This effect was likely due to a subset of bacteria that phase varied ON Opa expression in the parental and ΔopaBEGK Gc cultures. The latest wellness and longevity research. Human PMNs in suspension can bind and phagocytose unopsonized Gc only if the bacteria express OpaCEA proteins (28–32). The deletions were designed to replace most of the opa open reading frame (ORF) with an HpaI restriction site (underlined in the primer sequence in Table S2 in the supplemental material). Saba R, Störchel PH, Aksoy-Aksel A, Kepura F, Lippi G, Plant TD, Schratt GM. Absence of Opa protein expression in Opaless Gc. Interactions between Gc and human cells are modulated by a variety of bacterial surface structures, including type IV pili, lipooligosaccharide (LOS), porin, and opacity-associated (Opa) proteins (5). Die Verfügbarkeit der einzelnen Titel wird in der Trefferliste angezeigt. These in vivo observations indicate that factors other than PMNs select for bacterial expression of Opa proteins during acute infection. Screening and validation of opa gene deletions in FA1090 Gc.Deletion constructs were sequentially introduced into FA1090 Gc by natural transformation on a solid medium (43). . Since phagocytosis of Opaless Gc requires the PMN actin cytoskeleton, PMNs may be internalizing Opa− bacteria by actin-dependent macropinocytosis. Lun-Ven 9h30-19h [email protected] nous contacter. S2A in the supplemental material). © 2020 Visvesvaraya Technological University. Password Retrouver les marchés publics des collectivités locales, leurs établissements publics et des bailleurs sociaux. The remaining seven were then deleted to yield the complete opa deletion, here called Opaless. Ebenso Texte aus dem E-Repository der Hochschule und aus den Nationallizenzen. First, genetically opa-deficient Gc has no growth defect, and Gc growth in vitro is not affected by Opa expression. (B) Genomic DNA from parental, ΔopaBEGK, and Opaless Gc was used as the template for PCR, using primer sets homologous to the sequences flanking each of the 11 opa genes. Um effizient in Opac plus zu recherchieren, muss Ihr Rechner am Hochschulnetz angemeldet sein. The Neisseria gonorrhoeae (the gonococcus [Gc]) opacity-associated (Opa) proteins mediate bacterial binding and internalization by human epithelial cells and neutrophils (polymorphonuclear leukocytes [PMNs]). WeBoost - Agence Web. The parental bacterial lysate contained one or more heat-modifiable proteins that reacted with a pan-Opa antibody by Western blotting (Fig. These derivatives provide a robust system for assessing the role of Opa proteins in Gc biology. Gc constitutively expressing the OpaCEA OpaD recapitulated phenotypes associated with OpaD phase-ON Gc in induction of the PMN oxidative burst and bacterial sensitivity to PMNs. NLB promotes reading, learning and information literacy by providing a trusted, accessible and globally-connected library and information service through the National Library and a comprehensive network of Public Libraries. The three strains produced similarly sized colonies (Fig. Overview. $6.99 $ 6. Pendant la crise sanitaire, 70 % des parents ont ressenti plus de fatigue que d’habitude 22/12/2020 08:45:34. The presence of Gc at human mucosal surfaces initiates the recruitment of abundant polymorphonuclear leukocytes (neutrophils; PMNs) to the site of infection (4). L'aluminium est l'élément chimique de numéro atomique 13, de symbole Al. In each subsequent round of opa deletion, Southern blotting and/or PCR was used to verify that all opa deletions that were previously introduced were retained. Identification of surface proteins of, Up-regulation of human neutrophil receptors for, The specific innate immune receptor CEACAM3 triggers neutrophil bactericidal activities via a Syk kinase-dependent pathway, Opa (protein II) influences gonococcal organization in colonies, surface appearance, size and attachment to human fallopian tube tissues, Multiple gonococcal pilin antigenic variants are produced during experimental human infections, An alternative DNA structure is necessary for pilin antigenic variation in, Gene splicing and mutagenesis by PCR-driven overlap extension, Gonococci exit apically and basally from polarized epithelial cells and exhibit dynamic changes in type IV pili, Purification and visualization of lipopolysaccharide from Gram-negative bacteria by hot aqueous-phenol extraction, Loss of both Holliday junction processing pathways is synthetically lethal in the presence of gonococcal pilin antigenic variation, Experimental gonococcal genital tract infection and opacity protein expression in estradiol-treated mice, Anaerobic growth of gonococci does not alter their Opa-mediated interactions with human neutrophils, Investigation of oxidative stress defenses of, A strain-specific catalase mutation and mutation of the metal-binding transporter gene, Experimental gonococcal infection in male volunteers: cumulative experience with, Estradiol-treated female mice as surrogate hosts for, Construction of Opa-positive and Opa-negative