Helical Structure of the Needle of the Type III Secretion System of Shigella flexneri

Gram-negative bacteria commonly interact with animal and plant hosts using type III secretion systems (TTSSs) for translocation of proteins into eukaryotic cells during infection. 10 of the 25 TTSS-encoding genes are homologous to components of the bacterial flagellar basal body, which the TTSS needle complex morphologically resembles. This indicates a common ancestry, although no TTSS sequence homologues for the genes encoding the flagellum are found. We here present an ∼16-Å structure of the central component, the needle, of the TTSS. Although the needle subunit is significantly smaller and shares no sequence homology with the flagellar hook and filament, it shares a common helical architecture (∼5.6 subunits/turn, 24-Å helical pitch). This common architecture implies that there will be further mechanistic analogies in the functioning of these two bacterial systems. Gram-negative bacteria commonly interact with animal and plant hosts using type III secretion systems (TTSSs) for translocation of proteins into eukaryotic cells during infection. 10 of the 25 TTSS-encoding genes are homologous to components of the bacterial flagellar basal body, which the TTSS needle complex morphologically resembles. This indicates a common ancestry, although no TTSS sequence homologues for the genes encoding the flagellum are found. We here present an ∼16-Å structure of the central component, the needle, of the TTSS. Although the needle subunit is significantly smaller and shares no sequence homology with the flagellar hook and filament, it shares a common helical architecture (∼5.6 subunits/turn, 24-Å helical pitch). This common architecture implies that there will be further mechanistic analogies in the functioning of these two bacterial systems. type III secretion systems iterated helical real space reconstruction method x-ray fiber diffraction Gram-negative enteropathogenic bacteria cause a wide variety of diseases ranging from relatively harmless infections to life-threatening illnesses (1Dixon M.F. Underwood J.C.E. General and Systematic Pathology. 3rd Ed. Churchill Livingstone, London2000: 383Google Scholar). They account for more than 3 million deaths each year, mostly among children and immunocompromised adults in developing countries (2Donnenberg M.S. Nature. 2000; 406: 768-774Crossref PubMed Scopus (114) Google Scholar). Infections usually occur where hygiene is poor as the major route of infection with Salmonella, Escherichia, Yersinia, and Shigella spp. is the consumption of contaminated food (3N. N. Berkow R. Burs M. Beers M.H. Centenial Edition. The Merck Manual of Diagnosis and Therapy. Merck Publishing Group, London, United Kingdom2000: 1164-1166Google Scholar) and water (4Szewzyk U. Szewzyk R. Manz W. Schleifer K.H. Annu. Rev. Microbiol. 2000; 54: 81-127Crossref PubMed Scopus (440) Google Scholar). Despite significant differences among the distantly related genera, a common macromolecular system, the type III secretion system (TTSS1 or secreton), is the basis of each infectious cycle. This system consists of >20 proteins that form a macromolecular assembly, which delivers the bacterial virulence effectors not only across the bacterial inner and outer membranes but also directly into the host cell. The TTSS is well conserved among these bacteria, whereas the specific properties of the effectors and hence the resulting symptomatic effects on the host organism vary widely (5Hueck C.J. Microbiol. Mol. Biol. Rev. 1998; 62: 379-433Crossref PubMed Google Scholar). TTSSs are not constitutively active but are activated to secrete by signals that apparently vary among the different genera, yet they seem to ultimately derive from physical contact between the bacterium and its host cell. Understanding how an activation signal is transmitted in such a complex macromolecular assembly and how it results in secretion is one of the major questions to be answered. 10 of the 25 TTS-encoding genes show a strong similarity to those that encode the flagellar basal body, indicating a common ancestry (5Hueck C.J. Microbiol. Mol. Biol. Rev. 1998; 62: 379-433Crossref PubMed Google Scholar). Insights into the morphology of the system to date are derived from electron microscopy that reveals a supramolecular structure grossly resembling the flagellar hook-basal body complex (6Thomas D. Morgan D.G. DeRosier D.J. J. Bacteriol. 2001; 183: 6404-6412Crossref PubMed Scopus (78) Google Scholar, 7Blocker A. Gounon P. Larquet E. Niebuhr K. Cabiaux V. Parsot C. Sansonetti P. J. Cell Biol. 1999; 147: 683-693Crossref PubMed Scopus (390) Google Scholar, 8Kubori T. Matsushima Y. Nakamura D. Uralil J. Lara-Tejero M. Sukhan A. Galan J.E. Aizawa S. Science. 