The Type B Flagellin of Hypervirulent Clostridium difficile Is Modified with Novel Sulfonated Peptidylamido-glycans.

Glycosylation of flagellins is a well recognized property of many bacterial species. In this study, we describe the structural characterization of novel flagellar glycans from a number of hypervirulent strains of C. difficile We used mass spectrometry (nano-LC-MS and MS/MS analysis) to identify a number of putative glycopeptides that carried a variety of glycoform substitutions, each of which was linked through an initial N-acetylhexosamine residue to Ser or Thr. Detailed analysis of a LLDGSSTEIR glycopeptide released by tryptic digestion, which carried two variant structures, revealed that the glycopeptide contained, in addition to carbohydrate moieties, a novel structural entity. A variety of electrospray-MS strategies using Q-TOF technology were used to define this entity, including positive and negative ion collisionally activated decomposition MS/MS, which produced unique fragmentation patterns, and high resolution accurate mass measurement to allow derivation of atomic compositions, leading to the suggestion of a taurine-containing peptidylamido-glycan structure. Finally, NMR analysis of flagellin glycopeptides provided complementary information. The glycan portion of the modification was assigned as α-Fuc3N-(1→3)-α-Rha-(1→2)-α-Rha3OMe-(1→3)-ß-GlcNAc-(1→)Ser, and the novel capping moiety was shown to be comprised of taurine, alanine, and glycine. This is the first report of a novel O-linked sulfonated peptidylamido-glycan moiety decorating a flagellin protein.

Analyzing the modification of the Shewanella oneidensis MR-1 flagellar filament.

The unsheathed flagellar filament of Shewanella oneidensis MR-1 is composed of two highly homologous flagellins, FlaA, and the major structural unit, FlaB. We identified a gene cluster, SO_3261-SO_3265 (now sfmABCDE), that is required for the formation of a fully functional filament and for motility. The predicted function of the corresponding gene products strongly indicated a role in flagellin modification. Accordingly, loss of sfmABCDE results in a significant mass shift of both FlaA and FlaB. Mass spectroscopy analysis and single residue substitutions identified five serine residues in both flagellins that are modified via O-linkage. Modeling of the flagellin structures strongly suggests that at least four of the modified residues are exposed to the filament's surface. However, none of the five serine residues solely is crucial for function and assembly. Structural analysis of the flagellin modification revealed that it likely contains a nonulosonic acid (274 Da) linked to each glycosylated serine. The putative nonulosonic acid is further substituted with a 236 Da moiety which can carry additional methyl groups (250 Da, 264 Da). In addition, at least 5 lysine residues in FlaB and one in FlaA were found to be methylated. Based on homology comparisons we suggest that smfABCDE is required for species-specific flagellin modification in S. oneidensis MR-1.

Similarities and differences in the glycosylation mechanisms in prokaryotes and eukaryotes.

Recent years have witnessed a rapid growth in the number and diversity of prokaryotic proteins shown to carry N- and/or O-glycans, with protein glycosylation now considered as fundamental to the biology of these organisms as it is in eukaryotic systems. This article overviews the major glycosylation pathways that are known to exist in eukarya, bacteria and archaea. These are (i) oligosaccharyltransferase (OST)-mediated N-glycosylation which is abundant in eukarya and archaea, but is restricted to a limited range of bacteria; (ii) stepwise cytoplasmic N-glycosylation that has so far only been confirmed in the bacterial domain; (iii) OST-mediated O-glycosylation which appears to be characteristic of bacteria; and (iv) stepwise O-glycosylation which is common in eukarya and bacteria. A key aim of the review is to integrate information from the three domains of life in order to highlight commonalities in glycosylation processes. We show how the OST-mediated N- and O-glycosylation pathways share cytoplasmic assembly of lipid-linked oligosaccharides, flipping across the ER/periplasmic/cytoplasmic membranes, and transferring "en bloc" to the protein acceptor. Moreover these hallmarks are mirrored in lipopolysaccharide biosynthesis. Like in eukaryotes, stepwise O-glycosylation occurs on diverse bacterial proteins including flagellins, adhesins, autotransporters and lipoproteins, with O-glycosylation chain extension often coupled with secretory mechanisms.

Prevalencia de diabetes en pacientes internados: factores socioeconómicos-culturales; educación de la enfermedad y dificultades para el tratamiento / Prevalence of diabetes in hospitalised patients

Introducción: la diabetes mellitus determina elevada morbimortalidad. Los aspectos económico-educacionales inciden en su evolución. Actualmente no hay estudios que evalúen estos aspectos en nuestro medio. Objetivos: analizar la prevalencia de diabetes en pacientes internados, el factor económico-cultural, el nivel de información sobre diabetes y las dificultades para el tratamiento. Método: realizamos una encuesta a 345 pacientes internados, 194 del Hospital Maciel (HM) en octubre de 2002 y 151 del Centro de Asistencial del Sindicato Médico del Uruguay (CASMU) en marzo de 2001. Incluyó un cuestionario sobre aspectos básicos de la diabetes. Resultados: la prevalencia de diabetes en pacientes internados fue 19,9 por ciento en el HM y ed 13,9 por ciento en el CASMU, siendo esta diferencia significativa. El 29 por ciento de los pacientes del HM y 56 por ciento del CASMU presentaron escolaridades por encima del nivel primario. El nivel de salarios medio fue mayor en el CASMU, con valores de 763 dólares, respecto del HM, con 360 dólares. Superaron 55 por ciento de respuestas correctas del cuestionario sobre aspectos básicos de la diabetes 61,9 y 63,8 por ciento de las personas internadas en el HM y en el CASMU, respectivamente, siendo esta diferencia no significativa. Para la subpoblación diabética superaron dicho porcentaje 67,8 por ciento de pacientes del HM y 90,5 por ciento del CSMU, diferencia estadísticamente significativa. El 59 y 53 por ciento de los pacientes que refieren tener información sobre diabetes, la obtuvieron de medios de prensa; 60 por ciento de diabéticos del HM y 77 por ciento del CASMU señalan al factor económico como determinante de las dificultades para llevar a cabo un tratamiento adecuado. Conclusiones: la prevalencia de diabetes en pacientes internados es el doble o el triple (13,9-19,1 por ciento) de la que presenta nuestro país (7 por ciento). La población del CASMU tiene un nivel cultural y económico mayor que la del HM. Esta diferencia podría verse reflejada en relación con el nivel de información sobre diabetes solamente en la subpoblación de diabéticos. Los medios de prensa son el origen principal de información. La mayor dificultad para cumplir con el tratamiento es económica.