Antibody-Mediated Endocytosis of Polysialic Acid Enables Intracellular Delivery and Cytotoxicity of a Glycan-Directed Antibody-Drug Conjugate.

The specific targeting of differentially expressed glycans in malignant cells has emerged as an attractive anticancer strategy. One such target is the oncodevelopmental antigen polysialic acid (polySia), a polymer of α2,8-linked sialic acid residues that is largely absent during postnatal development but is re-expressed during progression of several malignant human tumors, including small-cell and non-small cell lung carcinomas, glioma, neuroblastoma, and pancreatic carcinoma. In these cancers, expression of polySia correlates with tumor progression and poor prognosis and appears to modulate cancer cell adhesion, invasiveness, and metastasis. To evaluate the potential of PolySia as a target for anticancer therapy, we developed a chimeric human polySia-specific mAb that retained low nanomolar (nmol/L) target affinity and exhibited exquisite selectivity for polySia structures. The engineered chimeric mAb recognized several polySia-positive tumor cell lines in vitro and induced rapid endocytosis of polySia antigens. To determine whether this internalization could be exploited for delivery of conjugated cytotoxic drugs, we generated an antibody-drug conjugate (ADC) by covalently linking the chimeric human mAb to the tubulin-binding maytansinoid DM1 using a bioorthogonal chemical reaction scheme. The resulting polySia-directed ADC demonstrated potent target-dependent cytotoxicity against polySia-positive tumor cells in vitro. Collectively, these results establish polySia as a valid cell-surface, cancer-specific target for glycan-directed ADC and contribute to a growing body of evidence that the tumor glycocalyx is a promising target for synthetic immunotherapies. SIGNIFICANCE: These findings describe a glycan-specific antibody-drug conjugate that establishes polySia as a viable cell surface target within the tumor glycocalyx.

A flow cytometric approach to engineering Escherichia coli for improved eukaryotic protein glycosylation.

A synthetic pathway for production of the eukaryotic trimannosyl chitobiose glycan (mannose3-N-acetylglucosamine2, Man3GlcNAc2) and its transfer to specific asparagine residues in target proteins was previously engineered in Escherichia coli, providing this simple microbe with the ability to perform a complex post-translational protein modification. Here, we leveraged a flow cytometric fluorescence-based assay to improve Man3GlcNAc2 glycan biosynthesis in E. coli cells. Specifically, pathway improvements were identified, including reducing pathway enzyme expression levels and overexpressing nucleotide sugar biosynthesis genes, which enhanced production of lipid-linked Man3GlcNAc2 by nearly 50-fold to 13.9 µg/L. In turn, cells producing higher levels of the Man3GlcNAc2 substrate yielded up to 10 times more glycosylated acceptor protein (to ~ 14 mg/L) than their non-optimized counterparts. These results demonstrate the use of flow cytometry screening as a powerful tool for interrogating the surfaces of glyco-engineered bacteria and identifying meaningful improvements in glycan biosynthesis. We anticipate this approach will enable further optimization of bacterial glycan biosynthesis pathways using new strain engineering tools from metabolic engineering and synthetic biology.

A library of chemically defined human N-glycans synthesized from microbial oligosaccharide precursors.

Synthesis of homogenous glycans in quantitative yields represents a major bottleneck to the production of molecular tools for glycoscience, such as glycan microarrays, affinity resins, and reference standards. Here, we describe a combined biological/enzymatic synthesis that is capable of efficiently converting microbially-derived precursor oligosaccharides into structurally uniform human-type N-glycans. Unlike starting material obtained by chemical synthesis or direct isolation from natural sources, which can be time consuming and costly to generate, our approach involves precursors derived from renewable sources including wild-type Saccharomyces cerevisiae glycoproteins and lipid-linked oligosaccharides from glycoengineered Escherichia coli. Following deglycosylation of these biosynthetic precursors, the resulting microbial oligosaccharides are subjected to a greatly simplified purification scheme followed by structural remodeling using commercially available and recombinantly produced glycosyltransferases including key N-acetylglucosaminyltransferases (e.g., GnTI, GnTII, and GnTIV) involved in early remodeling of glycans in the mammalian glycosylation pathway. Using this approach, preparative quantities of hybrid and complex-type N-glycans including asymmetric multi-antennary structures were generated and subsequently used to develop a glycan microarray for high-throughput, fluorescence-based screening of glycan-binding proteins. Taken together, these results confirm our combined synthesis strategy as a new, user-friendly route for supplying chemically defined human glycans simply by combining biosynthetically-derived precursors with enzymatic remodeling.

Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies.

The development of antibodies against specific glycan epitopes poses a significant challenge due to difficulties obtaining desired glycans at sufficient quantity and purity, and the fact that glycans are usually weakly immunogenic. To address this challenge, we leveraged the potent immunostimulatory activity of bacterial outer membrane vesicles (OMVs) to deliver designer glycan epitopes to the immune system. This approach involved heterologous expression of two clinically important glycans, namely polysialic acid (PSA) and Thomsen-Friedenreich antigen (T antigen) in hypervesiculating strains of non-pathogenic Escherichia coli. The resulting glycOMVs displayed structural mimics of PSA or T antigen on their surfaces, and induced high titers of glycan-specific IgG antibodies following immunization in mice. In the case of PSA glycOMVs, serum antibodies potently killed Neisseria meningitidis serogroup B (MenB), whose outer capsule is PSA, in a serum bactericidal assay. These findings demonstrate the potential of glycOMVs for inducing class-switched, humoral immune responses against glycan antigens.

Glycans-by-design: engineering bacteria for the biosynthesis of complex glycans and glycoconjugates.

There is an urgent need for new tools that enable better understanding of the structure, recognition, metabolism, and biosynthesis of glycans as well as the production of biologically important glycans and glycoconjugates. With the discovery of glycoprotein synthesis in bacteria and functional transfer of glycosylation pathways between species, Escherichia coli cells have become a tractable host for both understanding glycosylation and the underlying glycan code of living cells as well as for expressing glycoprotein therapeutics and vaccines. Here, we review recent efforts to harness natural biological pathways and engineer synthetic designer pathways in bacteria for making complex glycans and conjugating these to lipids and proteins. The result of these efforts has been a veritable transformation of bacteria into living factories for scalable, bottom-up production of complex glycoconjugates by design.

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.

2-Heptyl-4-quinolone, a precursor of the Pseudomonas quinolone signal molecule, modulates swarming motility in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen capable of group behaviors, including biofilm formation and swarming motility. These group behaviors are regulated by both the intracellular signaling molecule c-di-GMP and acylhomoserine lactone quorum-sensing systems. Here, we show that the Pseudomonas quinolone signal (PQS) system also contributes to the regulation of swarming motility. Specifically, our data indicate that 2-heptyl-4-quinolone (HHQ), a precursor of PQS, likely induces the production of the phenazine-1-carboxylic acid (PCA), which in turn acts via an as-yet-unknown downstream mechanism to repress swarming motility. We show that this HHQ- and PCA-dependent swarming repression is apparently independent of changes in global levels of c-di-GMP, suggesting complex regulation of this group behavior.

Modulation of Pseudomonas aeruginosa surface-associated group behaviors by individual amino acids through c-di-GMP signaling.

To colonize the cystic fibrosis lung, Pseudomonas aeruginosa establishes sessile communities referred to as biofilms. Although the signaling molecule c-di-GMP governs the transition from motile to sessile growth, the environmental signal(s) required to modulate biofilm formation remain unclear. Using relevant in vivo concentrations of the 19 amino acids previously identified in cystic fibrosis sputum, we demonstrated that arginine, ornithine, isoleucine, leucine, valine, phenylalanine and tyrosine robustly promoted biofilm formation in vitro. Among the seven biofilm-promoting amino acids, only arginine also completely repressed the ability of P. aeruginosa to swarm over semi-solid surfaces, suggesting that arginine may be an environmental cue favoring a sessile lifestyle. Mutating two documented diguanylate cyclases required for biofilm formation (SadC and RoeA) reduced biofilm formation and restored swarming motility on arginine-containing medium. Growth on arginine increased the intracellular levels of c-di-GMP, and this increase was dependent on the SadC and RoeA diguanylate cyclases. Strains mutated in sadC, roeA or both also showed a reduction in biofilm formation when grown with the other biofilm-promoting amino acids. Taken together, these results suggest that amino acids can modulate biofilm formation and swarming motility, at least in part, by controlling the intracellular levels of c-di-GMP.

