HR-MAS NMR reveals a pH-dependent LPS alteration by de-O-acetylation at abequose in the O-antigen of Salmonella enterica serovar Typhimurium.

NMR spectroscopy can detect biomolecules like lipopolysaccharide directly on the surface of the cell, thus avoiding isolation and purification, and providing a more realistic description than the one derived from in vitro studies. Here we present a high-resolution magic-angle spinning NMR study of the O-antigen of Salmonella enterica serovar Typhimurium (S. Typhimurium) performed directly on the cells showing the alteration of its acetylation state over time. The O-antigen region of S. Typhimurium consists of the repeating unit [→2)-α-d-Manp-(1→4)-α-l-Rhap-(1→3)-α-d-Galp-(1→] where Man stands for mannose, Rha for rhamnose, and Gal for galactose. Man is substituted with abequose (Abe) O-acetylated at carbon 2. Our studies revealed that the appearance of de-O-acetylated O-antigen in the stationary growth phase is due to the de-O-acetylation of already synthesized O-acetylated O-antigen and that this reaction is caused by the metabolism-induced basic pH of the growth medium. The labile O-acetylation of the O-antigen we observed in S. Typhimurium generates non-stoichiometric O-acetylation states and therefore changes the nature of an immunogenic epitope.

On-cell MAS NMR: physiological clues from living cells.

While structural information on biomolecules is mainly obtained from purified in vitro samples, NMR can also be applied in the context of entire cells or organisms. The present study describes maturation processes in living Salmonella enterica serovar Typhimurium, a prevalent cause for human gastroenteritis. In our physiological study, we follow the composition of the O-antigen on the outer bacterial membrane with high-resolution MAS NMR spectroscopy. We detect and characterize an evolution of the O-antigen composition, in particular of the O-acetylation state of the O-antigen, a factor that can play an important role in vaccine development.

Supported lipopolysaccharide bilayers.

In this report, the formation of supported lipopolysaccharide bilayers (LPS-SLBs) is studied with extracted native and glycoengineered LPS from Escherichia coli ( E. coli ) and Salmonella enterica sv typhimurium ( S. typhimurium ) to assemble a platform that allows measurement of LPS membrane structure and the detection of membrane tethered saccharide-protein interactions. We present quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence recovery after photobleaching (FRAP) characterization of LPS-SLBs with different LPS species, having, for example, different molecular weights, that show successful formation of SLBs through vesicle fusion on SiO(2) surfaces with LPS fractions up to 50 wt %. The thickness of the LPS bilayers were investigated with AFM force-distance measurements which showed only a slight thickness increase compared to pure POPC SLBs. The E. coli LPS were chosen to study the saccharide-protein interaction between the Htype II glycan epitope and the Ralstonia solanacearum lectin (RSL). RSL specifically recognizes fucose sugars, which are present in the used Htype II glycan epitope and absent in the epitopes LPS1 and EY2. We show via fluorescence microscopy that the specific, but weak and multivalent interaction can be detected and discriminated on the LPS-SLB platform.

Glycomimicry: display of fucosylation on the lipo-oligosaccharide of recombinant Escherichia coli K12.

We recently described the design of Escherichia coli K12 and Salmonella enterica sv Typhimurium to display the gangliomannoside 3 (GM3) antigen on the cell surface. We report here the fucosylation of modified lipooligosaccharide in a recombinant E.coli strain with a truncated lipid A core due to deletion of the core glycosyltransferases genes waaO and waaB. This truncated structure was used as a scaffold to assemble the Lewis Y motif by consequent action of the heterologously expressed ß-1,4 galactosyltransferase LgtE (Neisseria gonorrheae), the ß-1,3 N-acetylglucosaminyltransferase LgtA and the ß-1,3 galactosyltransferase LgtB from Neisseria meningitidis, as well as the α-1,2 and α-1,3 fucosyltransferases FutC and FutA from Helicobacter pylori. We show the display of the Lewis Y structure by immunological and chemical analysis.

Glycomimicry: display of the GM3 sugar epitope on Escherichia coli and Salmonella enterica sv Typhimurium.

Oligosaccharides present on the surface of pathogenic bacteria play an important role in their interaction with their host. Bacteria with altered cell surface structures can be used to study these interactions, and glycoengineering represents a tool to display a glycoepitope on a different bacterium. Here, we present non-pathogenic Escherichia coli and Salmonella enterica serovar Typhimurium expressing the sialyllactose oligosaccharide epitope of the ganglioside GM3. By expression of the galactosyltransferase LgtE and the sialic acid transferase Lst as well as the CMP-sialic acid synthetase SiaB from Neisseria gonorrhoeae and Neisseria meningitidis in engineered strains devoid of the sialic acid catabolism, the GM3 sugar epitope was displayed on these bacteria as demonstrated by live cell immunostaining and a detailed analysis of their lipooligosaccharides. These strains offer the possibility to investigate the role of sialic acid in the recognition of bacteria by the immune system in a non-pathogenic background.

O-antigen-negative Salmonella enterica serovar Typhimurium is attenuated in intestinal colonization but elicits colitis in streptomycin-treated mice.

Lipopolysaccharide (LPS) is a major constituent of the outer membrane and an important virulence factor of Salmonella enterica subspecies 1 serovar Typhimurium (serovar Typhimurium). To evaluate the role of LPS in eliciting intestinal inflammation in streptomycin-treated mice, we constructed an O-antigen-deficient serovar Typhimurium strain through deletion of the wbaP gene. The resulting strain was highly susceptible to human complement activity and the antimicrobial peptide mimic polymyxin B. Furthermore, it showed a severe defect in motility and an attenuated phenotype in a competitive mouse infection experiment, where the DeltawbaP strain (SKI12) was directly compared to wild-type Salmonella. Nevertheless, the DeltawbaP strain (SKI12) efficiently invaded HeLa cells in vitro and elicited acute intestinal inflammation in streptomycin-pretreated mice. Our experiments prove that the presence of complete LPS is not essential for in vitro invasion or for triggering acute colitis.

