Engineering Yarrowia lipolytica to Produce Glycoproteins Homogeneously Modified with the Universal Man3GlcNAc2 N-Glycan Core

Yarrowia lipolytica is a dimorphic yeast that efficiently secretes various heterologous proteins and is classified as “generally recognized as safe.” Therefore, it is an attractive protein production host. However, yeasts modify glycoproteins with non-human high mannose-type N-glycans. These structures reduce the protein half-life in vivo and can be immunogenic in man. Here, we describe how we genetically engineered N-glycan biosynthesis in Yarrowia lipolytica so that it produces Man3GlcNAc2 structures on its glycoproteins. We obtained unprecedented levels of homogeneity of this glycanstructure. This is the ideal starting point for building human-like sugars. Disruption of the ALG3 gene resulted in modification of proteins mainly with Man5GlcNAc2 and GlcMan5GlcNAc2 glycans, and to a lesser extent with Glc2Man5GlcNAc2 glycans. To avoid underoccupancy of glycosylation sites, we concomitantly overexpressed ALG6. We also explored several approaches to remove the terminal glucose residues, which hamper further humanization of N-glycosylation; overexpression of the heterodimeric Apergillus niger glucosidase II proved to be the most effective approach. Finally, we overexpressed an α-1,2-mannosidase to obtain Man3GlcNAc2 structures, which are substrates for the synthesis of complex-type glycans. The final Yarrowia lipolytica strain produces proteins glycosylated with the trimannosyl core N-glycan (Man3GlcNAc2), which is the common core of all complex-type N-glycans. All these glycans can be constructed on the obtained trimannosyl N-glycan using either in vivo or in vitro modification with the appropriate glycosyltransferases. The results demonstrate the high potential of Yarrowia lipolytica to be developed as an efficient expression system for the production of glycoproteins with humanized glycans

Treatment of enterohemorrhagic Escherichia coli (EHEC) infection and hemolytic uremic syndrome (HUS)

Verotoxigenic Escherichia coli (VTEC) are a specialized group of E. coli that can cause severe colonic disease and renal failure. Their pathogenicity derives from virulence factors that enable the bacteria to colonize the colon and deliver extremely powerful toxins known as verotoxins (VT) or Shiga toxins (Stx) to the systemic circulation. The recent devastating E. coli O104:H4 epidemic in Europe has shown how helpless medical professionals are in terms of offering effective therapies. By examining the sources and distribution of these bacteria, and how they cause disease, we will be in a better position to prevent and treat the inevitable future cases of sporadic disease and victims of common source outbreaks. Due to the complexity of pathogenesis, it is likely a multitargeted approach is warranted. Developments in terms of these treatments are discussed

Learning low-dimensional separable decompositions of MIMO non-linear systems

We present a new internal structure exploration method developed for the multiple-input multiple-output (MIMO) dynamical systems with finite memory and almost arbitrary non-linear characteristic. The proposed Double Separation Algorithm applies distance correlation screening for pre-selection of those system inputs that contribute to the consecutive outputs and, based on the first-stage inference outcomes, estimates projection coefficients sensitive to the existence of additive system sub-characteristics. In effect, the proposed approach allows for effective exploration of the internal system structure. A numerical experiment on an MIMO nonlinear finite impulse response (NFIR) system illustrates the ability of the proposed approach to indicate which of the system inputs contribute to which of the system outputs. The experiment also illustrates the ability of the approach to detect which of the nonlinear sub-characteristics, recovered in the first stage of the approach, can be separated into a sum of lower-dimensional sub-characteristics.</p

Maltose-binding protein is a potential carrier for oral immunizations

Maltose binding protein (MBP) is often fused to a relevant protein to improve its yield and facilitate its purification, but MBP can also enhance the immunogenicity of the fused proteins. Recent data suggest that MBP may potentiate antigen-presenting functions in immunized animals by providing intrinsic maturation stimuli to dendritic cells through TLR4. The aim of this study was to examine if an MBP-specific immune response can be elicited by oral administration of MBP. Therefore, in a first experiment the MBP specific immune response was analyzed after oral immunization with MBP or MBP+CT to piglets and both the systemic and mucosal immune responses were examined Although no high systemic response was observed in the MBP-group, a local mucosal IgM MBP-specific response in the jejunal Peyer's patches was observed. In the second experiment MBPFedF was orally administered to piglets. A significant systemic response against MBP and a weak response against FedF were found after oral administration of MBPFedF+CT. Also the presence of MBP-specific IgA ASC in the lamina propria indicates that a local intestinal immune response against MBP was induced. Our data suggests that MBP can cross the epithelial barrier reaching the gut-associated lymphoid tissue after oral administration to pigs, which implicates that MBP could act as a carrier and delivery system for fused proteins to target the vaccine antigens to intestinal immune cells.status: publishe