strains of, Macropinocytosis: an endocytic pathway for internalising large gulps, Antigonococcal activity of human neutrophil cathepsin G, Studies on gonococcus infection. Ebenso Texte aus dem E-Repository der Hochschule und aus den Nationallizenzen.Die Verfügbarkeit der einzelnen Titel wird in der Trefferliste angezeigt. Feedback on Strategic Plan, FET JU Congratulation message of Hon'ble MIC for the good academic attainments of the students of Jadavpur University in the 2018 All India GATE examination and also for performing exceedingly well in the Association of Indian University's East … Bottle Height: 2.75". Scale bar = 10 μm. Complementation of Opaless Gc with non-phase-variable OpaD restores Opa-dependent phenotypes in Gc. While it is possible that we selected for a mutation that allows for Gc growth in the absence of all Opa expression, opa-deficient derivatives of strain MS11 Gc and strain H44/76 Neisseria meningitidis also show no in vitro growth defect (19, 59). Julie Boyer. This observation suggests that the PMN survival defect of OpaD+ Gc is due to antigonococcal properties of PMNs other than cationic antimicrobial peptides. It is a branch library that services to students enrolled on various programs such as BSCS, BSIT, BSIS, BSBA, BSENT& BSA, that extends to faculty members, non-teaching staffs and administrators of Mapúa-Makati. One mutant clone served as the parent for the next round of transformation, until all 11 opa genes were sequentially deleted. Journal of Microbiology & Biology Education, Microbiology and Molecular Biology Reviews. To block actin-dependent phagocytosis, cytochalasin D (10 μg/ml) (Sigma) was added to PMNs 30 min prior to infection and kept in the infection medium for the duration of the experiment. 1B). Loss of Opa expression did not affect Gc growth. *, P < 0.025 (Student's two-tailed t test) for Opaless Gc ± cytochalasin D. Complementation of Opaless Gc with non-phase-variable OpaD restores Opa-dependent phenotypes in Gc.Opaless Gc was complemented with a constitutively expressed allele of the FA1090 opaD gene. Hinweise: Mit einem * können Sie beliebige Wortendungen mit suchen. In contrast, we reported that predominantly Opa− Gc can be internalized by adherent, IL-8-treated human PMNs (35). 2E to G). We do not retain these email addresses. Blown by UPC, this water pipe comes to you from Toluca Lake CA. Votre identifiant correspond à l'email que vous avez renseigné lors de l'abonnement. Statistics.Differences between Gc strains were compared using two-tailed Student's t test. 181 p. 22 cm Date: 2010 Availability: Copies available: College Library Filipiniana Section [ PE1130 .B928 2010 ] (3), Best Seller in Fabric & Textile Paints. From each transformation, 400 to 600 colonies were screened for the opa deletion by PCR, using the conditions listed in Table S3 in the supplemental material. Opa variation occurs at a rate of ∼10−3 to 10−4 variants/cell/generation in vitro (22, 23), and changes in Opa expression occur during experimental human male urethral infection and in the female mouse genital tract (24–26). Gc genomes contain approximately 11 opa loci, encoding 7 to 9 unique Opa proteins (7–9). NLB manages the National Library, 26 Public Libraries and the National Archives. We first determined how genetically opa-deficient Gc modulated the PMN oxidative burst. Given that opa phase variation can confound studies aimed at dissecting the role of Opa proteins in Gc pathogenesis, we reasoned that this issue could be circumvented by generating Gc in which Opa expression was constitutively absent or present. Communication Competence of Maritime Students of Lyceum of Batangas: Basis for a Proposed Development … Experiments were performed 3 times. One such pressure may come from the requirement for Gc to attach to the apical surface of the mucosal epithelium and be internalized by epithelial cells (63). Opa expression was validated by Western blotting with the 4B12 pan-Opa antibody (see below). Neisseria meningitidis is an inhabitant of the nasopharynx, from which it is transmitted from person to person or disseminates in blood and becomes a harmful pathogen. TISS is a unique institution that brings together high quality scholars and practitioners from Social, Economic, Political, Physical, Habitat, Engineering, Health, and Environmental Sciences to create interdisciplinary teaching and research programmes combined with field action to address the most critical current and emerging issues of the nation. 