Figure 1.
Proposed topologies for T3SS needle proteins in the needle assembly.
A, Model of Shigella flexneri T3SS needle based on a 7.7-Å cryoEM map, according to Ref. [19], PDB ID: 3J0R. The structure of the MxiH subunit protein contains an α-helix (S2-A38), a loop (K39-P44), an α-helix (Q45-Y50), a 14-residue-long β-hairpin (Q51–Q64) and an α-helix (S65-R83) with a kink at D73. The N-terminus faces inward to the lumen of the needle. B, Solid-state NMR atomic model of the Salmonella typhimurium T3SS needle according to Ref. [20], PDB ID: 2LPZ. The structure of the PrgI subunit protein comprises a rigid-extended conformation (T3-Y8), an α-helix (L9-A35) with a kink at N22, a loop (A36-P41), and an α-helix (A42-R80). The N-terminus is located at the surface of the needle. Top views of the needle assemblies are shown in the top corners. The structures in the bottom corners present the location of highly conserved residues that were identified from multiple sequence alignment (Fig. 2). The atoms of conserved amino acids are represented as spheres and are colored respectively blue or red depending if their amino acid belongs to the N- or the C-terminal region. Conserved residues are lining the lumen of the needle in the Salmonella model but are exposed to the extracellular milieu in the Shigella model.
Figure 2.
Multiple sequence alignment of T3SS needle proteins.
Conserved residues are colored according to amino acid type following the Clustal X color scheme [75]. Highly conserved residues (≥12 identical amino acids in both clusters) are highlighted with a star (N-terminal region: blue; C-terminal region: red). The numbering of residues corresponds to the sequence of MxiH. Database accession identifiers for the primary sequences are given in supplementary Table S3 in Text S1.
Figure 3.
Solid-state NMR spectroscopy of Shigella flexneri MxiH needles.
A, Transmission electron microscopy images of WT Shigella flexneri needles used for solid-state NMR measurements. B, 1D 13C cross-polarization spectra of [uniform-13C6]glucose (Glc)-labeled (black), [1-13C]Glc-labeled (green) and [2-13C]Glc-labeled (magenta) needles. Representative line-widths are indicated for two resonances (T23Cβ and N81Cα) for the sparsely-labeled samples. The free-induction decay of the signal was recorded for a total of 8192 scans, with signal remaining after an acquisition time of 60 ms. No apodization function was employed in the processing. C, N-Cα spectrum of [2-13C]Glc-labeled needles. The 2D spectrum correlates backbone amide nitrogen frequencies (δ115N) to backbone Cα frequencies (δ213C). N-Cα cross-peaks are numbered according to the MxiH amino acid sequence and N-Cδ cross-peaks of prolines are indicated. Unmarked cross-peaks correspond to sequential correlations. Spectra were recorded at a magnetic field of 21.1 T (850 MHz 1H resonance frequency) at 5.5°C.
Figure 4.
Identification of secondary structure elements.
A, Secondary structure elements identified in the cryoEM model of Fujii et al.; adapted from Fig. 1e in Ref. [19] where H1, H2, and H3 indicate the position of α-helices and “prot” that of the β-hairpin between H2 and H3. B, Secondary structure elements of MxiH needles determined by solid-state NMR (this study). C, Secondary chemical shifts (ΔδCα – ΔδCβ) of the Shigella flexneri MxiH protein in the assembled needle. Regions which do not present α-helical propensity are highlighted. D, Secondary chemical shifts of the Salmonella tympimurium PrgI protein in the assembled needle taken from Ref. [37].
Figure 5.
Structural model of the T3SS Shigella needle.
A, Top and B, side views of the T3SS needle assembly of Shigella flexneri. The homology model of the 29-subunit MxiH needle assembly generated by Rosetta modeling (cartoon representation) shows a correlation of 0.66 with the 7.7-Å cryoEM density map (grey surface). Using the nomenclature of Fujii et al., the EM density regions H1, H2 and H3 are indicated for the central subunit (i), as well as the kink in region H3. The first 11 N-terminal residues are not depicted due to the poor sequence homology of the rigid-extended segment (Fig. 2); however the protrusion in the EM map at subunit (i), labeled prot. and highlighted by a purple dashed contour trace, may be well explained by the N-terminus of subunit (i+5). The central subunit (i), colored as in Fig. 1B, forms a lateral interface with subunits (i±5), in red, and subunits (i±6), in dark blue. The axial interface is formed with subunits (i±11) shown in pink.
Figure 6.
Localization of the MxiH subunit N-terminus in vitro and in vivo.
A–C, Immunogold labeling of recombinant Shigella flexneri MxiH needles using a monoclonal Anti-His tag antibody. A, WT MxiH needles B, needles polymerized from N-terminal His tag fusion construct, mxiH-N(His), C, mxiH-N(His) needles labeled without primary Anti-His tag antibody. In B, some gold particles are indicated by arrows. Proteins expressed from a C-terminal His tag fusion construct did not polymerize. D, Effector protein secretion assay of Shigella strains used in Fig. E–G by addition of Congo Red (CR) followed by western blot analysis against IpaB, IpaC and DnaK. E–G, Non-polar Shigella mxiH-knockout cells (ΔmxiH) expressing E, wild-type mxiH, F, mxiH with N-terminal Strep-tag, mxiH-N(Strep) or G, MxiH with C-terminal Strep-tag, mxiH-C(Strep). In E and F, needles are indicated by arrows and two needles complexes are highlighted.
Table 1.
Structural statistics of different needle models.