , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Bacillus anthracis/genética , Bacillus anthracis/patogenicidade , Toxinas Bacterianas/genética , Toxinas Bacterianas/imunologia , Células Cultivadas , Conexina 43/metabolismo , Imunidade Inata/genética , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/microbiologia , Macrófagos Peritoneais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Proteína Oncogênica v-akt/antagonistas & inibidores , Receptores Purinérgicos P2X7/metabolismo , Virulência/genética , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Transcription factor NF-κB and its activating kinase IKKß are associated with inflammation and are believed to be critical for innate immunity. Despite the likelihood of immune suppression, pharmacological blockade of IKKß-NF-κB has been considered as a therapeutic strategy. However, we found neutrophilia in mice with inducible deletion of IKKß (Ikkß(Δ) mice). These mice had hyperproliferative granulocyte-macrophage progenitors and pregranulocytes and a prolonged lifespan of mature neutrophils that correlated with the induction of genes encoding prosurvival molecules. Deletion of interleukin 1 receptor 1 (IL-1R1) in Ikkß(Δ) mice normalized blood cellularity and prevented neutrophil-driven inflammation. However, Ikkß(Δ)Il1r1(-/-) mice, unlike Ikkß(Δ) mice, were highly susceptible to bacterial infection, which indicated that signaling via IKKß-NF-κB or IL-1R1 can maintain antimicrobial defenses in each other's absence, whereas inactivation of both pathways severely compromises innate immunity.
Assuntos
Infecções Bacterianas/imunologia , Células Progenitoras de Granulócitos e Macrófagos/metabolismo , Quinase I-kappa B/metabolismo , Interleucina-1beta/metabolismo , Neutrófilos/metabolismo , Animais , Contagem de Células , Processos de Crescimento Celular/genética , Sobrevivência Celular/genética , Células Cultivadas , Suscetibilidade a Doenças , Células Progenitoras de Granulócitos e Macrófagos/imunologia , Células Progenitoras de Granulócitos e Macrófagos/patologia , Quinase I-kappa B/genética , Quinase I-kappa B/imunologia , Imunidade Inata/genética , Interleucina-1beta/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise em Microsséries , Neutrófilos/imunologia , Neutrófilos/patologia , Receptores Tipo I de Interleucina-1/genética , Regulação para Cima/genéticaRESUMO
BACKGROUND: The recent resurgence of invasive group A streptococcal disease has been paralleled by the emergence of the M1T1 clone. Recently, invasive disease initiation has been linked to mutations in the covR/S 2-component regulator. We investigated whether a fitness cost is associated with the covS mutation that counterbalances hypervirulence. METHODS: Wild-type M1T1 group A Streptococcus and an isogenic covS-mutant strain derived from animal passage were compared for adherence to human laryngeal epithelial cells, human keratinocytes, or fibronectin; biofilm formation; and binding to intact mouse skin. Targeted mutagenesis of capsule expression of both strains was performed for analysis of its unique contribution to the observed phenotypes. RESULTS: The covS-mutant bacteria showed reduced capacity to bind to epithelial cell layers as a consequence of increased capsule expression. The covS-mutant strain also had reduced capacity to bind fibronectin and to form biofilms on plastic and epithelial cell layers. A defect in skin adherence of the covS-mutant strain was demonstrated in a murine model. CONCLUSION: Reduced colonization capacity provides a potential explanation for why the covS mutation, which confers hypervirulence, has not become fixed in the globally disseminated M1T1 group A Streptococcus clone, but rather may arise anew under innate immune selection in individual patients.