Production of secretory and extracellular N-linked glycoproteins in Escherichia coli.

The Campylobacter jejuni pgl gene cluster encodes a complete N-linked protein glycosylation pathway that can be functionally transferred into Escherichia coli. In this system, we analyzed the interplay between N-linked glycosylation, membrane translocation and folding of acceptor proteins in bacteria. We developed a recombinant N-glycan acceptor peptide tag that permits N-linked glycosylation of diverse recombinant proteins expressed in the periplasm of glycosylation-competent E. coli cells. With this "glycosylation tag," a clear difference was observed in the glycosylation patterns found on periplasmic proteins depending on their mode of inner membrane translocation (i.e., Sec, signal recognition particle [SRP], or twin-arginine translocation [Tat] export), indicating that the mode of protein export can influence N-glycosylation efficiency. We also established that engineered substrate proteins targeted to environments beyond the periplasm, such as the outer membrane, the membrane vesicles, and the extracellular medium, could serve as substrates for N-linked glycosylation. Taken together, our results demonstrate that the C. jejuni N-glycosylation machinery is compatible with distinct secretory mechanisms in E. coli, effectively expanding the N-linked glycome of recombinant E. coli. Moreover, this simple glycosylation tag strategy expands the glycoengineering toolbox and opens the door to bacterial synthesis of a wide array of recombinant glycoprotein conjugates.

Specific control of Pseudomonas aeruginosa surface-associated behaviors by two c-di-GMP diguanylate cyclases.

The signaling nucleotide cyclic diguanylate (c-di-GMP) regulates the transition between motile and sessile growth in a wide range of bacteria. Understanding how microbes control c-di-GMP metabolism to activate specific pathways is complicated by the apparent multifold redundancy of enzymes that synthesize and degrade this dinucleotide, and several models have been proposed to explain how bacteria coordinate the actions of these many enzymes. Here we report the identification of a diguanylate cyclase (DGC), RoeA, of Pseudomonas aeruginosa that promotes the production of extracellular polysaccharide (EPS) and contributes to biofilm formation, that is, the transition from planktonic to surface-dwelling cells. Our studies reveal that RoeA and the previously described DGC SadC make distinct contributions to biofilm formation, controlling polysaccharide production and flagellar motility, respectively. Measurement of total cellular levels of c-di-GMP in ∆roeA and ∆sadC mutants in two different genetic backgrounds revealed no correlation between levels of c-di-GMP and the observed phenotypic output with regard to swarming motility and EPS production. Our data strongly argue against a model wherein changes in total levels of c-di-GMP can account for the specific surface-related phenotypes of P. aeruginosa.

BifA, a cyclic-Di-GMP phosphodiesterase, inversely regulates biofilm formation and swarming motility by Pseudomonas aeruginosa PA14.

