CryoEM study of the adsorption of a phage to a type IV pili

Abstract

Xanthomonas citri pv. citri is the causative agent of citrus canker disease around the world, with important economic impacts in agriculture. This phytopathogen employs diverse mechanisms to colonize its host, survive in hostile environments, and acquire nutrients, including secretion systems, outer membrane vesicles, and extracellular appendages such as type IV pili (T4P). T4P are associated to "twitching" surface motility, biofilm formation, and adhesion to its host leaves. On the other hand, these structures may be used as receptors for bacteriophages, such as the podovírus ¦Xacm4-11, capable of infecting and lysing X. citri cells. The initial step of this infection is the recognition of the host by the interaction of the phage tail with the pilus, which is then retracted towards the cell surface. We plan to apply cryoelectron microscopy (cryoEM) to study this complex composed by different phage tail proteins attached to their receptor filament. To this end, T4P isolated from X. citri cells will be combined with samples containing the tail protein complex prepared by different approaches. First, the capsid may be removed by an EDTA treatment to release the tails. Alternatively, the proteins that assemble this pilus-interacting complex, already identified in the phage genome, may be cloned and heterologously expressed to produce of a purer sample with higher yields. In the end, we expect to obtain a structural model of this complex, providing crucial insights in the primordial step of the infection that may be applied in the future for phage engineering for microbial control. (AU)