Protein-protein interactions in the archaeal transcriptional machinery: binding studies of isolated RNA polymerase subunits and transcription factors.

Transcription in Archaea is directed by a pol II-like RNA polymerase and homologues of TBP and TFIIB (TFB) but the crystal structure of the archaeal enzyme and the subunits involved in recruitment of RNA polymerase to the promoter-TBP-TFB-complex are unknown. We described here the cloning expression and purification of 11 bacterially expressed subunits of the Pyrococcus furiosus RNAP. Protein interactions of subunits with each other and of archaeal transcription factors TFB and TFB with RNAP subunits were studied by Far-Western blotting and reconstitution of subcomplexes from single subunits in solution. In silico comparison of a consensus sequence of archaeal RNAP subunits with the sequence of yeast pol II subunits revealed a high degree of conservation of domains of the enzymes forming the cleft and catalytic center of the enzyme. Interaction studies with the large subunits were complicated by the low solubility of isolated subunits B, A', and A'', but an interaction network of the smaller subunits of the enzyme was established. Far-Western analyses identified subunit D as structurally important key polypeptide of RNAP involved in interactions with subunits B, L, N, and P and revealed also a strong interaction of subunits E' and F. Stable complexes consisting of subunits E' and F, of D and L and a BDLNP-subcomplex were reconstituted and purified. Gel shift analyses revealed an association of the BDLNP subcomplex with promoter-bound TBP-TFB. These results suggest a major role of subunit B (Rpb2) in RNAP recruitment to the TBP-TFB promoter complex.

EBV-associated neoplasms: alternative pathogenetic pathways.

We propose that there are two main classes of Epstein-Barr virus (EBV) associated lymphomas: primarily malignant Burkitt's Lymphoma (BL) and Hodgkin's Disease (HD), on one hand, and primarily benign lymphoproliferations, e.g., post-transplant lymphoproliferative disease (PTLD) on the other hand. PTLD may start as a benign lymphoproliferation which becomes malignant if out of T cell control for too long. Our discovery of a binding site for the oncoprotein c-Myc at a central position of the EBV genome favours a distinction of pathogenetic pathways or scenarios for the proposed lymphoma classes. In the first scenario nuclear maintenance of the EBV genome and activation of viral anti-apoptotic functions with the help of c-Myc are indispensable for the origin of malignant tumours (BL, HD) from the germinal centre B-cell. In the second scenario expression of the main viral transforming protein EBNA2 is essential for immortalisation and non-malignant morphological transformation of any (germinal centre derived or non-germinal centre) B-cell in the absence of T cell control. Although EBNA2 expression is permissible, under specific circumstances, in malignant B-cells, it is not required for oncogenesis.

A 30 kb region of the Epstein-Barr virus genome is colinear with the rearranged human immunoglobulin gene loci: implications for a "ping-pong evolution" model for persisting viruses and their hosts. A review.

The left part of the Epstein-Barr virus (EBV) genome exhibits a strong colinearity of structural and functional elements with the immunoglobulin (Ig) gene loci which is only partially reflected in nucleotide sequence homologies. We propose that this colinearity may be the result of an inter-dependent co-evolution of the immunoglobulin loci together with EBV. Our observation could help elucidating the mechanisms of somatic hypermutation, explaining the ability of EBV to accidentally cause tumors, and shedding more light on the general mechanisms of viral and organismal evolution. We suggest that persisting viruses served as a complement for the organismal germline like in a ping-pong game and outline The Ping-Pong Evolution Hypothesis.

The in vivo binding site for oncoprotein c-Myc in the promoter for Epstein-Barr virus (EBV) encoding RNA (EBER) 1 suggests a specific role for EBV in lymphomagenesis.

BACKGROUND: Epstein-Barr virus (EBV) was isolated in the 1960s from the African childhood tumor, Burkitt's Lymphoma (BL), characterized by the translocation of the c-myc gene into one of the immunoglobulin loci. Due to the extreme discrepancy between the widespread dissemination of EBV infection and the overall rarity of EBV-related tumors, it remains an open question whether EBV is really a human tumor virus, and if so, what specific contribution EBV may have to tumorigenesis. MATERIAL/METHODS: Protein binding at the EBER locus of EBV was analyzed by genomic footprinting electrophoretic mobility shift, reporter gene assay, and chromatin immunoprecipitation in a panel of six B-cell lines. RESULTS: Several novel in vivo protein binding sites were found in the EBER locus. Among those, a prominent binding site, 130 base pairs upstream of the EBER1 gene, contains two E-boxes providing a consensus sequence for binding of the transcription factor and oncoprotein c-Myc to the EBV genome. CONCLUSIONS: Based on the discovery of a binding site for c-Myc in the EBV genome, a new molecular model for the specific role of EBV as a causal factor in the origin of endemic Burkitt's Lymphoma is proposed. Translocated and deregulated c-myc directly activates and maintains the antiapoptotic functions of the EBER locus in a single EBV-infected B cell undergoing the germinal center (GC) reaction. With the balance shifted towards cell survival, the oncogenic potential of the pro-apoptotic c-Myc protein is unmasked in the translocated GC cell. This single translocated and surviving cell is the founder cell of an endemic BL. The new model reinstitutes EBV as a real human tumor virus.