Assuntos
Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidade , Animais , Aderência Bacteriana , Linhagem Celular , Células Epiteliais/microbiologia , Fibronectinas/metabolismo , Humanos , Queratinócitos/microbiologia , Camundongos , Fenótipo , Ligação Proteica , Pele/microbiologia , VirulênciaRESUMO
Group A Streptococcus is a leading human pathogen associated with a diverse array of mucosal and systemic infections. Cell wall anchored pili were recently described in several species of pathogenic streptococci, and in the case of GAS, these surface appendages were demonstrated to facilitate epithelial cell adherence. Here we use targeted mutagenesis to evaluate the contribution of pilus expression to virulence of the globally disseminated M1T1 GAS clone, the leading agent of both GAS pharyngitis and severe invasive infections. We confirm that pilus expression promotes GAS adherence to pharyngeal cells, keratinocytes, and skin. However, in contrast to findings reported for group B streptococcal and pneumococcal pili, we observe that pilus expression reduces GAS virulence in murine models of necrotizing fasciitis, pneumonia and sepsis, while decreasing GAS survival in human blood. Further analysis indicated the systemic virulence attenuation associated with pilus expression was not related to differences in phagocytic uptake, complement deposition or cathelicidin antimicrobial peptide sensitivity. Rather, GAS pili were found to induce neutrophil IL-8 production, promote neutrophil transcytosis of endothelial cells, and increase neutrophil release of DNA-based extracellular traps, ultimately promoting GAS entrapment and killing within these structures.
Assuntos
Neutrófilos/microbiologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/metabolismo , Animais , Adesão Celular , Células Epiteliais/microbiologia , Feminino , Fímbrias Bacterianas/microbiologia , Humanos , Interleucina-8/metabolismo , Macrófagos/microbiologia , Camundongos , Mutagênese , Fagocitose , Pele/microbiologia , Streptococcus pyogenes/patogenicidade , VirulênciaRESUMO
Group A Streptococcus (GAS) is a leading human bacterial pathogen capable of producing invasive infections even in previously healthy individuals. As frontline components of host innate defense, macrophages play a key role in control and clearance of GAS infections. We find GAS induces rapid, dose-dependent apoptosis of primary and cultured macrophages and neutrophils. The cell death pathway involves apoptotic caspases, is partly dependent on caspase-1, and requires GAS internalization by the phagocyte. Analysis of GAS virulence factor mutants, heterologous expression, and purified toxin studies identified the pore-forming cytolysin streptolysin O (SLO) as necessary and sufficient for the apoptosis-inducing phenotype. SLO-deficient GAS mutants induced less macrophage apoptosis in vitro and in vivo, allowed macrophage cytokine secretion, and were less virulent in a murine systemic infection model. Ultrastructural evidence of mitochondrial membrane remodeling, coupled with loss of mitochondrial depolarization and cytochrome c release, suggests a direct attack of the toxin initiates the intrinsic apoptosis pathway. A general caspase inhibitor blocked SLO-induced apoptosis and enhanced macrophage killing of GAS. We conclude that accelerated, caspase-dependent macrophage apoptosis induced by the pore-forming cytolysin SLO contributes to GAS immune evasion and virulence.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/imunologia , Macrófagos/citologia , Macrófagos/imunologia , Streptococcus pyogenes/imunologia , Estreptolisinas/farmacologia , Animais , Proteínas de Bactérias/farmacologia , Caspases/metabolismo , Linhagem Celular , Citocromos c/metabolismo , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/ultraestrutura , Streptococcus pyogenes/patogenicidade , Fatores de TempoRESUMO
Interleukin-8 (IL-8) promotes neutrophil-mediated host defense through its chemoattractant and immunostimulatory activities. The Group A Streptococcus (GAS) protease SpyCEP (also called ScpC) cleaves IL-8, and SpyCEP expression is strongly upregulated in vivo in the M1T1 GAS strains associated with life-threatening systemic disease including necrotizing fasciitis. Coupling allelic replacement with heterologous gene expression, we show that SpyCEP is necessary and sufficient for IL-8 degradation. SpyCEP decreased IL-8-dependent neutrophil endothelial transmigration and bacterial killing, the latter by reducing neutrophil extracellular trap formation. The knockout mutant lacking SpyCEP was attenuated for virulence in murine infection models, and SpyCEP expression conferred protection to coinfecting bacteria. We also show that the zoonotic pathogen Streptococcus iniae possesses a functional homolog of SpyCEP (CepI) that cleaves IL-8, promotes neutrophil resistance, and contributes to virulence. By inactivating the multifunctional host defense peptide IL-8, the SpyCEP protease impairs neutrophil clearance mechanisms, contributing to the pathogenesis of invasive streptococcal infection.
