1,720,994 research outputs found
Protein-Protein Interactions Required for CagA Recruitment and Secretion by the Helicobacter pylori Cag Type IV Secretion System
No full textHelicobacter pylori strains that contain the cag pathogenicity island (PAI) utilize the Cag type IV secretion system (T4SS) to deliver a bacterial effector protein (CagA) and non-protein substrates into human gastric cells. The Cag T4SS outer membrane core complex (OMCC) contains multiple copies of five proteins, two of which are species-specific proteins. Additional species-specific proteins encoded by the cag PAI are required for Cag T4SS activity, but their localization within the Cag T4SS is undefined. By using optimized mass spectrometric methods and modifications of a previously described OMCC immunopurification method, we have identified four cag PAI-encoded proteins (CagW, CagL, CagI, and CagH) that co-purify with the Cag T4SS OMCC. Analysis of immunopurified samples by size exclusion chromatography revealed that CagW, CagL, CagI and CagH co-elute with OMCC components. These four Cag proteins are copurified with the OMCC in immunopurifications from a Δcag3 mutant strain (lacking peripheral OMCC components), but not from a ΔcagX mutant strain (defective in OMCC assembly). Mutant strains with deletions of cagW, cagL, cagI, or cagH lacked T4SS activity but retained the ability to assemble OMCCs. Furthermore, by targeting several different Cag proteins and adding crosslinkers to the bacteria prior to immunopurification, we improved our ability to isolate multiple Cag proteins, providing further insight into protein-protein interactions among cag PAI-encoded proteins. We also generated H. pylori strains that produce CagA N-terminal fusion proteins (APEX2-CagA, GFP-CagA) and found that these fusion proteins retained the ability to interact with the Cag T4SS OMCC. In summary, the experiments described in my dissertation provide new insights into protein-protein interactions relevant to the structural organization of the Cag T4SS
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Revealing the Interactions of Clostridioides difficile Toxin B With Epithelial Receptors
No full textClostridioides difficile is a Gram-positive, spore-forming bacterium and the leading cause of hospital-acquired diarrhea in the United States. Upon colonizing the colon of susceptible hosts, C. difficile causes disease through two secreted protein toxins, toxin A (TcdA) and toxin B (TcdB). The glucosyltransferase activity of the toxins inactivates small Rho-family GTPases involved in cell signaling to promote inflammation, fluid secretion, and cell death. While multiple proteins have been identified to bind each toxin in vitro, the significance of these interactions remains unclear for mediating disease. In this dissertation, I investigated the roles of chondroitin sulfate proteoglycan 4 (CSPG4), Frizzled (FZD), and Nectin-3, three receptors proposed to mediate the endocytosis of TcdB. Using a rectal instillation mouse model of C. difficile toxins, I show that FZD interactions with TcdB are not essential for mediating pathologies. Furthermore, I show that ribotype 027 C. difficile, an epidemic C. difficile ribotype, produces a TcdB that is unable to interact with FZD proteins but is still capable of promoting fulminant disease in hosts. Mechanistically, I propose that TcdB promotes injuries to the colonic epithelium through interactions with Nectin-3 and CSPG4. By developing a method to visualize TcdB on cells using fluorescent microscopy, I demonstrate that both Nectin-3 and CSPG4 colocalize with TcdB on 18Co cells. On host colonic tissue, I observed novel localization of Nectin-3 within the brush border of epithelial cells and CSPG4 localization along the epithelial cell junction. As colonic epithelial cells do not express CSPG4, I propose that this CSPG4 signal is likely mediated by shed forms of CSPG4 produced by stromal cells along the crypt-surface axis. In support of this model, I show that shed forms of CSPG4 can be endocytosed by epithelial cells, a concept that TcdB can exploit to potentiate its activity. Collectively, my study provides new insights into how TcdB utilizes receptor interactions to intoxicate colonic epithelial cells to cause disease
Beyond Vacuolation: Helicobacter Pylori Vaca Toxin Activity In Vitro and In Vivo
No full textColonization of the human stomach with Helicobacter pylori strains producing active forms of a secreted toxin, VacA, is associated with an increased risk of peptic ulcer disease and gastric cancer compared to colonization with strains producing hypoactive forms of VacA. The goals of my project were to define cellular metabolic consequences of VacA intoxication and define consequences of VacA activity in vivo. Untargeted metabolomics analyses revealed that several hundred metabolites were significantly altered in VacA-treated gastroduodenal cells (AGS and AZ-521) compared with control cells. Pathway analysis indicated that VacA caused alterations in numerous metabolic pathways, including taurine and hypotaurine metabolism, purine metabolism, pyrimidine metabolism, and alanine, aspartate, and glutamate metabolism. Supplementation of the tissue culture medium with taurine or hypotaurine protected AZ-521 cells against VacA-induced cell death. Treatment of cells with purified active s1m1 forms of VacA, but not hypoactive s2m1 or Δ6-27 VacA-mutant proteins (defective in membrane channel formation), caused reductions in intracellular taurine and hypotaurine concentrations. VacA treatment also resulted in increased levels of lysophospholipids within cells and the release of lysophospholipids into the extracellular space. Comparative studies of an H. pylori wild-type strain and a vacA insertion mutant in the Mongolian gerbil model did not reveal significant differences in gastric colonization or gastric inflammation. However, detected numerous VacA-dependent metabolic changes in gastric tissues. These discoveries, described in my thesis, provide new insights into the effects of VacA on gastric epithelial cells and the gastric environment
