Christian Heiss

An engineered eukaryotic protein glycosylation pathway in Escherichia coli

We performed bottom-up engineering of a synthetic pathway in Escherichia coli for the production of eukaryotic trimannosyl chitobiose glycans and the transfer of these glycans to specific asparagine residues in target proteins. The glycan biosynthesis was enabled by four eukaryotic glycosyltransferases, including the yeast uridine diphosphate-N-acetylglucosamine transferases Alg13 and Alg14 and the mannosyltransferases Alg1 and Alg2. By including the bacterial oligosaccharyltransferase PglB from Campylobacter jejuni, we successfully transferred glycans to eukaryotic proteins.

Salmonella produces an O-antigen capsule regulated by AgfD and important for environmental persistence

In this study, we show that Salmonella produces an O-antigen capsule coregulated with the fimbria- and cellulose-associated extracellular matrix. Structural analysis of purified Salmonella extracellular polysaccharides yielded predominantly a repeating oligosaccharide unit similar to that of Salmonella enterica serovar Enteritidis lipopolysaccharide O antigen with some modifications. Putative carbohydrate transport and regulatory operons important for capsule assembly and translocation, designated yihU-yshA and yihVW, were identified by screening a random transposon library with immune serum generated to the capsule. The absence of capsule was confirmed by generating various isogenic Deltayih mutants, where yihQ and yihO were shown to be important in capsule assembly and translocation. Luciferase-based expression studies showed that AgfD regulates the yih operons in coordination with extracellular matrix genes coding for thin aggregative fimbriae and cellulose. Although the capsule did not appear to be important for multicellular behavior, we demonstrate that it was important for survival during desiccation stress. Since the yih genes are conserved in salmonellae and the O-antigen capsule was important for environmental persistence, the formation of this surface structure may represent a conserved survival strategy.

Phosphate incorporation during glycogen synthesis and Lafora disease.

Glycogen is a branched polymer of glucose that serves as an energy store. Phosphate, a trace constituent of glycogen, has profound effects on glycogen structure, and phosphate hyperaccumulation is linked to Lafora disease, a fatal progressive myoclonus epilepsy that can be caused by mutations of laforin, a glycogen phosphatase. However, little is known about the metabolism of glycogen phosphate. We demonstrate here that the biosynthetic enzyme glycogen synthase, which normally adds glucose residues to glycogen, is capable of incorporating the β-phosphate of its substrate UDP-glucose at a rate of one phosphate per approximately 10,000 glucoses, in what may be considered a catalytic error. We show that the phosphate in glycogen is present as C2 and C3 phosphomonoesters. Since hyperphosphorylation of glycogen causes Lafora disease, phosphate removal by laforin may thus be considered a repair or damage control mechanism.

The structure of Cryptococcus neoformans galactoxylomannan contains β-d-glucuronic acid

The structure of galactoxylomannan, a capsular polysaccharide from the opportunistic yeast Cryptococcus neoformans, was re-examined by NMR spectroscopy and GC-MS. The residue that is 3-linked to the side chain galactose and was previously assigned as beta-D-xylose [Vaishnav, V. V.; Bacon, B. E.; ONeill, M.; Cherniak, R. Carbohydr. Res.1998, 306, 315-330] was determined to be beta-D-glucuronic acid. A revised structure for this polymer is presented, along with a proposal that this compound be termed glucuronoxylomannogalactan (GXMGal).

Activation of iNKT cells by a distinct constituent of the endogenous glucosylceramide fraction

Significance Invariant natural killer T (iNKT) cells are a specialized subset of T cells that recognizes lipids, rather than peptides, as antigens. Recognition of both endogenous and exogenous lipids by iNKT cells contributes to immune responses during infection, cancer, autoimmune disease, and allergic disease. The endogenous lipids recognized by iNKT cells in most contexts, however, remain unclear. In this report, we characterize the lipid antigen activity found in mammalian milk and tissues. Our data suggest that activity is related to a minor component of the glucosylceramide fraction. Whether contributed from endogenous sources or from the diet, this rare, yet potent lipid activity may play an important role in driving immune responses. Invariant natural killer T (iNKT) cells are a specialized T-cell subset that recognizes lipids as antigens, contributing to immune responses in diverse disease processes. Experimental data suggests that iNKT cells can recognize both microbial and endogenous lipid antigens. Several candidate endogenous lipid antigens have been proposed, although the contextual role of specific antigens during immune responses remains largely unknown. We have previously reported that mammalian glucosylceramides (GlcCers) activate iNKT cells. GlcCers are found in most mammalian tissues, and exist in variable molecular forms that differ mainly in N-acyl fatty acid chain use. In this report, we purified, characterized, and tested the GlcCer fractions from multiple animal species. Although activity was broadly identified in these GlcCer fractions from mammalian sources, we also found activity properties that could not be reconciled by differences in fatty acid chain use. Enzymatic digestion of β-GlcCer and a chromatographic separation method demonstrated that the activity in the GlcCer fraction was limited to a rare component of this fraction, and was not contained within the bulk of β-GlcCer molecular species. Our data suggest that a minor lipid species that copurifies with β-GlcCer in mammals functions as a lipid self antigen for iNKT cells.