The intracellular signaling molecule, cyclic-di-GMP (c-di-GMP), has been shown to influence bacterial behaviors, including motility and biofilm formation. We report the identification and characterization of PA4367, a gene involved in regulating surface-associated behaviors in Pseudomonas aeruginosa. The PA4367 gene encodes a protein with an EAL domain, associated with c-di-GMP phosphodiesterase activity, as well as a GGDEF domain, which is associated with a c-di-GMP-synthesizing diguanylate cyclase activity. Deletion of the PA4367 gene results in a severe defect in swarming motility and a hyperbiofilm phenotype; thus, we designate this gene bifA, for biofilm formation. We show that BifA localizes to the inner membrane and, in biochemical studies, that purified BifA protein exhibits phosphodiesterase activity in vitro but no detectable diguanylate cyclase activity. Furthermore, mutational analyses of the conserved EAL and GGDEF residues of BifA suggest that both domains are important for the observed phosphodiesterase activity. Consistent with these data, the DeltabifA mutant exhibits increased cellular pools of c-di-GMP relative to the wild type and increased synthesis of a polysaccharide produced by the pel locus. This increased polysaccharide production is required for the enhanced biofilm formed by the DeltabifA mutant but does not contribute to the observed swarming defect. The DeltabifA mutation also results in decreased flagellar reversals. Based on epistasis studies with the previously described sadB gene, we propose that BifA functions upstream of SadB in the control of biofilm formation and swarming.

SadC reciprocally influences biofilm formation and swarming motility via modulation of exopolysaccharide production and flagellar function.

Pseudomonas aeruginosa has served as an important organism in the study of biofilm formation; however, we still lack an understanding of the mechanisms by which this microbe transitions to a surface lifestyle. A recent study of the early stages of biofilm formation implicated the control of flagellar reversals and production of an exopolysaccharide (EPS) as factors in the establishment of a stable association with the substratum and swarming motility. Here we present evidence that SadC (PA4332), an inner membrane-localized diguanylate cyclase, plays a role in controlling these cellular functions. Deletion of the sadC gene results in a strain that is defective in biofilm formation and a hyperswarmer, while multicopy expression of this gene promotes sessility. A DeltasadC mutant was additionally found to be deficient in EPS production and display altered reversal behavior while swimming in high-viscosity medium, two behaviors proposed to influence biofilm formation and swarming motility. Epistasis analysis suggests that the sadC gene is part of a genetic pathway that allows for the concomitant regulation of these aspects of P. aeruginosa surface behavior. We propose that SadC and the phosphodiesterase BifA (S. L. Kuchma et al., J. Bacteriol. 189:8165-8178, 2007), via modulating levels of the signaling molecule cyclic-di-GMP, coregulate swarming motility and biofilm formation as P. aeruginosa transitions from a planktonic to a surface-associated lifestyle.

Inverse regulation of biofilm formation and swarming motility by Pseudomonas aeruginosa PA14.

We previously reported that SadB, a protein of unknown function, is required for an early step in biofilm formation by the opportunistic pathogen Pseudomonas aeruginosa. Here we report that a mutation in sadB also results in increased swarming compared to the wild-type strain. Our data are consistent with a model in which SadB inversely regulates biofilm formation and swarming motility via its ability both to modulate flagellar reversals in a viscosity-dependent fashion and to influence the production of the Pel exopolysaccharide. We also show that SadB is required to properly modulate flagellar reversal rates via chemotaxis cluster IV (CheIV cluster). Mutational analyses of two components of the CheIV cluster, the methyl-accepting chemotaxis protein PilJ and the PilJ demethylase ChpB, support a model wherein this chemotaxis cluster participates in the inverse regulation of biofilm formation and swarming motility. Epistasis analysis indicates that SadB functions upstream of the CheIV cluster. We propose that P. aeruginosa utilizes a SadB-dependent, chemotaxis-like regulatory pathway to inversely regulate two key surface behaviors, biofilm formation and swarming motility.

Growing and analyzing static biofilms.

Many bacteria can exist as surface-attached aggregations known as biofilms. Presented in this unit are several approaches for the study of these communities. The focus here is on static biofilm systems, which are particularly useful for examination of the early stages of biofilm formation, including initial adherence to the surface and microcolony formation. Furthermore, most of the techniques presented are easily adapted to the study of biofilms under a variety of conditions and are suitable for either small- or relatively large-scale studies. Unlike assays involving continuous-flow systems, the static biofilm assays described here require very little specialized equipment and are relatively simple to execute. In addition, these static biofilm systems allow analysis of biofilm formation with a variety of readouts, including microscopy of live cells, macroscopic visualization of stained bacteria, and viability counts. Used individually or in combination, these assays provide useful means for the study of biofilms.