Unraveling Carbohydrate-Mediated Host-Pathogen Interactions: Surface Chemistry and Label-Free Biosensing

Abstract

Infectious diseases present a significant challenge to public health, and there is an urgent need to develop new strategies to address bacterial and viral pathogenesis. As the initial interaction between pathogen and the host cell is often mediated by a carbohydrate recognition event, a straightforward and effective strategy to prevent infection is to block host-pathogen protein-carbohydrate binding. By mimicking carbohydrate ligands present on the host mucosa, human milk glycans have been proven to function as soluble inhibitors capable of preventing a variety of pathogens from binding to the host. These glycans hold great promise to be employed as anti-infective prophylactics to combat infectious disease in both pediatric and adult populations. However, among the hundreds of carbohydrate structures found in human milk, it is challenging to establish the inhibitory relationship between discrete glycan entities and specific pathogens. To address this problem and accelerate the development of anti-infective glycans, we apply high-throughput and label-free biosensing platforms for characterizing the anti-viral activities of human milk glycans against norovirus and HIV. Surface plasmon resonance (SPR) and silicon photonic microring resonators are two types of label-free biosensors capable of quantifying binding events in real-time. However, the lack of efficient biofunctionalization strategies limits the application of these devices in characterizing carbohydrate-mediated interactions. In this study, reliable and versatile conjugation methods were developed to functionalize biosensors with carbohydrates. These methods utilize a homobifunctional linker, divinylsulfone (DVS), to conjugate natural sugars or carbohydrates modified with nucleophilic linkers (-NH2, -SH) onto the sensor surface. Compared to previous immobilization strategies, our DVS-based method greatly simplifies the process for carbohydrate immobilization and demonstrates high stability for biosensing. Next, the carbohydrate-functionalized biosensors were applied to study human milk glycan-norovirus interactions. We identified glycans that bind to two strains of norovirus and confirmed the activity of specific glycans against norovirus-host receptor interactions. Finally, the inhibitory mechanism of human milk glycosaminoglycans (GAGs) against HIV infection was investigated by SPR. The biosensing platforms and the binding studies in this dissertation advance our understanding of the anti-infective mechanism of human milk glycans and promote the application of glycan-based agents to prevent norovirus and HIV infection.