Structure of Enterohemorrhagic Escherichia coli O157:H7 Intimin Virulence Factor Bound to Nanobodies

Abstract

Enterohemorrhagic E. coli O157:H7 (EHEC) is a food and water-born pathogen that presents a significant risk to human health worldwide. EHEC naturally resides in the intestinal tract of cattle as normal flora but can be transmitted to humans when contaminated food is ingested. Once EHEC invades and colonizes the human intestinal tract, it secretes Shiga-like toxins causing serious and potentially fatal gastrointestinal diseases. Unlike most foodborne bacterial diseases, antibiotics are not recommended for treatment of EHEC as antibiotics can induce enterotoxin release, increasing the risk of hemolytic uremic syndrome. There is currently no effective treatment or vaccine against enterohemorrhagic E. coli. EHEC pathogenicity involves intimin, a virulence factor which mediates bacterial colonization of the host GI tract. Due to its critical role in EHEC pathogenesis, intimin has attracted attention as an antibacterial drug target. Inhibiting the interaction of intimin with its cognate bacterial secreted receptor Tir could neutralize bacterial colonization of the gastrointestinal tract. One novel solution for treating EHEC infection comes from a unique class of antibodies known as nanobodies. Nanobodies are the antigen binding domain of heavy chain antibodies produced by the Camelid family. Intimin specific nanobodies were generated by immunizing a llama followed by phage display and selection for high affinity intimin binding nanobodies. The resultant intimin-specific nanobodies neutralized EHEC in vitro. We hypothesize that nanobodies specifically bind the Tir-binding domain of intimin in order to neutralizing EHEC infection. The goal of this research project is to determine the complex crystal structure of the nanobodies bound to intimin. The structure of nanobodies-intimin would show their protein-protein interaction binding site revealing key amino acids involved in binding. These residues could also be involved in the binding of intimin to its natural ligand, Tir. Thus, revealing such residues could form the bases for identifying and developing novel therapeutics and biosensors for treating and preventing EHEC pathogenesis.We show through size exclusion chromatography that nanobodies bind intimin and form stable complexes. We have set up crystal trails of five nanobodies (Int1, Int2, Int3, Int4, and IntN1) complexed to intimin. Two of the five complexed proteins, Int2-intimin and Int3-intimn formed crystals. The crystal condition for the two protein complexes Int2 and Int3 bound to intimin were optimized by varying the concentration of precipitants and pH range. Crystals of the protein complexes were sent to the Canadian Light Source for X-ray diffraction data collection. Diffraction measurements of Int2-intimin and Int3-intimin complexes were collected and processed in space groups P1 and P222, respectively. Currently, we are analyzing the structure by molecular replacement.