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. 2010 Oct 12;107(41):17745-50.
doi: 10.1073/pnas.1008053107. Epub 2010 Sep 27.

Organization and coordinated assembly of the type III secretion export apparatus

Samuel Wagner  1 , Lisa Königsmaier, María Lara-Tejero, Matthew Lefebre, Thomas C Marlovits, Jorge E Galán
Affiliations

Organization and coordinated assembly of the type III secretion export apparatus

Samuel Wagner et al. Proc Natl Acad Sci U S A. .

Abstract

Type III protein secretion systems are unique bacterial nanomachines with the capacity to deliver bacterial effector proteins into eukaryotic cells. These systems are critical to the biology of many pathogenic or symbiotic bacteria for insects, plants, animals, and humans. Essential components of these systems are multiprotein envelope-associated organelles known as the needle complex and a group of membrane proteins that compose the so-called export apparatus. Here, we show that components of the export apparatus associate intimately with the needle complex, forming a structure that can be visualized by cryo-electron microscopy. We also show that formation of the needle complex base is initiated at the export apparatus and that, in the absence of export apparatus components, there is a significant reduction in the levels of needle complex base assembly. Our results show a substantial coordination in the assembly of the two central elements of type III secretion machines.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The export apparatus is intimately associated with the NC. (A) Export apparatus components cofractionate with the NC. Protein complexes of purified S. Typhimurium inner membrane fractions were solubilized with n-dodecyl-β-d-maltopyranoside (DDM) and separated by 2D BN-PAGE. Indicated proteins were identified by LC-MS/MS or Western blotting. (B) The export apparatus interacts with the needle complex. DDM-solubilized inner membrane fraction proteins interacting with C-terminally FLAG-tagged InvA or SpaSN258A were immunoprecipitated, separated by BN-PAGE, and identified by LC-MS/MS.
Fig. 2.
Fig. 2.
Export apparatus components associate with one another in the absence of the needle complex. (A) The export apparatus components SpaP and SpaR form a complex in the absence of the NC. DDM-solubilized inner membrane fraction proteins of a S. Typhimurium ΔprgH ΔprgI ΔprgJ ΔprgK mutant strain expressing FLAG-epitope tagged SpaP, InvA, or SpaSN258A (as indicated) were immunoprecipitated with an anti-FLAG antibody. Precipitated proteins were separated by 2D BN-PAGE, and indicated proteins were identified by LC-MS/MS. (B) The levels of SpaP, SpaR, and SpaS (but not InvA) are significantly reduced in the absence of some export apparatus components. Purified inner membrane fractions from wild-type S. Typhimurium (wt) or the ΔspaP, ΔspaQ, ΔspaR, ΔspaS, or ΔinvA mutants expressing FLAG-epitope tagged InvA, SpaP, SpaR, or SpaSN258A (as indicated) were analyzed by Western blotting (Upper). The intensity of the bands was quantified using the Odyssey imaging system (Li-Cor), and values represent the mean ± SD of three independent experiments (Lower). Values were standardized relative to a loading control (PrgH) and across different samples relative to wild type, which was assigned an arbitrary value of 1.
Fig. 3.
Fig. 3.
Hierarchy in the recruitment to the needle complex of export apparatus components. Protein complexes of purified inner membrane fractions of wild type (wt) and the indicated mutants of S. Typhimurium expressing FLAG-epitope tagged InvA or SpaP were solubilized with DDM, separated by BN-PAGE, and analyzed by Western blotting using antibodies directed to the NC base or FLAG (to detect either SpaP or InvA; A). The intensity of the bands corresponding to SpaP or InvA associated with the NC was quantified as above (B), and values represent the mean ± SD of three independent experiments. Values were standardized relative to the levels of the NC and across different samples relative to wild type, which was assigned an arbitrary value of 1. The total levels of SpaP-FLAG and InvA-FLAG in the different mutants are shown in Fig. 2B.
Fig. 4.
Fig. 4.
The export apparatus is required for efficient NC base assembly. (A and B) Efficient NC base assembly requires SpaP, SpaQ, SpaR, and SpaS but not InvA. DDM-solubilized protein complexes of purified inner membrane fractions of wild type (wt) and the indicated mutants of S. Typhimurium were separated by BN-PAGE and analyzed by Western blotting (A). The total levels of PrgH and PrgK in the different mutants are shown in A Lower. The quantification of the relative levels of NC in the different mutants is shown (B). The intensity of the bands was quantified as above, and values represent the mean ± SD of three independent experiments. Values were standardized across different samples relative to wild type, which was considered 100%. (C–E). The export apparatus components SpaP, SpaQ, SpaR, and SpaS promote NC base assembly. (C) The experimental setup for these experiments involved the use of engineered strains of S. Typhimurim in which expression of the base components is under the control of an arabinose-inducible promoter, whereas the expression of either spaPQRS or invA is under the control of a rhamnose-inducible promoter (details in Material and Methods). Base expression was induced by the