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Role of the CagY (VirB10) Antenna Projection in Helicobacter pylori Cag Type IV Secretion System Activity and Assembly
No full textHelicobacter pylori strains containing the cag pathogenicity island (PAI) are associated with the development of gastric adenocarcinoma and peptic ulcer disease. The cag PAI encodes a secreted effector protein (CagA) and a type IV secretion system (Cag T4SS). The Cag T4SS outer membrane core complex (OMCC) is composed of an outer membrane cap (OMC), a periplasmic ring (PR), and a stalk. The OMC contains multiple copies of CagY/VirB10, CagX/VirB9, CagT/VirB7, CagM, and Cag3. The PR contains only CagY and CagX, and the stalk's composition is undefined. The OMC contains a ring-like structure known as the antenna region, which is formed by 14 CagY (VirB10) proteins. Individual CagY components of the antenna region (antenna projections, AP) contain two α-helices connected by an unstructured loop. This dissertation reports an investigation of the role of the CagY AP in Cag T4SS function, along with an investigation of its role in OMCC assembly. We found that a H. pylori CagY(∆AP) mutant lacks Cag T4SS activity, and mutants with shortened CagY AP loop lengths exhibit reduced T4SS activity. To test the hypothesis that CagY AP interactions with the outer membrane contribute to OMCC assembly, we analyzed the composition and structure of OMCCs isolated from the CagY(∆AP) mutant. Mass spectrometry analyses indicated that these OMCCs contained all 5 protein components; in contrast, Cag T4SS complexes isolated from a ΔcagT mutant lacked CagT and Cag3, and complexes from a ΔcagM mutant lacked CagM, CagT, and Cag3. Cryo-EM analysis indicated that the OMCCs isolated from the CagY(∆AP) mutant have a structure nearly identical to that of wild-type OMCCs; in contrast, OMCCs from ΔcagT or ΔcagM mutants have disorganized OMCs but stable PRs. These results indicate that CagY and CagX components are sufficient for maintaining a stable PR, but a well-structured OMC requires the presence of CagM and CagT. Collectively, these results provide new insights into functional properties of CagY in Cag T4SS activity and the roles of individual OMCC components in Cag T4SS structural organization
INVESTIGATION OF THE HELICOBACTER PYLORI CAG TYPE IV SECRETION SYSTEM IN VITRO AND IN VIVO
No full textHelicobacter pylori colonizes the stomach in about half of the world’s population. Gastric colonization with H. pylori strains containing the cag pathogenicity island (cag PAI) is a risk factor for gastric adenocarcinoma and peptic ulcer disease. The cag PAI encodes a type IV secretion system (T4SS) that mediates delivery of effector molecules into gastric epithelial cells. The Cag T4SS spans the bacterial cell envelope and is composed of at least 15 components, including three ATPases - Cagα, Cagβ, and CagE. In this dissertation, I show that these ATPases have non-redundant functional properties, based on co-culture experiments with H. pylori mutant strains and gastric epithelial cells. Experiments using a Mongolian gerbil model of H. pylori-induced gastric carcinogenesis show that Cag T4SS activity contributes to the development of gastric cancer and help to define the stages of H. pylori infection when Cag T4SS activity contributes to cancer pathogenesis. Finally, by using imaging mass spectrometry methods, I show that H. pylori infection of the Mongolian gerbil stomach can lead to alterations in gastric lipid composition, a finding that lays the foundation for identifying biomarkers of gastric atrophy and premalignant changes
Regulatory Mechanisms Governing Cell Wall Biogenesis in, and Host Immune Modulation Against, Acinetobacter baumannii
No full textThe emergence of multidrug-resistant Acinetobacter baumannii presents a critical threat to global health, driven by its capacity to evade antimicrobial therapies and persist in hostile environmental conditions. This dissertation explores the interconnected roles of bacterial zinc (Zn) homeostasis, cell wall biogenesis, and host immune responses, revealing how metal regulation influences both A. baumannii pathogenesis and host defense mechanisms. Central to this investigation are COG0523 family proteins, key mediators of Zn-dependent processes essential for bacterial fitness and virulence. ZigA coordinates Zn homeostasis with cell envelope integrity by interacting with the lytic transglycosylase SltB, linking metal availability to peptidoglycan remodeling. MigC, another COG0523 protein, functions as a Zn-dependent inhibitor of MurD, a critical enzyme in peptidoglycan synthesis. This regulation ensures cell wall integrity and bacterial survival under Zn-limiting conditions, while loss of MigC sensitizes A. baumannii to β-lactam antibiotics, identifying a novel therapeutic vulnerability. On the host side, Zn sequestration emerges as a pivotal factor in immune defense. The Zn-binding protein calprotectin, particularly its S100A9 component, regulates immune homeostasis during A. baumannii infection. S100A9 deficiency appears to disrupt this balance by enhancing ZNRF1 protein degradation activity, potentially reducing the abundance of its targets like Cav1, which may promote immune cell apoptosis, thereby contributing to increased bacterial colonization and tissue damage. Together, these findings elucidate the complex interplay between bacterial metal homeostasis, cell wall dynamics, and host immune regulation. By identifying critical regulatory nodes and potential therapeutic targets, this dissertation establishes a framework for novel strategies to combat multidrug-resistant A. baumannii, contributing to broader efforts in addressing the global challenge of antimicrobial resistance
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