Understanding the mechanism of action of a metal-independent bacterial glycosyltransferase for potential use in the treatment of infectious disease

Humans have developed mechanisms to resist viral and bacterial pathogens through changes and individual variations in carbohydrate structures that are synthesized by a family of enzymes (glycosyltranferases). This project will investigate members of this family from bacteria and mammals, to determine why they differ in requiring (mammals) and not requiring (bacteria) metal ions for activity. The carbohydrates produced by the enzymes in some intestinal bacteria help us resist infections by blocking toxins and viruses and may lead to bacteria‑based therapies.

The carbohydrates (glycans) that cover all cell surfaces mediate cellular interactions, signalling, infectious processes and immunity. The members of 96 families of glycosyltransferases (GTs) mold cellular glycan landscapes, but we have limited knowledge of their structures and mechanisms. Humans have 4 genes for family 6 GTs (GT6); 3 encode inactive enzymes while the fourth is polymorphic, generating the intense phenotypic variation of the ABO blood group antigens. The loss and variation in GT6 activities impact susceptibility and resistance to pathogenic viruses and bacterial toxins, through mechanisms that are partly linked to co‑evolution of GT6 in humans and gastrointestinal (GI) microbiomes. Specific examples include: (a) the loss or exposure of receptors for pathogens, (b) production of antibodies targeting missing GT6 glycans that protect against enveloped viruses carrying glycans from another species or an individual with a different array of GT6; these antibodies are elicited through exposure to GT6 glycans displayed by GI bacteria, (c) binding and blocking viral pathogens and toxins that use GT6 glycans as receptors, by GI bacteria displaying the same glycan. Examples of pathogenic agents that bind GT6 glycans are viruses that are major causes of childhood gastroenteritis [rotavirus (RV) and norovirus (NV)] and toxins from a bacterium [Clostridium difficile (CD)] responsible for nosocomial GI infections that are a major burden to the health care system. We suggest that bacteria displaying GT6 glycans may provide a novel approach for treating viral and CD infections (CDI), reflected in the success of fecal transplants for treating intransigent GI infections.

In an continuing highly productive collaboration with Professor Keith Brew (FAU, USA) which has so far resulted in 19 publications (and 38 PDB entries) we have investigated a GT6 from Bacteroides ovatus (BoGT6a) uncovering its close similarity in structure and function to mammalian GT6 along with a profound functional difference in metal‑dependence. While mammalian GT6 require an essential divalent metal cofactor that mediates interactions with an UDP‑sugar substrate, BoGT6a is metal‑independent, reflecting substitutions in a metal‑binding Asp‑x‑Asp (DxD) sequence motif. Metal‑independence is shared by GT6s from other Gram‑negative GI residents, but other bacteria, including an emerging human pathogen, Parachlamydia acanthamoebae (Pa), have metal‑dependent GT6.

The goal of this PhD project is to understand better the structure, mechanisms and specificity of bacterial GT6, because of their roles in resistance to infectious diseases and to explore the application of specific GI bacteria carrying GT6 for treating CDI.

Training:
The PhD project would involve structural studies on BoGT native protein and mutants with several substrates and substrate analogues in order to understand the catalytic mechanism and specificity. During the study, the student will receive training in protein expression, purification, structural studies using X-ray crystallography, protein engineering, structure analysis using computational and biophysical methods.

Collaboration:
The research project is a collaborative effort between Professor K. Ravi Acharya (University of Bath, UK) and Professor Keith Brew (Florida Atlantic University, Boca Raton, USA).