Mutation of cysteine-295 to alanine in secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus affects the enantioselectivity and substrate specificity of ketone reductions.

The mutation of Cys-295 to alanine in Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase (SADH) was performed to give C295A SADH, on the basis of molecular modeling studies utilizing the X-ray crystal structure coordinates of the highly homologous T. brockii secondary alcohol dehydrogenase (1YKF.PDB). This mutant SADH has activity for 2-propanol comparable to wild-type SADH. However, the C295A mutation was found to cause a significant shift of enantioselectivity toward the (S)-configuration in the reduction of some ethynylketones to the corresponding chiral propargyl alcohols. This result confirms our prediction that Cys-295 is part of a small alkyl group binding pocket whose size determines the binding orientation of ketone substrates, and, hence, the stereochemical configuration of the product alcohol. Furthermore, C295A SADH has much higher activity towards t-butyl and some alpha-branched ketones than does wild-type SADH. The C295A mutation does not affect the thioester reductase activity of SADH. The broader substrate specificity and altered stereoselectivity for C295A SADH make it a potentially useful tool for asymmetric reductions.

Listeria monocytogenes exopolysaccharide: origin, structure, biosynthetic machinery and c-di-GMP-dependent regulation

Elevated levels of the second messenger c‐di‐GMP activate biosynthesis of an unknown exopolysaccharide (EPS) in the food‐borne pathogen Listeria monocytogenes. This EPS strongly protects cells against disinfectants and desiccation, indicating its potential significance for listerial persistence in the environment and for food safety. We analyzed the potential phylogenetic origin of this EPS, determined its complete structure, characterized genes involved in its biosynthesis and hydrolysis and identified diguanylate cyclases activating its synthesis. Phylogenetic analysis of EPS biosynthesis proteins suggests that they have evolved within monoderms. Scanning electron microscopy revealed that L.u2009monocytogenesu2005EPS is cell surface‐bound. Secreted carbohydrates represent exclusively cell‐wall debris. Based on carbohydrate composition, linkage and NMR analysis, the structure of the purified EPS is identified as a β‐1,4‐linked N‐acetylmannosamine chain decorated with terminal α‐1,6‐linked galactose. All genes of the pssA‐E operon are required for EPS production and so is a separately located pssZ gene. We show that PssZ has an EPS‐specific glycosylhydrolase activity. Exogenously added PssZ prevents EPS‐mediated cell aggregation and disperses preformed aggregates, whereas an E72Q mutant in the presumed catalytic residue is much less active. The diguanylate cyclases DgcA and DgcB, whose genes are located next to pssZ, are primarily responsible for c‐di‐GMP‐dependent EPS production.

Development of capsular polysaccharide-based glycoconjugates for immunization against melioidosis and glanders.

Burkholderia pseudomallei and Burkholderia mallei, the etiologic agents of melioidosis and glanders, respectively, cause severe disease in humans and animals and are considered potential agents of biological warfare and terrorism. Diagnosis and treatment of infections caused by these pathogens can be challenging and, in the absence of chemotherapeutic intervention, acute disease is frequently fatal. At present, there are no human or veterinary vaccines available for immunization against these emerging/re-emerging infectious diseases. One of the long term objectives of our research, therefore, is to identify and characterize protective antigens expressed by B. pseudomallei and B. mallei and use them to develop efficacious vaccine candidates. Previous studies have demonstrated that the 6-deoxy-heptan capsular polysaccharide (CPS) expressed by these bacterial pathogens is both a virulence determinant and a protective antigen. Consequently, this carbohydrate moiety has become an important component of the various subunit vaccines that we are currently developing in our laboratory. In the present study, we describe a reliable method for isolating CPS antigens from O-polysaccharide (OPS) deficient strains of B. pseudomallei; including a derivative of the select agent excluded strain Bp82. Utilizing these purified CPS samples, we also describe a simple procedure for covalently linking these T-cell independent antigens to carrier proteins. In addition, we demonstrate that high titer IgG responses can be raised against the CPS component of such constructs. Collectively, these approaches provide a tangible starting point for the development of novel CPS-based glycoconjugates for immunization against melioidosis and glanders.