Assuntos
Interações Hospedeiro-Patógeno , Interleucina-8/metabolismo , Neutrófilos/imunologia , Peptídeo Hidrolases/metabolismo , Infecções Estreptocócicas/imunologia , Streptococcus pyogenes/enzimologia , Animais , Células Cultivadas , Células Endoteliais/microbiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/microbiologia , Peptídeo Hidrolases/genética , Pele/microbiologia , Infecções Estreptocócicas/microbiologia , Streptococcus/enzimologia , Streptococcus/genética , Streptococcus/fisiologia , Streptococcus pyogenes/genética , VirulênciaRESUMO
Macrophage activation relies on complex intracellular signaling processes that integrate the need for rapid inflammatory responses to pathogens with the need to resolve inflammation without permanent harm to normal tissues. Patterns of aberrant macrophage activation characterize and sustain disorders of chronic inflammation, infection, and cancer. New studies now show a role for the NF-kappaB activator IKKbeta in promoting an alternative, immunosuppressive pattern of macrophage activation, which limits the cell's tumoricidal and bactericidal capacities. As cancers and pathogens may have evolved multiple mechanisms to manipulate macrophages for their own survival, is there anything we can do about it?
Assuntos
Quinase I-kappa B/fisiologia , Inflamação/fisiopatologia , Ativação de Macrófagos/fisiologia , Macrófagos/fisiologia , NF-kappa B/fisiologia , Diferenciação Celular/fisiologia , Humanos , Infecções/fisiopatologia , Interleucinas/fisiologia , Macrófagos/patologia , Complexo Principal de Histocompatibilidade , Neoplasias/patologia , Neoplasias/fisiopatologia , Fagócitos/fisiologiaRESUMO
Streptococcus iniae is a major fish pathogen producing invasive infections that result in economic losses in aquaculture. Development of in vitro models of S. iniae virulence may provide insight to the pathogenesis of infection in vivo. Three S. iniae strains (K288, 94-426, and 29178) were tested for virulence in a hybrid-striped bass (HSB) model using intraperitoneal injection. S. iniae strains K288 and 94-426 caused high levels of mortality in HSB (lethal dose 2x10(5)CFU) while strain 29178 was avirulent even upon IP challenge with 1000-fold higher inocula. In vitro assays were developed to test for the presence of characteristics previously associated with virulence in other species of pathogenic Streptococcus in animals and humans. In vitro differences relevant to virulence were not detected for beta-hemolysin activity, sensitivity to antimicrobial peptides, or adherence and invasion of epithelial cell layers. However, in whole-blood killing assays, the pathogenic strains were resistant to blood clearance, while 29178 was cleared (P<0.001) and more sensitive to complement (P<0.001). The avirulent strain 29178 was most efficiently phagocytosed and was most susceptible to intracellular killing (P<0.01) by the carp leukocyte cell line (CLC). When exposed to reactive oxygen species, strain 29178 was most susceptible. When the oxidative burst of CLC cells was inhibited, intracellular survival of 29178 was rescued fivefold, while no significant enhancement in survival of K288 or 94-426 was detected. Our results indicate that resistance to phagocytosis, oxidative killing, and associated phagocytic clearance is a significant factor in S. iniae virulence.
