Glycoconjugates are positioned at the functional interface between hosts and microbes. Many bacterial pathogens of public health importance translocate protein toxins during infection, a process which plays an essential role in virulence. In particular, bacterial AB5 toxins, which are comprised of an enzymatic ‘A’ subunit(s) and a homopentamer of glycan receptor-binding ‘B’ subunit, play a crucial role in inducing disease signs and symptoms, some of which are life-threatening. Valuable tools, such as glycan microarrays and lectins, serve as the workhorses for determining the glycan-binding specificity of bacterial toxins, but they have limitations in further discriminating a glycan receptor(s) of physiological significance. Establishing a toolbox of customized, genetically engineered host cell lines can help overcome this limitation by discriminating functionally important glycan binder(s) in toxin-mediated virulence during bacterial infection. In the proposed study, we will generate a series of CRISPR/Cas9-assisted knockout cell lines of human intestinal epithelial cells possessing an impaired α2-3 or α2-6 sialosides biosynthesis. To increase the rigor of the proposed research outcome, we will also generate human intestinal epithelial cell lines that overexpress either functional or catalytically inactive enzymes at various stages in α2-3 a nd α 2-6 sialosides biosynthesis. We will use the engineered cells to test our hypothesis with the support of strong preliminary data that glycoconjugates are at the interface of co-adaptation (or co-evolution) of Salmonella and its secreted toxins. Glyco-tools to be developed in the proposed study will greatly help in demonstrating and highlighting the remarkable co-evolution of bacterial toxins for their specific need in Salmonella virulence/pathogenesis. These cell lines are expected to be highly beneficial to other investigators in the glycoscience community.