Outer membrane vesicles displaying engineered glycotopes elicit protective antibodies

Significance Conjugate vaccines have proven to be an effective and safe strategy for reducing the incidence of disease caused by bacterial pathogens. However, the manufacture of these vaccines is technically demanding, inefficient, and expensive, thereby limiting their widespread use. Here, we describe an alternative methodology for generating glycoconjugate vaccines whereby recombinant polysaccharide biosynthesis is coordinated with vesicle formation in nonpathogenic Escherichia coli, resulting in glycosylated outer membrane vesicles (glycOMVs) that can effectively deliver pathogen-mimetic glycotopes to the immune system. An attractive feature of our approach is the fact that different plasmid-encoded polysaccharide biosynthetic pathways can be readily transformed into E. coli, enabling a “plug-and-play” platform for the on-demand creation of glycOMV vaccine candidates that carry heterologous glycotopes from numerous pathogenic bacteria. The O-antigen polysaccharide (O-PS) component of lipopolysaccharides on the surface of gram-negative bacteria is both a virulence factor and a B-cell antigen. Antibodies elicited by O-PS often confer protection against infection; therefore, O-PS glycoconjugate vaccines have proven useful against a number of different pathogenic bacteria. However, conventional methods for natural extraction or chemical synthesis of O-PS are technically demanding, inefficient, and expensive. Here, we describe an alternative methodology for producing glycoconjugate vaccines whereby recombinant O-PS biosynthesis is coordinated with vesiculation in laboratory strains of Escherichia coli to yield glycosylated outer membrane vesicles (glycOMVs) decorated with pathogen-mimetic glycotopes. Using this approach, glycOMVs corresponding to eight different pathogenic bacteria were generated. For example, expression of a 17-kb O-PS gene cluster from the highly virulent Francisella tularensis subsp. tularensis (type A) strain Schu S4 in hypervesiculating E. coli cells yielded glycOMVs that displayed F. tularensis O-PS. Immunization of BALB/c mice with glycOMVs elicited significant titers of O-PS–specific serum IgG antibodies as well as vaginal and bronchoalveolar IgA antibodies. Importantly, glycOMVs significantly prolonged survival upon subsequent challenge with F. tularensis Schu S4 and provided complete protection against challenge with two different F. tularensis subsp. holarctica (type B) live vaccine strains, thereby demonstrating the vaccine potential of glycOMVs. Given the ease with which recombinant glycotopes can be expressed on OMVs, the strategy described here could be readily adapted for developing vaccines against many other bacterial pathogens.

Exploiting enzyme specificities in digestions of chondroitin sulfates A and C: production of well-defined hexasaccharides

Interactions between proteins and glycosaminoglycans (GAGs) of the extracellular matrix are important to the regulation of cellular processes including growth, differentiation and migration. Understanding these processes can benefit greatly from the study of protein-GAG interactions using GAG oligosaccharides of well-defined structure. Materials for such studies have, however, been difficult to obtain because of challenges in synthetic approaches and the extreme structural heterogeneity in GAG polymers. Here, it is demonstrated that diversity in structures of oligosaccharides derived by limited enzymatic digestion of materials from natural sources can be greatly curtailed by a proper selection of combinations of source materials and digestive enzymes, a process aided by an improved understanding of the specificities of certain commercial preparations of hydrolases and lyases. Separation of well-defined oligosaccharides can then be accomplished by size-exclusion chromatography followed by strong anion-exchange chromatography. We focus here on two types of chondroitin sulfate (CS) as starting material (CS-A, and CS-C) and the use of three digestive enzymes with varying specificities (testicular hyaluronidase and bacterial chondroitinases ABC and C). Analysis using nuclear magnetic resonance and mass spectrometry focuses on isolated CS disaccharides and hexasaccharides. In all, 15 CS hexasaccharides have been isolated and characterized. These serve as useful contributions to growing libraries of well-defined GAG oligosaccharides that can be used in further biophysical assays.