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. 2013 Jan;20(1):99-104.
doi: 10.1038/nsmb.2452. Epub 2012 Dec 9.

Architecture of the major component of the type III secretion system export apparatus

Patrizia Abrusci  1 , Marta Vergara-Irigaray, Steven Johnson, Morgan D Beeby, David R Hendrixson, Pietro Roversi, Miriam E Friede, Janet E Deane, Grant J Jensen, Christoph M Tang, Susan M Lea
Affiliations

Architecture of the major component of the type III secretion system export apparatus

Patrizia Abrusci et al. Nat Struct Mol Biol. 2013 Jan.

Abstract

Type III secretion systems (T3SSs) are bacterial membrane-embedded nanomachines designed to export specifically targeted proteins from the bacterial cytoplasm. Secretion through T3SS is governed by a subset of inner membrane proteins termed the 'export apparatus'. We show that a key member of the Shigella flexneri export apparatus, MxiA, assembles into a ring essential for secretion in vivo. The ring-forming interfaces are well-conserved in both nonflagellar and flagellar homologs, implying that the ring is an evolutionarily conserved feature in these systems. Electron cryo-tomography revealed a T3SS-associated cytoplasmic torus of size and shape corresponding to those of the MxiA ring aligned to the secretion channel located between the secretion pore and the ATPase complex. This defines the molecular architecture of the dominant component of the export apparatus and allows us to propose a model for the molecular mechanisms controlling secretion.

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Figures

Figure 1
Figure 1. MxiAC crystallize as a nonameric ring
(a) Representation of MxiAC ring structure as surface and cartoon, top and lateral views colored by chain. (b) Boundaries of MxiAC subdomains: SD1 (residues: 356-428, 478-493) red; SD2 (residues: 429-477) yellow; SD3 (residues: 494-583) blue; SD4 (residues: 584-686) cyan. The inner ring surface is indicated as a dashed line. (c) Immunoblotting of MxiAC cross-linked by DTBP.
Figure 2
Figure 2. In vitro and in vivo analysis of the MxiA ring
(a) Close-up of interface between monomers highlights intermolecular salt bridging residues with their side chains represented as sphere. (b) Immunodetection by western blots of IpaBC in bacterial supernatants (upper panel) and pellet (lower panel) for complemented mxiA− Shigella strains upon CR stimulation. Complementation with M5, R545A, several triple and quadruple mutations fail to restore secretion, despite the fact that all complemented strains express secretion substrates at normal level (lower panel) and the expression of all MxiA variants is comparable to the WT level with the only exception of the mutant E502A-K504A-R560A (anti-His strip, pellet panel).
Figure 3
Figure 3. Altering the inner and outer surfaces of the MxiA ring
(a) Surface of the outer SD2 is represented in yellow mesh surface whilst K519, R523 and K562 are represented in shades of blue. Close-up of the inner surface of the ring on the right. (b) Detection of IpaB, C and D in bacterial supernatants (upper panel) and pellet (lower panel) by immunoblotting upon Congo Red stimulation for complemented mxiA− Shigella strains. Double and triple mutants fail to restore secretion although expression of these variants in the bacterial pellet is comparable to the WT level (His strip, pellet panel). (c) Histogram of the percentage of IpaB, C and D secreted as fraction of the WT. Data are calculated from three independent experiments and the standard error of the mean is represented as error bars. Significant differences are detected using a one way ANOVA and pairwise comparisons made using the Holm-Sidak test. Differences are considered statistically significant for P<0.05; (**) indicates P<0.005. (d) Invasion assay performed on HeLa epithelial cells. The deletion of the SD2 greatly decreases the invasion efficiency, in accordance with the reduction of the IpaC secretion. K562A does not affect the invasion ability. Results are normalized to M90T wild type strain (100%). Data represent the means of six independent experiments performed in triplicate (error bars represent the standard deviation). Significant differences are detected using a Student’s t-test. Differences are considered statistically significant for P <0.05; (**) means P<0.01; (***) means P<0.001.
Figure 4
Figure 4. MxiAC surface conservation
(a) Surface residues are colored in accordance with evolutionary conservation (high=purple, low=cyan) among amino acid sequences from 30 members of the FlhA family. These figures were prepared using ConSurf (http://consurf.tau.ac.il/). The black line marks the footprint of the interface between monomers in MxiAC, Salmonella FlhAC (3A5I) and Bacillus FlhAC (3MIX) crystal structures. (b) MxiAC monomers (left) are shown reoriented to match the molecular packing found in the Salmonella FhlAC (middle) and Bacillus FlhAC (right) crystal structures to highlight that both flagellar MxiA counterparts associate with the same interface as MxiAC.
Figure 5
Figure 5. Model for MxiA architecture within the T3SS
(a) The dimensions of the nonameric MxiA ring fit the inner-membrane proximal density observed in in situ cryotomograms of flagellar T3SS from Treponema primitia and Samonella enterica . Both models are represented as side and top view. (b) in situ subtomogram averages identifying the location of MxiAC homolog in the Campylobacter jejuni flagellar T3SS: (left) wild-type, (middle) truncation of the cytoplasmic domain of MxiA homolog FlhA, (right) truncation of cytoplasmic domain of Spa40 homolog FlhB. Red arrow indicates the toroidal density. Yellow arrow indicates the density of the ATPase. 20 nm scale bar is shown. (c) Correlation between stator protein length and C-ring diameter in selected cryotomograms of flagellar motors,. R2=0.8115. (d) A model for the intact MxiA (including linker and transmembrane domains) is positioned in overlaid cryotomograms (Treponema-grey, Salmonella-yellow). The EM reconstruction of the Shigella flexneri T3SS (cyan) is also overlaid for comparison. The MxiA transmembrane ring is modeled using bacterorhodopsin (PDB_ID:2WJK) and the linker between the cytoplasmic and transmembrane rings is modeled as an arbitrary 36-amino acid helix. This model is completed with the hexameric FliI (PDB_ID:2DPY) located as previously determined, FliJ (PDB_ID:3AJW) and a model of FliH.
Figure 6
Figure 6. Mechanistic model of T3SS secretion
(a) On the left panel, slab view of the exporting cage loaded with substrate (IpaD, PDB ID: 2J0O). MxiA model is represented as dark red surface with residues crucial for the function of the export-ring pore, highlighted in yellow. IpaD structure is in dark gray. On the right panel, slab view of the needle with a traversing helix. (b) 3-view cartoon of key steps in T3SS secretion: 1) chaperone-effector complex is recruited to the ATPase level; 2) the ATPase complex ‘strips’ the chaperone from the exporting substrate and allowing the partially folded substrate to enter the exporting cage; 3) proton motive force mediated secretion of the unfolded substrate through the hollow needle. This simplified diagram does not show the other export apparatus components (Spa24, Spa9, Spa29 and Spa40), although they act to nucleate the MxiA assembly in the nascent export apparatus or MxiK which is thought to aid assembly of Spa33. See text and Supplementary Table 1 for details.
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