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. 2006 Aug 15;103(33):12529-33.
doi: 10.1073/pnas.0602689103. Epub 2006 Aug 3.

Molecular model of a type III secretion system needle: Implications for host-cell sensing

Janet E Deane  1 , Pietro Roversi, Frank S Cordes, Steven Johnson, Roma Kenjale, Sarah Daniell, Frank Booy, William D Picking, Wendy L Picking, Ariel J Blocker, Susan M Lea
Affiliations

Molecular model of a type III secretion system needle: Implications for host-cell sensing

Janet E Deane et al. Proc Natl Acad Sci U S A. .

Abstract

Type III secretion systems are essential virulence determinants for many Gram-negative bacterial pathogens. The type III secretion system consists of cytoplasmic, transmembrane, and extracellular domains. The extracellular domain is a hollow needle protruding above the bacterial surface and is held within a basal body that traverses both bacterial membranes. Effector proteins are translocated, via this external needle, directly into host cells, where they subvert normal cell functions to aid infection. Physical contact with host cells initiates secretion and leads to formation of a pore, thought to be contiguous with the needle channel, in the host-cell membrane. Here, we report the crystal structure of the Shigella flexneri needle subunit MxiH and a complete model for the needle assembly built into our three-dimensional EM reconstruction. The model, combined with mutagenesis data, reveals that signaling of host-cell contact is relayed through the needle via intersubunit contacts and suggests a mode of binding for a tip complex.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Crystal structure of the MxiH monomer and comparison with related proteins. (A) Sequence of full-length MxiH and the truncated, His-tagged construct used in this study (different residues are highlighted in orange). Residues for which mutagenesis data are discussed are colored according to the following proposed role: signaling (green) and tip interaction (blue). (B) Ribbon diagram of MxiH molecule A colored N (blue) to C (red) termini. Views rotated by 90° about the long axis of the molecule are shown. The head (residues 26–57) and tail are labeled. (C) SHARP/SOLOMON solvent-flattened electron density for the PSNP loop is shown at 1.0 σ. The final model for this region of molecule A is shown as a stick representation colored by atom type (C, green; O, red; N, blue). (D) Change in the relative domain arrangement of MxiH molecules A (red) and B (blue). Molecules are aligned over the tail region. (E) Ribbon diagrams of the D0 domain of flagellin (19) (cyan), the ordered (chaperone-bound) region of EspA (20) (orange), and YscE (21) (magenta). Figures were prepared by using PyMOL (31).
Fig. 2.
Fig. 2.
Docking of the atomic model of MxiH into the EM density of the Shigella T3SS needle. (A) Molecule A of MxiH (ribbon) with the modeled N-terminal helix (cylinder) is shown as two views rotated by 90° about the long axis of the molecule. (B) End-on view of a 40-Å-thick section of the assembled needle. Each MxiH monomer is shown as in A and colored differently, starting from red and circling the needle to purple. EM density is shown as a blue mesh. (C) Stereo diagram of the side view of the assembled needle, colored as for B. Note that B and C are not shown at the same scale, and the needle assembly has an exterior diameter of ≈70 Å (16).
Fig. 3.
Fig. 3.
Interactions between subunits in the assembled needle. (A) A monomer of MxiH (ribbon diagram, red) is surrounded by seven identical subunits (shown as colored surface representations) within the needle assembly (Upper). The C terminus of MxiH, magnified (Lower), with the five C-terminal residues colored yellow, makes direct contact with three surrounding monomers (shown as ribbon diagrams in blue, green, and purple). (B) The tail region of MxiH (blue for the monomer above and red for the central monomer) contacts the head region of the monomer below (light red for the central monomer and light blue for the monomer below). Mutations that cause severe defects in hemolysis/invasion despite normal needle assembly (11) are shown in gray (D73A, D75A, I78A, I79A, and Q80A) (Upper). The magnification of the interface (as a ribbon diagram) (Lower) is rotated to show the patch of residues (black, L30, L34, A38, and Y50) on the head (light blue) that contact the residues listed above (colored gray on the red ribbon diagram).
Fig. 4.
Fig. 4.
Characterization of the putative tip-interaction interface of a T3SS needle. (A) Surface representation of the side view of the T3SS needle with each monomer colored differently, starting from red and circling the needle to purple. Residues likely to effect interactions with the tip complex are highlighted as follows: P44 and Q51 in Shigella (white) and the equivalent of D46 in Yersinia (gray). (B) View and coloring as for A, with residues conserved between Shigella MxiH and Yersinia YscF highlighted. Residues conserved in the head domain: L37, P41, N43, P44, L46, L47, A48, and Q51 (white); in the tail domain, N62, S65, V68, K72, D73, I78, Q80, and F82 (gray) are shown for the top circle of the needle. (C Left) Ribbon diagram of LcrV (30) colored N (blue) to C (red) termini. (C Right) Overlay of the C-terminal helices of MxiH (red, residues 45–75) and LcrV (blue, residues 287–317), with all but the overlaid region made transparent to aid visualization. (D) Model of an LcrV tip complex (surface representation, gray) onto the tip of a T3SS needle.
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