Type IV pili (T4P) in Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) represent the simplest of all pilus systems whereby all the proteins needed for pilus assembly are encoded within a single operon. These systems are unusual for their lack of a retraction ATPase and each operon encodes only one minor pilin instead of several. How can pili retract without a retraction ATPase? The only minor pilin in the operon is key to understanding retraction in these systems. V. cholerae T4P, the toxin co-regulated pilus (TCP), produces the minor pilin TcpB which shares N-terminal homology with the major pilin but possess a larger C-terminal domain. TCP can assemble in a ∆tcpB mutant but at much lower levels than the wild type strain. We show that the minor pilin is required for efficient pilus assembly and pilus-related functions. We quantified the stoichiometry between the major and minor pilins and established this ratio is critical to maintaining optimal pilus functions. We show by immunodetection and immunogold electron microscopy that the minor pilins are incorporated into surface-displayed pili at low levels. Moreover, we determined minor pilin incorporation at the base of an assembling filament is necessary to induce pilus retraction and this mechanism is mediated by a conserved glutamate at position 5. This residue is conserved in all major pilins and some minor pilins, and is hypothesized to be critical for stabilizing pilin:pilin interactions by charge complementarity during assembly when new pilins are incorporated at the base of the filament. We characterized the ETEC minor pilins and achieved similar findings. We propose a new model to explain pilus extension and retraction by which the minor pilins have dual functions in priming pilus assembly as the first subunit in assembly and inducing retraction by incorporating into assembling filaments to stall assembly and cause spontaneous depolymerization of the pilin subunits. Our results have implications in understanding pilus dynamics in the more complex T4P systems and in the related bacterial virulence factor, the type II secretion system.
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Thesis advisor: Craig, Lisa
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