A systems approach to analyzing bacterial glycans and glycan-binding proteins

textCarbohydrates are prominent and accessible polymers found on the cell-surface and represent the first interface with the extracellular environment. The enormous diversity of cell-surface carbohydrates is recognized as a high-density coding language that mediates molecular recognition between cells. In the case of bacteria-host interactions, both bacterial glycans and glycan-binding proteins (lectins) are important in fine-tuning the biological response. The nature of these interactions is unique, with affinity and specificity determined by multiple low-affinity binding events between lectins and carbohydrates. Thus, the analysis of lectin-glycan interactions on a systems level is an important and necessary step towards understanding the role of glycosylation in bacteria-host communication. This dissertation describes work pioneering the application of lectin microarrays to the analysis of bacterial glycosylation. Lectin microarrays represent a new glycomics approach for the high-throughput analysis of bacterial glycans. This approach is non-destructive and allows rapid profiling of intact bacterial surfaces. Lectin microarray analysis of fluorescently-labeled bacteria provides a visual binding pattern representing the glycosylation profile of the cell-surface. These lectin-binding profiles can be compared and used to distinguish bacterial strains based on glycosylation. In the course of examining a pathogenic strain, we discover a major limitation of the lectin microarray technology. The majority of plant lectins present on the array are post-translationally modified with carbohydrates, leading to potential false positives due to binding by bacterial lectins. Unexpectedly, this limitation provides the rationale for creating a recombinant lectin panel using bacteria as the source. We define the glycan-binding specificity of the bacterial lectins using both carbohydrate microarrays and ELISA, leading to the discovery of new specificities. The recombinant panel is then used to create the first recombinant lectin microarray, demonstrating that bacterial lectins in an array format are capable of analyzing glycosylated samples. Therefore, the studies on bacterial glycans have led to the development of new tools for carbohydrate analysis, a necessary step towards understanding the structure-function relationship of carbohydrates in nature.Biochemistr