RESUMEN
In Burkholderia-Riptortus symbiosis, the host bean bug Riptortus pedestris harbors Burkholderia symbionts in its symbiotic organ, M4 midgut, for use as a nutrient source. After occupying M4, excess Burkholderia symbionts are moved to the M4B region, wherein they are effectively digested and absorbed. Previous studies have shown that M4B has strong symbiont-specific antibacterial activity, which is not because of the expression of antimicrobial peptides but rather because of the expression of digestive enzymes, mainly cathepsin L protease. However, in this study, inhibition of cathepsin L activity did not reduce the bactericidal activity of M4B, indicating that there is an unknown digestive mechanism that renders specifically potent bactericidal activity against Burkholderia symbionts. Transmission electron microscopy revealed that the lumen of symbiotic M4B was filled with a fibrillar matter in contrast to the empty lumen of aposymbiotic M4B. Using chromatographic and electrophoretic analyses, we found that the bactericidal substances in M4B existed as high-molecular-weight (HMW) complexes that were resistant to protease degradation. The bactericidal HMW complexes were visualized on non-denaturing gels using protein- and polysaccharide-staining reagents, thereby indicating that the HMW complexes are composed of proteins and polysaccharides. Strongly stained M4B lumen with Periodic acid-Schiff (PAS) reagent in M4B paraffin sections confirmed HMW complexes with polysaccharide components. Furthermore, M4B smears stained with Periodic acid-Schiff revealed the presence of polysaccharide fibers. Therefore, we propose a key digestive mechanism of M4B: bacteriolytic fibers, polysaccharide fibers associated with digestive enzymes such as cathepsin L, specialized for Burkholderia symbionts in Riptortus gut symbiosis.
Asunto(s)
Burkholderia , Heterópteros , Animales , Catepsina L/metabolismo , Catepsina L/farmacología , Simbiosis/fisiología , Ácido Peryódico/metabolismo , Ácido Peryódico/farmacología , Insectos , Heterópteros/microbiología , Bacterias , Polisacáridos/metabolismo , Burkholderia/fisiologíaRESUMEN
Increased lung vascular permeability is an important contributor to respiratory failure in acute lung injury (ALI). We found that a function-blocking antibody against the integrin alphavbeta5 prevented development of lung vascular permeability in two different models of ALI: ischemia-reperfusion in rats (mediated by vascular endothelial growth factor [VEGF]) and ventilation-induced lung injury (VILI) in mice (mediated, at least in part, by transforming growth factor-beta [TGF-beta]). Knockout mice homozygous for a null mutation of the integrin beta5 subunit were also protected from lung vascular permeability in VILI. In pulmonary endothelial cells, both the genetic absence and blocking of alphavbeta5 prevented increases in monolayer permeability induced by VEGF, TGF-beta, and thrombin. Furthermore, actin stress fiber formation induced by each of these agonists was attenuated by blocking alphavbeta5, suggesting that alphavbeta5 regulates induced pulmonary endothelial permeability by facilitating interactions with the actin cytoskeleton. These results identify integrin alphavbeta5 as a central regulator of increased pulmonary vascular permeability and a potentially attractive therapeutic target in ALI.
Asunto(s)
Barrera Alveolocapilar/metabolismo , Permeabilidad Capilar/fisiología , Endotelio Vascular/metabolismo , Integrinas/metabolismo , Receptores de Vitronectina/metabolismo , Amidas/farmacología , Animales , Anticuerpos/inmunología , Barrera Alveolocapilar/efectos de los fármacos , Permeabilidad Capilar/efectos de los fármacos , Bovinos , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Humanos , Integrinas/antagonistas & inhibidores , Integrinas/inmunología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Enfermedades Pulmonares/inducido químicamente , Enfermedades Pulmonares/patología , Ratones , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Arteria Pulmonar/citología , Arteria Pulmonar/efectos de los fármacos , Piridinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Vitronectina/antagonistas & inhibidores , Receptores de Vitronectina/inmunología , Daño por Reperfusión , Fibras de Estrés/efectos de los fármacos , Ventiladores Mecánicos , Quinasas Asociadas a rhoRESUMEN
Several bacterial genera express proteins that contain collagen-like regions, which are associated with variable (V) non-collagenous regions. The streptococcal collagen-like proteins, Scl1 and Scl2, of group A Streptococcus (GAS) are members of this 'prokaryotic collagen' family, and they too contain an amino-terminal non-collagenous V region of unknown function. Here, we use recombinant rScl constructs, derived from several Scl1 and Scl2 variants, and affinity chromatography to identify Scl ligands present in human plasma. First, we show that Scl1, but not Scl2, proteins from different GAS serotypes bind the same ligand identified as apolipoprotein B (ApoB100), which is a major component of the low-density lipoprotein (LDL). Scl1 binding to purified ApoB100 and LDL is specific and concentration-dependent. Furthermore, the non-collagenous V region of the Scl1 protein is responsible for LDL/ApoB100 binding because only those rScls, constructed by domain swapping, which contain the V region from Scl1 proteins, were able to bind to ApoB100 and LDL ligands, and this binding was inhibited by antibodies directed against the Scl1-V region. Electron microscopy images of Scl1-LDL complexes showed that the globular V domain of Scl1 interacted with spherical particles of LDL. Importantly, live M28-type GAS cells absorbed plasma LDL on the cell surface and this binding depended on the surface expression of the Scl1.28, but not Scl2.28, protein. Phylogenetic analysis showed that the non-collagenous globular domains of Scl1 and Scl2 evolved independently to form separate lineages, which differ in amino acid sequence, and these differences may account for the variations in binding patterns of Scl1 and Scl2 proteins. Present studies provide insight into the structure-function relationship of the Scl proteins and also underline the importance of lipoprotein binding by GAS.
