Glycans in infection: challenge and opportunity

Glycoconjugate vaccines are an important and successful means for prevention of infectious disease, including pneumoniae, meningitidis and salmonellosis. Understanding at atomic level the binding between microbial carbohydrates and specific functional monoclonal antibodies can direct vaccine design, particularly when synthetic carbohydrates are used. Recently, Dr Adamo has applied structural studies to identify the minimal epitope of group B Streptococcus type III polysaccharide (GBS PSIII). GBS PSIII is a leading cause of invasive infections in pregnant women, newborns, and elderly people, and the capsule is a major virulence factor targeted for vaccine development. GBS PSIII epitope has been historically considered the prototype of a complex conformational carbohydrate epitope. Through an integrated approach based on competitive ELISA/Surface Plasmon Resonance/Saturation Transfer NMR/X-ray he has elucidated a structural epitope consisting of a hexasaccharide constituted of a single repeating unit, and the glucosamine moiety of the next consecutive repeat unit. Based on this data, a conjugate vaccine from the short hexasaccharide epitope was prepared and elicited in mice functional antibodies comparably to a polysaccharide conjugate. Likewise, structural studies carried out for serotypes Ia and Ib showed that the polymeric nature of the polysaccharide can strongly impact epitope presentation.

A similar approach allowed the structural epitope of Neisseria meningitidis serogroup A and X, which are responsible for epidemic meningitis in the sub-Saharan region of Africa known as meningitis belt, to be mapped. Studies are ongoing to gain this type of information also on structurally similar sialylated W and Y polysaccharides. Structural data can be exploited to guide synthetic carbohydrate vaccine design. 

Staphylococcus aureus is a commensal bacterium that colonises about 30% of the population without any harmful effects. However, S. aureus also represents a major public health concern, due to its ability to cause a wide range of clinical infections combined with the alarming development of antibiotic resistance, which limits treatment options. One of the prime targets for the development of new therapeutic interventions against S. aureus are the wall teichoic acids (WTAs). WTAs are cell wall expressed glycopolymers that are critical for bacterial physiology, antibiotic resistance and colonisation. S. aureus WTAs display structural variation through glycosylation, resulting in three main glycotypes, which differently impact host-pathogen interaction. Through a multidisciplinary approach, including bacterial genetics, microbiology, immunology, and glycobiology, my group aims to unravel the molecular interplay between WTA glycotypes and host immunity to advance the development of new antimicrobial therapies. This presentation will highlight our findings related to the interaction of WTA glycotypes with innate receptors and human antibodies, which are critical components for front-line defense at barrier sites and long-term protection, respectively. 

In this talk Dr van Kasteren will focus on his recent work looking at carbohydrate-lectin interactions at the single molecule level on living cells. These interactions play an important role in a wide diversity of biological processes, but are hard to quantify due to the low affinities of the interactions and the overlapping specificities of different lectins. To study this in more detail, particularly on immune cells, he has developed a point-accumulation in nanoscale topography (PAINT)-based super-resolution microscopy method that can be used to capture the weak glycan-lectin interactions at the single molecule level in living cells (glyco-PAINT). This has allowed Dr van Kasteren to obtain on rates and off rates for lectins, and he is now using this to dissect the role of glycan interactions on downstream immune activation events. Overall, Glyco-PAINT represents a powerful approach to study weak glycan-lectin interactions on the surface of living cells that can be potentially extended to a variety of lectin-sugar interactions.

Streptococcus agalactiae (Group B Strep, GBS), is one of the most common perinatal pathogens responsible for causing premature birth. Current therapeutic techniques aimed to ameliorate invasive GBS infections can result in complications in both the child and mother. To this end, the need for novel therapeutic options is urgent. Human milk oligosaccharides (HMOs) have been previously shown to possess antiadhesive and antimicrobial properties. To interrogate these characteristics, Professor Townsend examined HMO-mediated outcomes in both in vivo and ex vivo models of GBS infection using a murine model of ascending GBS infection, an EpiVaginalTM human organoid tissue model, and ex vivo human gestational membranes. Treatment with HMOs resulted in diminished adverse pregnancy outcomes, decreased GBS adherence to gestational tissues, decreased colonisation within the reproductive tract, and reduced in proinflammatory immune responses to GBS infection.

