Molecular basis of usher pore gating in Escherichia coli pilus biogenesis

Abstract

Extracellular fibers called chaperone-usher pathway pili are critical virulence factors in a wide range of Gram-negative pathogenic bacteria that facilitate binding and invasion into host tissues and mediate biofilm formation. Chaperone-usher pathway ushers, which catalyze pilus assembly, contain five functional domains: a 24-stranded transmembrane β-barrel translocation domain (TD), a β-sandwich plug domain (PLUG), an N-terminal periplasmic domain, and two Cterminal periplasmic domains (CTD1 and 2). Pore gating occurs by a mechanism whereby the PLUG resides stably within the TD pore when the usher is inactive and then upon activation is translocated into the periplasmic space, where it functions in pilus assembly. Using antibiotic sensitivity and electrophysiology experiments, a single salt bridge was shown to function in maintaining the PLUG in the TD channel of the P pilus usher PapC, and a loop between the 12th and 13th beta strands of the TD (β12-13 loop) was found to facilitate pore opening. Mutation of the β12-13 loop resulted in a closed PapC pore, which was unable to efficiently mediate pilus assembly. Deletion of the PapH terminator/anchor resulted in increased OM permeability, suggesting a role for the proper anchoring of pili in retaining OM integrity. Further, we introduced cysteine residues in the PLUG and N-terminal periplasmic domains that resulted in a FimD usher with a greater propensity to exist in an open conformation, resulting in increased OM permeability but no loss in type 1 pilus assembly. These studies provide insights into the molecular basis of usher pore gating and its roles in pilus biogenesis and OM permeability.outer membrane usher | macromolecular assembly | bacterial pathogenesis

Electrostatic networks control plug stabilization in the PapC usher

“…Also within the translocation domain are a hairpin loop between β-strands 5 and 6 (the β5-6 hairpin), which dips toward the barrel lumen, and the only α-helix in the translocation domain, which caps the β5-6 hairpin from the extracellular side. These two elements are believed to confer stability to the surrounding region, particularly to the plug domain, as their removal causes increased pore permeability (Volkan et al, 2013), channel opening (Mapingire et al, 2009) and decreased interactions between conserved residues (Farabella et al, 2014). Specific communities of amino acids in the α-helix and the β5-6 hairpin were proposed to function in the transmission of a putative allosteric signal that triggers plug displacement (Farabella et al, 2014).…”

Comparative Transcriptome Analysis of Vibrio splendidus JZ6 Reveals the Mechanism of Its Pathogenicity at Low Temperatures

“…The pilus is a critical virulence factor in mediating the attachment and invasion of bacteria into host cells, while the flagellum is mainly responsible for cell motility. It has been reported that some pilus proteins could negatively regulate the expression of flagellar proteins and the process of flagellar biosynthesis (40)(41)(42). In Vibrio species, FlhG is a typical pilus assembly protein with a function as an antiactivator for flagellar biosynthesis, and its high expression would impact the synthesis of the flagellum (43).…”

Innovative Solutions to Sticky Situations: Antiadhesive Strategies for Treating Bacterial Infections

“…After translocation into the periplasmic space, the PD mediates a high-affinity interaction with the NTD of the usher (41, 44). Thus, the PD gates the translocation domain such that, in the absence of pili, the PD prevents large molecules from flowing freely across the outer membrane (45). The PD, NTD, CTD1, and CTD2 work together in the assembly function of this molecular machine.…”

“…(45,(47)(48)(49). Pilus DSE occurs at the usher when the chaperone is displaced, and an incoming subunit's N-terminal extension zips into the previously chaperone-bound groove of a nascently incorporated subunit at the growing terminus of the pilus (Fig.…”