Protein sialylation is a post-translational modification produced by members of a family of sialyltransferases. We recently discovered that the resulting sialic acid linkages are involved in the regulation of glycoprotein abundance and function in an intrinsic homeostatic mechanism that is targeted by multiple pathogens. Environmental factors are dominant in the origins of the human Inflammatory Bowel Diseases (IBDs) and seasonal bacterial infections have been implicated. We therefore developed a mouse model of repeated human food poisoning comprised of recurrent low-titer non-lethal gastric infections of the Gram-negative bacterial pathogen Salmonella enterica Typhimurium (ST), a leading cause of human foodborne illness worldwide. In this unique model, the ST pathogen was rapidly cleared by the host, however a progressively severe and persistent colitis developed similar to Ulcerative Colitis (UC). We demonstrated that pathogenesis was linked to the disabling of a protective mechanism in the host involving the anti-inflammatory Intestinal Alkaline Phosphatase (IAP) glycoprotein enzyme produced by enterocytes. Recurrent ST infections resulted in Toll-like receptor-4 (Tlr4) induction of neuraminidase (Neu) activity and host Neu3 abundance with nascent IAP de-sialylation and endocytic degradation, thereby reducing IAP half-life, abundance, and function. IAP deficiency was similarly acquired in mice lacking the ST3Gal6 sialyltransferase resulting in spontaneous colitis. The disease mechanism in both cases was Tlr4-dependent and linked to reduced dephosphorylation and detoxification of the lipopolysaccharide-phosphate produced by commensal bacteria of the colon. In humans, similar modulation of IAP and Neu3 have been reported in colitis, and genetic deficiency of IAP causes colitis, supporting the rationale for ongoing clinical trials of IAP augmentation and neuraminidase inhibition. However, the key involvement of Neu3 remains to be established. Research proposed herein will directly test the role of host Neu3 in the onset and progression of colitis among Neu3-null mice. In addition, the possibility that other enteric Gram-negative pathogens similarly provoke IAP deficiency will be investigated with the development of other recurrent non-lethal gastric infection models using related Gram-negative enteric pathogens including the hypervirulent Salmonella enterica Choleraesuis serovar. We have recently measured elevated Neu activity and Neu3 protein abundance in the colon during recurrent ST infection associated with the de-sialylation of Mucin? 2 (Muc2), the major glycoprotein component of the protective mucin barrier. The mechanism of erosion of the mucin barrier in colitis remains unknown but plays a large role in pathogenesis. We have recently linked erosion of the mucin barrier to reductions of Muc2 protein sialylation, likely by ST3Gal6. Remarkably, Neu treatment increases Muc2 proteolysis with reduced Muc2 abundance. Research proposed herein will determine the role of Muc2 sialylation by ST3Gal6 in generating and maintaining the protective mucin barrier. Together these studies will provide important advances pertaining to the prevention and treatment of colitis.
Recent discoveries have revealed that the half-lives and functions of sialylated glycoproteins at the cell surface can be regulated by their rates of de-sialylation with increased protein age. The neuraminidases responsible thereby control glycoprotein abundance and function among various cell types including epithelial cells of the intestine where they may participate in the onset of colitis. The research proposed in this application will determine whether Neu3 induction is involved in the onset of colitis in a mouse model of recurrent human food poisoning and will explore the role of protein sialylation in maintenance and erosion of the mucin barrier during intestinal inflammation and disease.