4.4 out of 5 stars 2,707. The product was cloned into pCR-Blunt TOPO (Invitrogen) or pSmart (Lucigen) and transformed into chemically competent TOP10 E. coli, following the manufacturers' instructions, except for opaC, where the overlap extension product was confirmed by DNA sequencing and then directly transformed into Gc (see below). Targeting of the Arpc3 actin nucleation factor by miR-29a/b regulates dendritic spine morphology. (ii) Constitutively expressed OpaD+ Gc (OpaD+nv).The CTCTT repeat-rich region of FA1090 opaD was replaced with a sequence encoding the same amino acids but unable to undergo phase variation (see Fig. MarketingTracer SEO Dashboard, created for webmasters and agencies. Parental and ΔopaBEGK Gc induced minor amounts of ROS production from PMNs, but the oxidative burst was smaller and delayed compared to PMNs infected with S. aureus (Fig. Opa+ Gc engagement of CEACAM1 also leads to B cell death (67). Prior to this work, a derivative of FA1090 in which all 11 opa genes had been deleted, the ΔopaA-K mutant, was constructed (50, 53). Opacity-associated phenotypes and growth characteristics of Opa-deficient Gc.Parental Gc and opa-deleted progeny were examined for Opa expression by Western blotting and colony photoopacity. This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Mapúa University, Muralla St., Intramuros, Manila 1002 Philippines © 2013-2019 All Rights Reserved We thank Christof Hauck, Ann Jerse, and Michael Apicella for providing antibodies and the University of Virginia Lymphocyte Culture Center for production of the 4B12 monoclonal antibody. Using Opaless and OpaD+nv Gc, we found that constitutively Opa− and Opa+ Gc behave similarly to their phase-varying counterparts in survival after challenge with primary human PMNs and in modulating the PMN oxidative burst. Opaless Gc and an intermediate isolate in which the genes encoding translucent Opa proteins were deleted (ΔopaBEGK) behaved similarly to Opa phase-OFF bacteria in in vitro growth and survival after exposure to primary human PMNs. In contrast, the Opaless FA1090 derivative described in this work grew with similar kinetics to the opa-intact parent. The use of these derivatives provides a robust and flexible system for quantifying the effect of Opa proteins on bacteria-bacteria and bacteria-host interactions. Notably, unopsonized Opaless Gc was internalized by adherent, chemokine-primed, primary human PMNs, by an actin-dependent process. As support for this possibility, PMNs that are attached to a substrate and primed with chemokines, like the PMNs in our assay system, show increased motility and membrane ruffling, features that enhance macropinocytosis (60). OpaD+nv and OpaD+var Gc induced similar amounts of ROS production in PMNs, whereas Opaless Gc did not induce detectable ROS (Fig. Occurrence of color/opacity colonial variants in clinical cultures, Opa binding to cellular CD66 receptors mediates the transcellular traversal of, Neisserial binding to CEACAM1 arrests the activation and proliferation of CD4+ T lymphocytes, Submission, Review, & Publication Processes, http://exon.gatech.edu/metagenome/CodonUsageDatabase/display.cgi?accession=NC_002946, Constitutively Opa-Expressing and Opa-Deficient Neisseria gonorrhoeae Strains Differentially Stimulate and Survive Exposure to Human Neutrophils. Opa+ Gc may show more sensitivity to killing by PMN serine proteases, which have antigonococcal activity in vitro and in PMN phagosomes (48, 61), as Opa expression makes Gc more sensitive to the serine proteases trypsin and chymotrypsin (50). 3C. (ii) Liquid medium.Gc was grown in GCBL modified with Kellogg's supplements I and II and 0.042% sodium bicarbonate as previously described (33). Other options New and used from $6.99. 5B). Get it as soon as Thu, Nov 19. Enseignement Formation et Conseil chez LB FORMATION Montpellier. Search. --HappyCamper 1 July 2005 02:55 (UTC) To me it looks kinda like a sycamore. Your session has expired. 5B). Therefore, the ability to phase vary Opa protein expression allows Gc to fine-tune its interactions with epithelial and immune cells as needed during the course of infection. Pädagogische Hochschulbibliothek Ludwigsburg. Insert-containing plasmids were isolated from E. coli using the QiaPrep miniprep kit (Qiagen), and the correct opa deletion sequence was verified by DNA sequencing (Genewiz). Results are the averages ± standard errors from 11 to 14 replicate samples per strain. 3A. 4). These results indicate that opa-deficient Gc, like Opa phase-OFF parental Gc, retains the ability to suppress PMN ROS production. 3A). In addition, antibodies have been raised against individual FA1090 Opa proteins (23, 24). (B) Opaless Gc, OpaD+var Gc, and OpaD+nv Gc were exposed to adherent, IL-8-primed PMNs, and bacterial survival was calculated as described for Fig. XV. Opa proteins share a conserved β-barrel structure but differ in the sequence of extracellular loops, which dictate Opa receptor specificity (10–12). PMN preparations routinely contained >95% granulocytes, as assessed by phase-contrast microscopy, and were >99% viable, as assessed by trypan blue exclusion. 4D). One clone with the opa deletion was transformed with the next opa deletion construct, until all 11 opa genes were replaced in the genome. While translucent colonies were occasionally detected in the OpaD+var Gc population, OpaD+nv Gc colonies were uniformly opaque (Fig. TOP10 E. coli (Invitrogen) was grown in Luria-Bertani (LB) (BD Difco) broth or agar supplemented with 50 μg/ml kanamycin (Sigma-Aldrich) at 37°C for plasmid propagation.