Assuntos
Bass/microbiologia , Doenças dos Peixes/microbiologia , Fagocitose , Infecções Estreptocócicas/veterinária , Streptococcus/patogenicidade , Animais , Linhagem Celular , Contagem de Colônia Microbiana/veterinária , Hemólise , Injeções Intraperitoneais/veterinária , Distribuição Aleatória , Especificidade da Espécie , Infecções Estreptocócicas/microbiologia , Fatores de VirulênciaRESUMO
Serum opacity factor (SOF) is a unique multifunctional virulence determinant expressed at the surface of Streptococcus pyogenes and has been shown to elicit protective immunity against GAS infection in a murine challenge model. SOF consists of two distinct domains with different binding capacities: an N-terminal domain that binds apolipoprotein AI and a C-terminal repeat domain that binds fibronectin and fibrinogen. The capacity of SOF to opacify serum by disrupting the structure of high density lipoproteins may preclude its use as a vaccine antigen in humans. This study generated mutant forms of recombinant SOF with reduced (100-fold) or abrogated opacity factor (OF) activity, for use as vaccine antigens. However, alterations introduced into the N-terminal SOF peptide (SOFDeltaFn) by mutagenesis to abrogate OF activity, abolish the capacity of SOF to protect against lethal systemic S. pyogenes challenge in a murine model. Mutant forms of purified SOFDeltaFn peptide were also used to assess the contribution of OF activity to the pathogenic processes of cell adhesion and cell invasion. Using latex beads coated with full-length SOF, SOFDeltaFn peptide, or a peptide encompassing the C-terminal repeats (FnBD), we demonstrate that adhesion to HEp-2 cells is mediated by both SOFDeltaFn and FnBD. The HEp-2 cell binding displayed by the N-terminal SOFDeltaFn peptide is independent of OF activity. We demonstrate that while the N terminus of SOF does not directly mediate intracellular uptake by epithelial cells, this domain enhances epithelial cell uptake mediated by full-length SOF, in comparison to the FnBD alone.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Células Epiteliais/imunologia , Peptídeo Hidrolases/imunologia , Infecções Estreptocócicas/imunologia , Vacinas Estreptocócicas/imunologia , Streptococcus pyogenes/imunologia , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/farmacologia , Apolipoproteína A-I/genética , Apolipoproteína A-I/imunologia , Aderência Bacteriana/genética , Aderência Bacteriana/imunologia , Proteínas de Bactérias/genética , Linhagem Celular , Modelos Animais de Doenças , Células Epiteliais/microbiologia , Fibrinogênio/genética , Fibrinogênio/imunologia , Fibronectinas/genética , Fibronectinas/imunologia , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Peptídeo Hidrolases/genética , Peptídeos/genética , Peptídeos/imunologia , Peptídeos/farmacologia , Ligação Proteica/genética , Ligação Proteica/imunologia , Estrutura Terciária de Proteína/genética , Infecções Estreptocócicas/genética , Infecções Estreptocócicas/prevenção & controle , Vacinas Estreptocócicas/genética , Vacinas Estreptocócicas/farmacologia , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidadeRESUMO
Antibiotics are designed to support host defense in controlling infection. Here we describe a paradoxical inhibitory effect of bacteriostatic antibiotics on key mediators of mammalian innate immunity. When growth of species including Escherichia coli and Staphylococcus aureus is suppressed by chloramphenicol or erythromycin, the susceptibility of the bacteria to cathelicidin antimicrobial peptides or serum complement was markedly diminished. Survival of the bacteria in human whole blood, human wound fluid, or a mouse wound infection model was in turn increased after antibiotic-induced bacteriostasis. These findings provide a further rationale against the indiscriminate use of antibiotics.
Assuntos
Anti-Infecciosos/farmacologia , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Catelicidinas , Cloranfenicol/farmacologia , Proteínas Inativadoras do Complemento/farmacologia , Eritromicina/farmacologia , Feminino , Humanos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Group A Streptococcus (GAS) is a leading human pathogen associated with a wide spectrum of mucosal and invasive infections. GAS expresses a large number of virulence determinants whose expression is under the control of several transcriptional regulatory networks. Here we performed the first mutational analysis of a genetic locus immediately upstream of the streptolysin S biosynthetic operon in several GAS genome sequences, including that of the M1T1 serotype, the leading isolates associated with serious invasive disease. The locus consists of a predicted RofA-like stand-alone transcriptional regulator (RALP3) and the largest open reading frame in the GAS genome, encoding a predicted LPXSG motif cell wall-anchored protein we have named LSA (for "large surface-anchored" protein). Comparative reverse transcription-PCR analysis of wild-type M1T1 GAS and an isogenic RALP3-deficient mutant identifies RALP3 as a global transcriptional regulator affecting expression of numerous virulence factor genes, including those for strong repression of the hyaluronic acid capsule and cysteine protease production. RALP3 contributed to GAS epithelial cell invasion and bloodstream survival. LSA was found to be under negative regulation by RALP3 and to influence GAS-epithelial cell interactions and GAS antimicrobial peptide sensitivity. Isogenic M1T1 GAS mutants lacking either RALP3 or LSA were attenuated in a murine model of systemic infection, indicating that this locus plays a role in the virulence potential of the organism.