Asunto(s)
Proteínas Bacterianas/metabolismo , Colágeno/metabolismo , Lipoproteínas LDL/metabolismo , Streptococcus pyogenes/metabolismo , Apolipoproteína B-100 , Apolipoproteínas B/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Sitios de Unión , Cromatografía de Afinidad , Colágeno/química , Colágeno/genética , Humanos , Lipoproteínas LDL/sangre , Lipoproteínas LDL/aislamiento & purificación , Microscopía Electrónica/métodos , Datos de Secuencia Molecular , Filogenia , Estructura Terciaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The streptococcal collagen-like proteins Scl1 and Scl2 are prokaryotic members of a large protein family with domains containing the repeating amino acid sequence (Gly-Xaa-Yaa)(n) that form a collagen-like triple-helical structure. Here, we test the hypothesis that Scl variant might interact with mammalian collagen-binding integrins. We show that the recombinant Scl protein p176 promotes adhesion and spreading of human lung fibroblast cells through an alpha2beta1 integrin-mediated interaction as shown in cell adhesion inhibition assays using anti-alpha2beta1 and anti-beta1 integrins monoclonal antibodies. Accordingly, C2C12 cells stably expressing alpha2beta1 integrin as the only collagen-binding integrin show productive cell adhesion activities on p176 that can be blocked by an anti-alpha2beta1 integrin antibody. In addition, p176 promotes tyrosine phosphorylation of p125(FAK) of C2C12 cells expressing alpha2beta1 integrin, whereas parental cells do not. Furthermore, C2C12 adhesion of human lung fibroblast cells to p176 induces phosphorylation of p125FAK, p130CAS, and p68Paxillin proteins. In a domain swapping experiment, we show that integrin binds to the collagenous domain of the Scl protein. Moreover, the recombinant inserted domain of the alpha2 integrin interacts with p176 with a relatively high affinity (K(D) = 17 nm). Attempts to identify the integrin sites in p176 suggest that more than one site may be involved. These studies, for the first time, suggest that the collagen-like proteins of prokaryotes retained not only structural but also functional characteristics of their eukaryotic counterparts.
Asunto(s)
Proteínas Bacterianas/metabolismo , Colágeno/metabolismo , Integrina alfa2beta1/metabolismo , Isoformas de Proteínas/metabolismo , Streptococcus pyogenes/metabolismo , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/ultraestructura , Sitios de Unión , Adhesión Celular/fisiología , Células Cultivadas , Colágeno/química , Colágeno/genética , Colágeno/ultraestructura , Proteína Sustrato Asociada a CrK , Proteínas del Citoesqueleto/metabolismo , Fibroblastos/química , Fibroblastos/metabolismo , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Humanos , Integrina alfa2beta1/genética , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Pulmón/citología , Ratones , Datos de Secuencia Molecular , Paxillin , Fosfoproteínas/metabolismo , Fosforilación , Unión Proteica , Conformación Proteica , Isoformas de Proteínas/genética , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína p130 Similar a la del RetinoblastomaRESUMEN
Endorepellin, the COOH-terminal domain of the heparan sulfate proteoglycan perlecan, inhibits several aspects of angiogenesis. We provide evidence for a novel biological axis that links a soluble fragment of perlecan protein core to the major cell surface receptor for collagen I, alpha2beta1 integrin, and provide an initial investigation of the intracellular signaling events that lead to endorepellin antiangiogenic activity. The interaction between endorepellin and alpha2beta1 integrin triggers a unique signaling pathway that causes an increase in the second messenger cAMP; activation of two proximal kinases, protein kinase A and focal adhesion kinase; transient activation of p38 mitogen-activated protein kinase and heat shock protein 27, followed by a rapid down-regulation of the latter two proteins; and ultimately disassembly of actin stress fibers and focal adhesions. The end result is a profound block of endothelial cell migration and angiogenesis. Because perlecan is present in both endothelial and smooth muscle cell basement membranes, proteolytic activity during the initial stages of angiogenesis could liberate antiangiogenic fragments from blood vessels' walls, including endorepellin.