The structure of the Salmonella typhimurium type III secretion system needle shows divergence from the flagellar system

Abstract

The type III secretion system (T3SS) is essential for the infectivity of many pathogenic Gram-negative bacteria. The T3SS contains proteins that form a channel in the inner and outer bacterial membranes, as well as an extracellular needle that is used for transporting and injecting effector proteins into a host cell. The homology between the T3SS and the bacterial flagellar system has been firmly established, based upon both sequence similarities between respective proteins in the two systems and the structural homology of higher-order assemblies. It has previously been shown that the Shigella flexneri needle has a helical symmetry of approximately 5.6 subunits/turn, which is quite similar to that of the most intensively studied flagellar filament (from Salmonella typhimurium), which has approximately 5.5 subunits/turn. We now show that the Sa. typhimurium needle, expected by homology arguments to be more similar to the Sa. typhimurium flagellar filament than is the needle from Shigella, actually has approximately 6.3 subunits/turn. It is not currently understood how host cell contact, made at the tip of the needle, is communicated to the secretory system at the base. In contrast to the Sa. typhimurium flagellar filament, which shows a nearly crystalline order, the Sa. typhimurium needle has a highly variable symmetry, which could be used to transmit information about host cell contact.

Fig. 1

(a) Cryo-EM image of bundles of needles. (b) averaged power spectrum generated from 14 non-overlapping boxes (512×512 px, 5.9 Å/px) taken from bundles such as in (a). (c) STEM mass histogram, with a gaussian curve fitted. (d) negatively stained needles. (e) averaged negative stain power spectrum from 259 non-overlapping boxes, each 480 px long (1.33 Å/px). (f) overall IHRSR three-dimensional negative stain reconstruction derived from 5,420 segments (120 px long, 5.32 Å/px), filtered to 24 Å resolution. This reconstruction converged to a helical symmetry of 57° rotation and an axial rise of 4.2 Å, which corresponds to 6.3 subunits per turn of a 26.5 Å pitch helix. (g) Cryo-EM of isolated filaments. (h) averaged cryo-EM power spectrum from 850 non-overlapping boxes, each 480 px long (1.33 Å/px). (i) overall IHRSR cryo-EM three-dimensional reconstruction derived from 22,834 segments at (120 px long, 2.66 Å/px), filtered to 24 Å resolution. This converged to the same symmetry as the negative stain reconstruction (57°/4.2 Å).

Fig. 2

(a) reconstruction of the frozen-hydrated filaments filtered to 18 Å resolution, using 9,366 segments from the total 22,834 segments. The sorting was based on the variable twist and axial rise while keeping the n=+7 layer line fixed at 1/(38 Å). This class from the center of the distribution yielded a symmetry of 57°/4.2 Å). Residues 18 to 60 (blue ribbons) from the solution structure of the S. typhymurium needle subunit (pdb entry 2JOW) were manually docked into the reconstruction. (b) side view of the atomic model of S. flexneri T3SS needle (pdb entry 2V6L), with one of the protomers shown in green, and (c) top view of this model. (d) the atomic model of the S. flexneri T3SS needle in (b) cut along the plane perpendicular to the helical axis (black line). One of the protomers (green) was aligned with residues 18–60 of the S. typhymurium needle subunit (blue), positioned as fit to the reconstruction shown in (a). Despite the difference in symmetry, the paired helices of the needle protomers from both bacteria are located at the same radius and have the same orientation. (e) the atomic model of S. flexneri T3SS needle in (c) cut along the plane parallel to the helical axis (black line). It can be seen in this view, as well, that the paired helices in the models for both bacteria are located at the same radius and have the same orientation. (f) The reconstruction in (a) cut along the plane parallel to the helical axis. When the subunit from the atomic model of the S. flexneri needle (green) is docked into the reconstruction of the Salmonella needle, the N-terminal part of it sticks out from the volume (red arrowhead).