BamA is required for autotransporter secretion.

BACKGROUND: In Gram-negative bacteria, type Va and Vc autotransporters are proteins that contain both a secreted virulence factor (the "passenger" domain) and a ß-barrel that aids its export. While it is known that the folding and insertion of the ß-barrel domain utilize the ß-barrel assembly machinery (BAM) complex, how the passenger domain is secreted and folded across the membrane remains to be determined. The hairpin model states that passenger domain secretion occurs independently through the fully-formed and membrane-inserted ß-barrel domain via a hairpin folding intermediate. In contrast, the BamA-assisted model states that the passenger domain is secreted through a hybrid of BamA, the essential subunit of the BAM complex, and the ß-barrel domain of the autotransporter. METHODS: To ascertain the models' plausibility, we have used molecular dynamics to simulate passenger domain secretion for two autotransporters, EspP and YadA. RESULTS: We observed that each protein's ß-barrel is unable to accommodate the secreting passenger domain in a hairpin configuration without major structural distortions. Additionally, the force required for secretion through EspP's ß-barrel is more than that through the BamA ß-barrel. CONCLUSIONS: Secretion of autotransporters most likely occurs through an incompletely formed ß-barrel domain of the autotransporter in conjunction with BamA. GENERAL SIGNIFICANCE: Secretion of virulence factors is a process used by practically all pathogenic Gram-negative bacteria. Understanding this process is a necessary step towards limiting their infectious capacity.

Presence of substrate aids lateral gate separation in LptD.

Lipopolysaccharides (LPS) provide the outer membrane (OM) of Gram-negative bacteria with a strong protective barrier. The periplasm-spanning Lpt machinery is responsible for the transport of LPS molecules across the periplasm, culminating in insertion by the outer-membrane proteins LptD and LptE. In order to elucidate the mechanisms of LPS insertion by LptDE, we performed over 14 microseconds of equilibrium molecular dynamics simulations. Bilayer-dependent differences in the fluctuations and secondary structure of LptD's extracellular loops are observed for a pure DMPE membrane vs. a model of the OM. Furthermore, LptD's periplasmic N-terminal domain is highly dynamic, which may help to maintain the integrity of the periplasm-spanning complex amidst relative motion of the inner-membrane and outer-membrane anchored domains. In addition, our simulations demonstrate that binding of LPS substrate activates a switching between the associated and dissociated states of two lumenal loops at the interface between the ß-barrel and the N-terminal domain as well as LptD's lateral gate on the microsecond timescale, neither of which is observed for the apo state. Placement of a substrate LPS molecule also causes an increase in the average separation of the LptD lateral gate strands and a lowering of the energetic barrier to lateral gate opening.