The F18 fimbrial adhesin FedF is highly conserved among F18+Escherichia coli isolates

F18+Escherichia coli cause postweaning diarrhoea and oedema disease in newly weaned piglets. Protection against these diseases can be established by preventing the fimbrial adhesion of these bacteria to the enterocytes of the porcine intestine. To test a vaccine against F18+E. coli consisting of the adhesin of F18 fimbriae, FedF, the conservation of the FedF subunit had to be examined. Therefore, the fedF sequence of 37 F18+E. coli isolates from different countries was determined and compared to the fedF gene of the F18ab reference strain F107/86. The amino acid sequence of the mature FedF from the individual F18+E. coli isolates was 96-100% identical to that from E. coli F107/86, but the overall homology was 90.4%. Hyper variable regions were not found in the FedF sequence. The FedF sequence was conserved over the different countries and between the two antigenic variants, F18ab and F18ac, suggesting that F18ab and F18ac strains have the same receptor. Furthermore, the conserved C-terminal region in the FedF adhesin suggests that the F18 fimbriae, in analogy with type 1 and P pili, are assembled by a donor strand mechanism. In conclusion, the reported conservation of FedF supports the usefulness of the fimbrial adhesin as a subunit vaccine against F18+E. coli infection.status: publishe

Monoclonal antibodies reveal a weak interaction between the F18 fimbrial adhesin FedF and the major subunit FedA

F18(+) Escherichia coli can cause post-weaning diarrhoea and oedema disease in pigs. These diseases are responsible for substantial economic losses, but a vaccine is not available. A good knowledge of the characteristic of the fimbriae is useful for the development of a vaccine composed of the fimbrial virulence factor. F18 fimbriae are composed of the major subunit FedA and the minor subunits FedE and the adhesin FedF In the present study monoclonal antibodies (mAbs) against FedA and FedF were produced. In addition to their diagnostic value, these mAbs revealed a weaker interaction between FedA and FedF compared to the subunit-subunit interactions in other fimbriae, like type I and P pili. Further experiments are needed to investigate if this weak interaction could be one of the reasons for the slow colonisation of the small intestinal mucosa by F18(+) E. coli. (c) 2006 Elsevier B.V. All rights reserved.status: publishe

The age-dependent expression of the F18(+) E.coli receptor on porcine gut epithelial cells is positively correlated with the presence of histo-blood group antigens

F18(+) Escherichia coli have the ability to colonize the gut and cause oedema disease or post-weaning diarrhoea by adhering to specific F18 receptors (F18R) on the porcine epithelium. Although it is well established that a DNA polymorphism on base pair 307 of the FUT1 gene, encoding an alpha(1,2)fucosyltransferase, accounts for the F18R phenotype, the F18R nature is not elucidated yet. The aim of the present study was to investigate the correlation between the presence of H-2 histo-blood group antigens (HBGAs) or its derivative A-2 HBGAs on the porcine gut epithelium and F18(+) E. coli adherence. A significant positive correlation was found between expression of both the H-2 (r = 0.586, P < 0.01) and A-2 (r = 0.775, P < 0.01) HBGAs and F18(+) E. coli adherence after examination of 74 pigs aged from 0 to 23 weeks. The majority of the genetically resistant pigs (FUT1M307(A/A)) showed no HBGA expression (91.7%) and no F18(+) E. coli adherence (83.3%). In addition, it was found that F18R expression levels rise with increasing age during the first 3 weeks after birth and that F18R expression is maintained in older pigs (3-23 weeks old). Taken together, these data suggest that, apart from H-2 HBGAs, A-2 HBGAs might be involved in F18(+) E. coli adherence. (c) 2007 Elsevier V.B. All rights reserved.status: publishe