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. 2000 Aug 1;19(15):3876-87.
doi: 10.1093/emboj/19.15.3876.

Supramolecular structure of the Shigella type III secretion machinery: the needle part is changeable in length and essential for delivery of effectors

K Tamano  1 , S Aizawa, E Katayama, T Nonaka, S Imajoh-Ohmi, A Kuwae, S Nagai, C Sasakawa
Affiliations

Supramolecular structure of the Shigella type III secretion machinery: the needle part is changeable in length and essential for delivery of effectors

K Tamano et al. EMBO J. .

Abstract

We investigated the supramolecular structure of the SHIGELLA: type III secretion machinery including its major components. Our results indicated that the machinery was composed of needle and basal parts with respective lengths of 45.4 +/- 3.3 and 31.6 +/- 0.3 nm, and contained MxiD, MxiG, MxiJ and MxiH. spa47, encoding a putative F(1)-type ATPase, was required for the secretion of effector proteins via the type III system and was involved in the formation of the needle. The spa47 mutant produced a defective, needle-less type III structure, which contained MxiD, MxiG and MxiJ but not MxiH. The mxiH mutant produced a defective type III structure lacking the needle and failed to secrete effector proteins. Upon overexpression of MxiH in the mxiH mutant, the bacteria produced type III structures with protruding dramatically long needles, and showed a remarkable increase in invasiveness. Our results suggest that MxiH is the major needle component of the type III machinery and is essential for delivery of the effector proteins, and that the level of MxiH affects the length of the needle.

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Figures

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Fig. 1. Electron micrograph of osmotically shocked M94 cells. Cells were negatively stained with 2% phosphotungstic acid pH 7.0 and observed under TEM. The open arrowheads indicate the type III secretion complexes on the bacterial envelope and the closed arrowhead denotes the bleb-like moiety associated with the tip of the complex, reminiscent of the secreted proteins through the type III secretion complexes. Scale bar, 100 nm.
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Fig. 2. Purification of type III secretion complexes from M94. (A) Each sample of partially purified type III secretion complexes was separated by 15% SDS–PAGE and the gel was stained with CBB or immuno blotted with antibodies specific for MxiD, MxiG and MxiJ. (B) A partially purified sample of type III secretion complexes from M94 was fractionated by 40% CsCl gradient centrifugation. Each fraction was separated by 15% SDS–PAGE and visualized by silver staining (upper part) or immunoblotted with antibodies specific for MxiD and MxiG (lower part).
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Fig. 3. Electron micrograph of purified type III secretion complexes from M94. (A) Purified type III secretion complexes at a low magnification. The sample was obtained from the lower fraction of a 40% CsCl gradient centrifugation, which corresponds to fraction 6 in Figure 2B. The sample was stained with 2% phosphotungstic acid pH 7.0 and observed by TEM. Scale bar, 100 nm. (B) Type III secretion complexes at a high magnification. N indicates the needle, while B indicates the basal part of the type III secretion complex. The open arrowhead denotes upper (or outer) rings, while the closed arrowhead denotes the lower (or inner) rings. Scale bar, 10 nm. (C) Measurement of the size of type III secretion complexes. The purified type III secretion complexes under TEM with TMV (300 nm long, 20 nm wide, arrowheads). Scale bar, 100 nm. (D) Proposed size of the type III secretion complex of S.flexneri. The size of each portion was measured based on the 28 best preserved type III secretion complexes in comparison with the scale of TMV.
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Fig. 4. Effect of the spa47 mutation on the formation of type III secretion complexes. (A) Genomic organization of the spa operon and construction of an spa47::aphA-3 mutant. (B) Electron micrographs of the purified type III secretion complexes from the spa47 mutant. Scale bar, 20 nm. (C) Pattern on the 20% SDS–polyacrylamide gel and immunoblot with antibodies specific for MxiD, MxiG and MxiJ of partially purified type III secretion complexes of M94, the spa47 mutant (spa47–) and non-invasive strains del-17 and YSH6200. The gel was stained with CBB.
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Fig. 5. Analysis of the mxiH mutant and its complemented mutant for the type III secretion complexes. (A) Genomic organization of the mxi operon and construction of the mxiH::aphA-3 mutant. (B) Pattern on the 20% SDS–polyacrylamide gel (upper part) and immunoblot (lower part) with antibodies specific for MxiD, MxiG and MxiJ of partially purified type III secretion complexes of M94, the mxiH mutant (mxiH–), the mxiH mutant harboring pKT001 (mxiH–/mxiH), del-17 and YSH6200. The gel was stained with CBB. (C) Electron micrographs of the purified type III secretion complexes from the mxiH mutant. Scale bar, 20 nm.
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Fig. 6. Effect of overexpression of MxiH in Shigella on the length of type III secretion needles. (A) Electron micrograph of the purified type III secretion complexes from the mxiH mutant harboring pKT001 (cloned mxiH gene). Bacteria were grown in L-broth and induced with 1 mM IPTG. Scale bar, 100 nm. (B) Distribution of needle lengths in the type III secretion complexes purified from the mxiH mutant harboring pKT001 grown in L-broth with 1 mM IPTG. (C) Electron micrographs of the mxiH mutant harboring pKT001 grown in L-broth with 1 mM IPTG (a) or without IPTG (b). Arrowheads are indicative of the long needle structures in type III secretion complexes protruding from the cell surface. Scale bars, 100 nm.
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Fig. 7. Effect of the mxiH mutation on secretion of effector proteins and invasion of HeLa cells. (A) Immunoblot analysis of secreted proteins from M94, spa47–, mxiH–, mxiH– harboring pKT001 (mxiH–/mxiH) or the S325 strain by addition of Congo red (0.003% final concentration) with antibodies specific for IpaB, IpaC and IpaD. S325 is a mxiA::Tn5 mutant used for negative control of Ipa secretion. WC, CS and PS denote the whole-cell lysate, Congo red-induced supernatant and supernatant in PBS, respectively. (B) Band pattern on a 10% SDS–polyacrylamide gel of secreted proteins from M94, spa47–, mxiH–, mxiH– harboring pKT001 (mxiH–/mxiH) or the S325 strain by addition of Congo red. The gel was silver stained. (C) Invasiveness of the mxiH mutant (mxiH–) or the mxiH mutant harboring pKT001 (mxiH–/mxiH) in HeLa cells by the gentamicin protection assay.
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Fig. 8. A schematic representation of the interaction between the extended needles of the Shigella type III secretion machinery and the target host cell. The length of the wild-type needle of the type III secretion machinery of S.flexneri was estimated as 45.4 ± 3.3 nm (Figure 3D), while the maximum distance between the contact bacterium and the target epithelial cells for efficiently delivering the effector proteins into the host cell was proposed to be ∼100 nm (Blocker et al., 1999). Although Shigella expressing wild-type type III machinery may be too short to achieve direct contact, because the length of type III needles can be extended by elevated MxiH (Figure 6), wild-type Shigella may have the ability to extend long needles by controlling mxiH expression under in vivo conditions.
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References

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