Assuntos
Proteínas de Bactérias/metabolismo , Óperon/genética , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidade , Estreptolisinas/metabolismo , Animais , Cápsulas Bacterianas/metabolismo , Proteínas de Bactérias/genética , Linhagem Celular , Sobrevivência Celular , Meios de Cultura , Cisteína Endopeptidases/metabolismo , Feminino , Humanos , Ácido Hialurônico/metabolismo , Camundongos , Estreptolisinas/genética , VirulênciaRESUMO
The TGFbeta family member Nodal has been implicated in heart induction through misexpression of a dominant negative version of the type I Nodal receptor (Alk4) and targeted deletion of the co-receptor Cripto in murine ESCs and mouse embryos; however, whether Nodal acts directly or indirectly to induce heart tissue or interacts with other signaling molecules or pathways remained unclear. Here we present Xenopus embryological studies demonstrating an unforeseen role for the DAN family protein Cerberus within presumptive foregut endoderm as essential for differentiation of cardiac mesoderm in response to Nodal. Ectopic activation of Nodal signaling in non-cardiogenic ventroposterior mesendoderm, either by misexpression of the Nodal homologue XNr1 together with Cripto or by a constitutively active Alk4 (caAlk4), induced both cardiac markers and Cerberus. Mosaic lineage tracing studies revealed that Nodal/Cripto and caAlk4 induced cardiac markers cell non-autonomously, thus supporting the idea that Cerberus or another diffusible factor is an essential mediator of Nodal-induced cardiogenesis. Cerberus alone was found sufficient to initiate cardiogenesis at a distance from its site of synthesis. Conversely, morpholino-mediated specific knockdown of Cerberus reduced both endogenous cardiomyogenesis and ectopic heart induction resulting from misactivation of Nodal/Cripto signaling. Since the specific knockdown of Cerberus did not abrogate heart induction by the Wnt antagonist Dkk1, Nodal/Cripto and Wnt antagonists appear to initiate cardiogenesis through distinct pathways. This idea was further supported by the combinatorial effect of morpholino-medicated knockdown of Cerberus and Hex, which is required for Dkk1-induced cardiogenesis, and the differential roles of essential downstream effectors: Nodal pathway activation did not induce the transcriptional repressor Hex while Dkk-1 did not induce Cerberus. These studies demonstrated that cardiogenesis in mesoderm depends on Nodal-mediated induction of Cerberus in underlying endoderm, and that this pathway functions in a pathway parallel to cardiogenesis initiated through the induction of Hex by Wnt antagonists. Both pathways operate in endoderm to initiate cardiogenesis in overlying mesoderm.
Assuntos
Diferenciação Celular/fisiologia , Indução Embrionária/fisiologia , Coração/embriologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mesoderma/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Animais , Linhagem da Célula/fisiologia , Primers do DNA , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteína Nodal , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas de Xenopus/genéticaRESUMO
Serum opacity factor (SOF) is a bifunctional cell surface protein expressed by 40-50% of group A streptococcal (GAS) strains comprised of a C-terminal domain that binds fibronectin and an N-terminal domain that mediates opacification of mammalian sera. The sof gene was recently discovered to be cotranscribed in a two-gene operon with a gene encoding another fibronectin-binding protein, sfbX. We compared the ability of a SOF(+) wild-type serotype M49 GAS strain and isogenic mutants lacking SOF or SfbX to invade cultured HEp-2 human pharyngeal epithelial cells. Elimination of SOF led to a significant decrease in HEp-2 intracellular invasion while loss of SfbX had minimal effect. The hypoinvasive phenotype of the SOF(-) mutant could be restored upon complementation with the sof gene on a plasmid vector, and heterologous expression of sof49 in M1 GAS or Lactococcus lactis conferred marked increases in HEp-2 cell invasion. Studies using a mutant sof49 gene lacking the fibronectin-binding domain indicated that the N-terminal opacification domain of SOF contributes to HEp-2 invasion independent of the C-terminal fibronectin binding domain, findings corroborated by observations that a purified SOF N-terminal peptide could promote latex bead adherence to HEp-2 cells and inhibit GAS invasion of HEp-2 cells in a dose-dependent manner. Finally, the first in vivo studies to employ a single gene allelic replacement mutant of SOF demonstrate that this protein contributes to GAS virulence in a murine model of necrotizing skin infection.