BamA is required for autotransporter secretion

Abstract

Background: In Gram-negative bacteria, type Va and Vc autotransporters are proteins that contain both a secreted virulence factor (the "passenger" domain) and a β-barrel that aids its export. While it is known that the folding and insertion of the β-barrel domain utilize the β-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 β-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 β-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 β-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 β-barrel is more than that through the BamA β-barrel.

Conclusions: Secretion of autotransporters most likely occurs through an incompletely formed β-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.

Keywords: Autotransporters; Membrane proteins; Molecular dynamics; Secretion systems.

Figure 1:

Comparison between the original YadA-M (PDB: 2LME, [45]) structure (A) and the generated hairpin intermediate (B). The hairpin structure is constructed by stretching the linker and the passenger domain. Lipopolysaccharides in the outer leaflet are shown in pink (with sugar groups in licorice, polar headgroups in light pink and lipid tails in magenta). Phospholipids in the inner leaflet are shown in blue (with lipid tails in cyan and polar heads in dark blue). YadA β-strands are semi-transparent to clearly show the α-helical domain in panel A and the hairpin folding intermediate in panel B. The individual YadA monomers are shown in green, orange, and grey.

Figure 2:

Passenger domain hairpin folding intermediates result in β-barrel expansion during equilibration. (A) Channel coordinate vs. pore radius for the original structures and the hairpin structures in three different replicas. The dotted lines on the top and the bottom indicate the top and bottom positions of the β-barrel. The blue line is a channel profile for the crystal structure, and the red line is at the end of the simulation. (B) Channel coordinate position relative to the β-barrel of YadA. Throughout the 250 ns simulation, the pore radii of the WT replicas remain relatively unchanged, while those of the WT _hairpin replicas expand by 3 to 4 Å.

Figure 3:

The A354P mutation drives the YadA helices apart. (A) How the relative angles were measured. First, the principal axes of the C _α atoms of the helices are calculated (as represented by purple and cyan arrows). Then, the angle between the principal axes is measured. A larger angle indicates a greater separation of the helices. The C _α atoms of A354 are indicated in red. (B) Average of relative angles formed between the principal axes of each helix pair. The data shown are running averages taken over 10 ns. Black and red lines represent the WT and A354P, and the error bars are represented in blue and green, respectively.

Figure 4:

Comparison between the linker-stretched and linker-helix structures. (A) EspP structure [9] with the hairpin (red) made by stretching the linker and passenger domain. (B) The same structure but with only the passenger domain stretched to make a hairpin (red). Lipopolysaccharides in the outer leaflet are shown in pink (with sugar groups in licorice, polar headgroups in light pink and lipid tails in magenta), and phospholipids in the inner leaflet are shown in blue (with lipid tails in cyan and polar heads in dark blue). Some β-strands are semi-transparent to clearly show the differences between the hairpin domains.

Figure 5:

The linker-helix EspP structure either unfolds the linker region or disrupts its β-barrel to secrete its passenger domain. (A) The EspP β-barrel pore sizes for both the linker-stretched and linker-helix EspP structure for three different replicas. The black line represents the initial pore size. The blue, red and orange lines represent the final pore size for each simulation. While both the linker-stretched replicas and one linker-helix do not demonstrate a significant pore size change over time, the other linker-helix replicas show pore expansion over time. (B) The channel coordinate position relative to the EspP β-barrel. (C) The mid-secretion structures of linker-native - 1 and linker-native - 2 (in red and in orange, respectively). In the initial 50 ns of simulation, linker-native - 1 unfolds the folded linker region and preserves its β-barrel structure (red). Linker-native - 2 and linker-native - 3 keep its linker region folded while disrupting the β-barrel structure (orange).

Figure 6:

Comparison of force vs. time of linker-stretched hairpin structure and BamA β-barrel with EspP passenger domain. The black line shows the average force vs. time of the secretion of linker stretched hairpin structure from three replicas and the green line shows that of the BamA β-barrel with the EspP passenger domain from three replicas. Both lines are running average of the raw data taken over 10 ns. The error bars for linker-stretched hairpin structure and BamA β-barrel with EspP passenger domain are shown in blue and red, respectively.

Figure 7:

Preferred model for passenger domain transport. A passenger domain hairpin intermediate forms in a hybrida BamA-AT β-barrel (left panel), and secretion of the passenger domain occurs before the release and closure of the AT β-barrel domain to form the fully folded AT (right panel). The BamA and AT β-barrels are semi-transparent to clearly show the AT passenger domain.