Activation mechanisms of the innate immune system: structure-function studies of interactions between death domains of MyD88 and IRAK proteins

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 27 de Noviembre de 2012

Surface Glucan Structures in Aeromonas spp.

Aeromonas spp. are generally found in aquatic environments, although they have also been isolated from both fresh and processed food. These Gram-negative, rod-shaped bacteria are mostly infective to poikilothermic animals, although they are also considered opportunistic pathogens of both aquatic and terrestrial homeotherms, and some species have been associated with gastrointestinal and extraintestinal septicemic infections in humans. Among the different pathogenic factors associated with virulence, several cell-surface glucans have been shown to contribute to colonization and survival of Aeromonas pathogenic strains, in different hosts. Lipopolysaccharide (LPS), capsule and α-glucan structures, for instance, have been shown to play important roles in bacterial-host interactions related to pathogenesis, such as adherence, biofilm formation, or immune evasion. In addition, glycosylation of both polar and lateral flagella has been shown to be mandatory for flagella production and motility in different Aeromonas strains, and has also been associated with increased bacterial adhesion, biofilm formation, and induction of the host proinflammatory response. The main aspects of these structures are covered in this review

The Complete Structure of the Core Oligosaccharide from Edwardsiella tarda EIB 202 Lipopolysaccharide

The chemical structure and genomics of the lipopolysaccharide (LPS) core oligosaccharide of pathogenic Edwardsiella tarda strain EIB 202 were studied for the first time. The complete gene assignment for all LPS core biosynthesis gene functions was acquired. The complete structure of core oligosaccharide was investigated by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry MSn, and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry. The following structure of the undecasaccharide was established: The heterogeneous appearance of the core oligosaccharide structure was due to the partial lack of β-d-Galp and the replacement of α-d-GlcpNAcGly by α-d-GlcpNGly. The glycine location was identified by mass spectrometry

Structural characterization of core Region in Erwinia amylovora lipopolysaccharide.

Erwinia amylovora (E. amylovora) is the first bacterial plant pathogen described and demonstrated to cause fire blight, a devastating plant disease affecting a wide range of species including a wide variety of Rosaceae. In this study, we reported the lipopolysaccharide (LPS) core structure from E. amylovora strain CFBP1430, the first one for an E. amylovora highly pathogenic strain. The chemical characterization was performed on the mutants waaL (lacking only the O-antigen LPS with a complete LPS-core), wabH and wabG (outer-LPS core mutants). The LPSs were isolated from dry cells and analyzed by means of chemical and spectroscopic methods. In particular, they were subjected to a mild acid hydrolysis and/or a hydrazinolysis and investigated in detail by one and two dimensional Nuclear Magnetic Resonance (NMR) spectroscopy and ElectroSpray Ionization Fourier Transform-Ion Cyclotron Resonance (ESI FT-ICR) mass spectrometry

Polar flagella glycosylation in Aeromonas: genomic characterization and involvement of a specific glycosyltransferase (Fgi-1) in heterogeneous flagella glycosylation

Polar flagella from mesophilic Aeromonas strains have previously been shown to be modified with a range of glycans. Mass spectrometry studies of purified polar flagellins suggested the glycan typically includes a putative pseudaminic acid like derivative; while some strains are modified with this single monosaccharide, others modified with a heterologous glycan. In the current study, we demonstrate that genes involved in polar flagella glycosylation are clustered in highly polymorphic genomic islands flanked by pseudaminic acid biosynthetic genes (pse). Bioinformatic analysis of mesophilic Aeromonas genomes identified three types of polar flagella glycosylation islands (FGIs), denoted Group I, II and III. FGI Groups I and III are small genomic islands present in Aeromonas strains with flagellins modified with a single monosaccharide pseudaminic acid derivative. Group II were large genomic islands, present in strains found to modify polar flagellins with heterogeneous glycan moieties. Group II, in addition to pse genes, contained numerous glycosyltransferases and other biosynthetic enzymes. All Group II strains shared a common glycosyltransferase downstream of luxC that we named flagella glycosylation island 1, fgi-1, in A. piscicola AH-3. We demonstrate that Fgi-1 transfers the first sugar of the heterogeneous glycan to the pseudaminic acid derivative linked to polar flagellins and could be used as marker for polysaccharidic glycosylation of Aeromonas polar flagella

Structural and functional analysis of APOA5 mutations identified in patients with severe hypertriglyceridemia

During the diagnosis of three unrelated patients with severe hypertriglyceridemia, three APOA5 mutations [p.(Ser232_Leu235)del, p.Leu253Pro, and p.Asp332ValfsX4] were found without evidence of concomitant LPL, APOC2, or GPIHBP1 mutations. The molecular mechanisms by which APOA5 mutations result in severe hypertriglyceridemia remain poorly understood, and the functional impairment/s induced by these specific mutations was not obvious. Therefore, we performed a thorough structural and functional analysis that included follow-up of patients and their closest relatives, measurement of apoA-V serum concentrations, and sequencing of the APOA5 gene in 200 nonhyperlipidemic controls. Further, we cloned, overexpressed, and purified both wild-type and mutant apoA-V variants and characterized their capacity to activate LPL. The interactions of recombinant wild-type and mutated apoA-V variants with liposomes of different composition, heparin, LRP1, sortilin, and SorLA/LR11 were also analyzed. Finally, to explore the possible structural consequences of these mutations, we developed a three-dimensional model of full-length, lipid-free human apoA-V. A complex, wide array of impairments was found in each of the three mutants, suggesting that the specific residues affected are critical structural determinants for apoA-V function in lipoprotein metabolism and, therefore, that these APOA5 mutations are a direct cause of hypertriglyceridemia.</p

Functional genomics of the Aeromonas salmonicida lipopolysaccharide O-antigen and A-layer from typical and atypical strains

The A. salmonicida A450 LPS O-antigen, encoded by the wb salmo gene cluster, is exported through an ABC-2 trans porter-dependent pathway. It re presents the first example of an O-antigen LPS polysaccharide with three different monosaccharides in their repeating unit assembled by this pathway. Until now, only repeating units with one or two different monosaccharides have been described. F unctional genomic analysis of this wb salmo region is mostly in agreement with the LPS O-antigen structure of acetylated L -rhamnose (Rha), D -glucose (Glc), and 2-amino-2-deoxy- D -mannose (ManN). Between genes of the wb salmo we found the genes responsible for the biosynthe sis and assembly of the S-layer (named A-layer in these strains). Through comparative genomic analysis and in-frame deletions of some of the genes, we concluded that all the A. salmonicida typical and at ypical strains, other than A. salmonicida subsp. pectinolytica strains, shared the same wb salmo and presence of A-layer. A. salmonicida subsp. pectinolytica strains lack wb salmo and A-layer, two major virulence factors, and th is could be the reason they ar e the only ones not found as fish pathogens

The Complete Structure of the Core Oligosaccharide from Edwardsiella tarda EIB 202 Lipopolysaccharide

The chemical structure and genomics of the lipopolysaccharide (LPS) core oligosaccharide of pathogenic Edwardsiella tarda strain EIB 202 were studied for the first time. The complete gene assignment for all LPS core biosynthesis gene functions was acquired. The complete structure of core oligosaccharide was investigated by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry MSn, and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry. The following structure of the undecasaccharide was established: The heterogeneous appearance of the core oligosaccharide structure was due to the partial lack of β-d-Galp and the replacement of α-d-GlcpNAcGly by α-d-GlcpNGly. The glycine location was identified by mass spectrometry