addition of arabinose. The inducer was washed away, and expression of the base components was blocked by the addition of the repressor fucose. Sixty minutes later (indicated as time 0 in the legend), expression of export apparatus components (either SpaPFLAGQRS or InvAFLAG) was induced by the addition of rhamnose. Samples were harvested 30 min and 60 min thereafter, and DDM-solubilized proteins of whole-cell lysates were separated by BN-PAGE and analyzed by Western blotting with antibodies directed to NC base components (PrgH and PrgK) or FLAG (to detect SpaP or InvA, as indicated). SDS/PAGE of whole-cell lysates shows the absolute levels of the base components PrgH and PrgK, which remained constant throughout the experiment. D Left shows results in which expression of the base was followed by expression of SpaPQRS, whereas D Right shows results in which expression of the base was followed by expression of InvA. To minimize variation when comparing NC-associated proteins and PrgH monomers, we divided the same sample after solubilization and addition of Coomassie and loaded them onto two different gels (D). The total levels of PrgH and PrgK in different samples are shown in the bottom row (D). Quantification of these results is shown in E and F. The intensity of the bands was quantified as above, and values represent the mean ± SD of three independent experiments. The maximal values for bases/NC and PrgH protomers, respectively, were assigned an arbitrary value of 1. The proportion of bases and assembled NCs of the total base/NC signal (D) is shown in F. As an example, Inset in F shows, for a time point (D), the region corresponding to NC (green) or bases (red) in the BN-PAGE analysis.
Fig. 5.
Fig. 5.
NC base assembly requires the predeployment of the export apparatus. (A) Outline of the experimental setup to ensure that expression of the base components preceded expression of the export components (indicated as base > SpaPFLAGQRS or base > InvAFLAG in B) or vice versa (indicated as SpaPFLAGQRS > base or InvAFLAG > base in B). (B and C) Export apparatus components do not efficiently incorporate into preassembled bases. The incorporation of SpaP (B Left) or InvA (B Right) into preformed NC bases was examined by BN-PAGE of DDM-solubilized proteins of whole-cell lysates followed by Western blotting analysis with specific antibodies and an Odyssey (Li-Cor) infrared imaging system to simultaneously detect the indicated proteins. The merger of the two detection channels is shown in color [red, bases/NC (PrgH/PrgK); green, InvA or SpaP]. The total levels of PrgH and SpaP/InvA in the different samples are shown in Lower (B). (C) Quantification of the results shown in B. The intensity of the bands was quantified as above, and values represent the mean ± SD of three independent experiments. Incorporation of InvA or SpaP into preexisting bases, de novo assembled bases from free preexisting protomers, or de novo assembled bases from de novo synthesized protomers was determined as indicated in SI Materials and Methods. Values were standardized relative to the NC and across different samples relative to wild type, which was assigned an arbitrary value of 1.
Fig. 6.
Fig. 6.
Components of the export apparatus form a defined substructure in the NC base. (A) Diagram of the S. Typhimurium NC base depicting the different substructures. (B) Total class averages of single particle analysis of cryo-electron microscopy images and density differences between the averaged images of wild-type NCs and the indicated mutant strains. (C) Longitudinal sections through the center of base complexes from wild type or the indicated mutant strains lacking SpaP, SpaQ, SpaR, SpaS, and InvA or only SpaP reveal the absence of the cup and socket substructures observed in the NC base obtained from wild type. The difference images of sections from either of the mutant strains and wild type are very similar, indicating that removal of SpaP alone is sufficient to generate this structural phenotype. The cut-away view and blow-up view of an overlay of wild-type complexes (gray/mesh) and complexes from the ΔspaPQRS ΔinvA mutant strain at a distance that allows the visualization of the entire cup and socket substructures in 3D emphasizes the difference between the complexes analyzed. (D and E) Highly purified NC bases contain SpaP and SpaR but no InvA or SpaS. NC bases either prepared in an identical manner to those used in cryo-electron microscopy studies or from purified inner membrane fractions were analyzed by SDS/PAGE (D) or BN-PAGE (E) followed by Western blotting with antibodies to detect the indicated proteins. (F) Model for the coordinated assembly of the T3SS export apparatus and NC base. SpaP forms a stable complex with SpaQ and SpaR, which is likely to be the nucleation point for PrgH/PrgK NC base ring assembly. The presence of SpaS further enhances the assembly of the PrgH/PrgK ring, although it does not seem to be essential. The presence of InvA is not required for efficient NC base formation or for the stability and/or recruitment of any of the other export apparatus components. These results suggest that InvA is the last component recruited to the export apparatus. However, InvA is not efficiently recruited into preassembled bases.
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References

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