All most all eukaryotic cell surface and secreted proteins are modified by covalently-linked glycans which are essential mediators of biological processes such as protein folding, cell signalling, fertilisation, embryogenesis, and the proliferation of cells and their organisation into specific tissues. Overwhelming data supports the relevance of glycosylation in pathogen recognition, inflammation, innate immune responses, the development of autoimmune diseases, and cancer. It has, however, been difficult to explore biological properties of individual glycans because these bio-molecules are not readily available. To address this challenge, we have developed chemo-enzymatic methodologies that make it possible to prepare large libraries of highly complex glycans found on cells of interest. The synthetic approaches were employed to prepare a collection of glycans found on the upper airway of humans that were printed as a microarray to probe receptor specificities of several respiratory viruses. To validate the array data, cell surface engineering strategies were developed to place synthetic glycans on the surface of cells for gain of function studies. The technological platform made it possible to examine the evolution of receptor specificities of influenza H3N2 viruses and beta-corona viruses. Furthermore, it was employed to determine receptor specificities of Lassa virus. Knowledge of glycan receptor usage of zoonotic pathogens is making it possible to implement surveillance strategies to prevent future pandemics.

Glycosylation is one of the most structurally and functionally diverse co- and post-translational modifications in a cell. This process is characterised by the addition and removal of glycans, especially to proteins and lipids, which have important implications in several biological processes. In mammals, the repeated enzymatic addition of a sialic acid unit to underlying sialic acids by polysialyltransferases, including ST8Sia2, leads to the formation of a sugar polymer called polysialic acid (polySia). The functional relevance of polySia has been extensively demonstrated in the nervous system. However, the role of polysialylation in infection is still little explored. Previous reports have shown that Trypanosoma cruzi (T. cruzi), a flagellated parasite that causes Chagas disease, changes host glycoproteins' sialylation. To understand the role of host polySia during T. cruzi infection, Associate Professor Palmisano used a combination of in silico and experimental tools. We observed that T. cruzi reduces the expression of the ST8Sia2 and the polysialylation of target substrates. He also found that chemical inhibition of ST8Sia2 reduced the parasite load in host cells. These findings suggest a novel approach to interfere with parasite infections through modulation of host polysialylation.

Urinary tract infections (UTI) belong to the most common infections worldwide and are predominantly caused by uropathogenic E. coli (UPEC). UPEC initiate the infection cycle by adhering to high-mannose glycoproteins on urothelial cells through their lectin FimH. Preventing the initial adhesion by blocking FimH offers a promising alternative to the conventional antibiotic treatment, which has become increasingly inefficacious due to antibiotic resistance.

The FimH-mannose interaction is shear stress dependent, meaning that FimH mediates medium binding at low shear stress and stronger binding at high shear stress. In the urinary tract, the shear stress-induced switch from medium- to the high-affinity becomes an effective tool for UPEC to evade clearance by the bulk flow of urine. By studying bacterial binding in a cell motility assay, we were able to show that these variants were highly mobile on a mannosylated surface, allowing optimal colonisation in the bladder. However, by switching to the high-affinity conformation, they can resist upcoming shear stress and are thus protected from being washed out through urination. In contrast, a variant locked in the high-affinity state, which is often used for in vitro and in vivo studies, formed long-lived interactions with mannose also in the absence of shear stress, rendering bacteria immobile.

It becomes evident that dynamic FimH variants are pathophysiologically favoured and represent the dominant therapeutic target. To further elucidate this conformational issue, antagonists with high affinities to both affinity states were synthesised and broadly characterised by extended ITC studies as well as in a mouse model.