Abstract
Bacterial type II secretion systems (T2SSs) translocate virulence factors, toxins and enzymes across the cell outer membrane. Here we use negative stain and cryo-electron microscopy to reveal the core architecture of an assembled T2SS from the pathogen Klebsiella pneumoniae. We show that 7 proteins form a ~2.4 MDa complex that spans the cell envelope. The outer membrane complex includes the secretin PulD, with all domains modelled, and the pilotin PulS. The inner membrane assembly platform components PulC, PulE, PulL, PulM and PulN have a relative stoichiometric ratio of 2:1:1:1:1. The PulE ATPase, PulL and PulM combine to form a flexible hexameric hub. Symmetry mismatch between the outer membrane complex and assembly platform is overcome by PulC linkers spanning the periplasm, with PulC HR domains binding independently at the secretin base. Our results show that the T2SS has a highly dynamic modular architecture, with implication for pseudo-pilus assembly and substrate loading.
Highlights
Bacterial type II secretion systems (T2SSs) translocate virulence factors, toxins and enzymes across the cell outer membrane
Using affinity chromatography tags positioned on the cytoplasmic ATPase PulE and the periplasmic pilotin PulS, a complex containing seven components was purified by two successive pulldowns
In the absence of glutaraldehyde, the outer membrane complex (OMC) sometimes separated from the assembly platform (AP) and yielded top views, which confirmed PulD and PulS in 1:1 stoichiometric ratio[7] with C15 symmetry (Fig. 1c)
Summary
Bacterial type II secretion systems (T2SSs) translocate virulence factors, toxins and enzymes across the cell outer membrane. The outer membrane complex includes the secretin PulD, with all domains modelled, and the pilotin PulS. Symmetry mismatch between the outer membrane complex and assembly platform is overcome by PulC linkers spanning the periplasm, with PulC HR domains binding independently at the secretin base. 1234567890():,; The bacterial T2SS is found in human pathogens such as Acinetobacter baumannii, Chlamydia trachomatis, Escherichia coli and Vibrio cholerae[1] It secretes a broad repertoire of substrates including digestive enzymes and infective agents like the cholera and heat-labile (LT) toxins[2]. The protein GspD forms a 15-fold rotationally symmetric pore termed the secretin that inserts into the outer membrane (OM) and provides a conduit for substrate into the external environment. Combined with the polytopic membrane protein GspF12 and the ATPase GspE, these proteins form an assembly platform (AP) for the pseudo-pilus[13]. The homologous ATPases PilB and PilT in the closely related type IV pilus (T4P) system function as hexamers[15,16]
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