1998; 280: 602-605Crossref PubMed Scopus (698) Google Scholar). The major TTSS structure seen is termed the needle complex and spans the inner and outer bacterial membranes with a basal body into which a needle (∼70 Å in diameter traversed by a central channel 20–30-Å wide) is inserted that protrudes ∼500 Å into the extracellular space (8Kubori T. Matsushima Y. Nakamura D. Uralil J. Lara-Tejero M. Sukhan A. Galan J.E. Aizawa S. Science. 1998; 280: 602-605Crossref PubMed Scopus (698) Google Scholar, 9Blocker A. Jouihri N. Larquet E. Gounon P. Ebel F. Parsot C. Sansonetti P. Allaoui A. Mol. Microbiol. 2001; 39: 652-663Crossref PubMed Scopus (289) Google Scholar). The activation of the Shigella TTSSs for secretion seems to require the contact of the needle tip with the host cell-limiting membrane (7Blocker A. Gounon P. Larquet E. Niebuhr K. Cabiaux V. Parsot C. Sansonetti P. J. Cell Biol. 1999; 147: 683-693Crossref PubMed Scopus (390) Google Scholar). It is particularly difficult to understand how such physical contact leads to a change at the cytoplasmic face of the basal body ∼800 Å away and results in secretion of proteins through the structure. Gross structural homologies can be added to the genetic resemblance to the flagella noted above. Common ancestry has been used to gain insights into the functioning of the TTSSs on the basis of the well studied flagellar system (10Blocker A. Komoriya K. Aizawa S. Proc. Natl. Acad. Sci. U. S. A. 2003; (in press)PubMed Google Scholar). However, TTSS homologues for several of the key flagellum-building blocks, notably hook protein and flagellin, are lacking. Their absence combined with a lack of any detailed knowledge of the TTSS structures involved means that the hypothesis of the common mode of biological function remains unproven. This hypothesis implies that the needle of the Shigella TTSS will be made up by a helical arrangement of the 10-kDa protein MxiH (previously identified to be the major needle component (9Blocker A. Jouihri N. Larquet E. Gounon P. Ebel F. Parsot C. Sansonetti P. Allaoui A. Mol. Microbiol. 2001; 39: 652-663Crossref PubMed Scopus (289) Google Scholar, 11Tamano K. Aizawa S. Katayama E. Nonaka T. Imajoh-Ohmi S. Kuwae A. Nagai S. Sasakawa C. EMBO J. 2000; 19: 3876-3887Crossref PubMed Scopus (196) Google Scholar)) with an architecture similar to that shared by both the flagellar hook and filament. However, MxiH shows no sequence homology to either the hook or filament subunits and is in fact a much smaller protein being approximately one-fifth the molecular weight of either of the flagellar proteins. Homology between these two systems leads to the hypothesis (10Blocker A. Komoriya K. Aizawa S. Proc. Natl. Acad. Sci. U. S. A. 2003; (in press)PubMed Google Scholar) that signal transduction will occur via changes in the architecture of the needle because it is known that the flagellar filament is able to switch between different helical forms (12Samatey F.A. Imada K. Nagashima S. Vonderviszt F. Kumasaka T. Yamamoto M. Namba K. Nature. 2001; 410: 331-337Crossref PubMed Scopus (384) Google Scholar, 13Yamashita I. Hasegawa K. Suzuki H. Vonderviszt F. Mimori-Kiyosue Y. Namba K. Nat. Struct. Biol. 1998; 5: 125-132Crossref PubMed Scopus (108) Google Scholar). To test this predicted homology, we have used x-ray fiber diffraction and electron microscopy-based three-dimensional reconstruction techniques to determine the structure of the Shigella flexneri needle at ∼16 Å. We find that as the hypothesis predicts, the MxiH subunits making up the needle are arranged in a helical fashion to form an extended cylindrical structure with a central channel. The parameters that describe the geometry of the helix are very similar to those of the flagellar hook (14Morgan D.G. Macnab R.M. Francis N.R. Derosier D.J. J. Mol. Biol. 1993; 229: 79-84Crossref PubMed Scopus (41) Google Scholar) and filament (15Mimori Y. Yamashita I. Murata K. Fujiyoshi Y. Yonekura K. Toyoshima C. Namba K. J. Mol. Biol. 1995; 249: 69-87Crossref PubMed Scopus (118) Google Scholar), providing strong support for the hypothesis that these two systems share a common architecture and thus common functional mechanisms. The Shigella flexneri serotype 2a non-polar null mutant for mxiH− strain harboring pKT001, which encodes the cloned mxiH gene under an isopropyl-1-thio-औ-d-galactopyranoside-inducible promoter (a gift of Prof. Chihiro Sasakawa, Tokyo University, Tokyo, Japan) (11Tamano K. Aizawa S. Katayama E. Nonaka T. Imajoh-Ohmi S. Kuwae A. Nagai S. Sasakawa C. EMBO J. 2000; 19: 3876-3887Crossref PubMed Scopus (196) Google Scholar) hereafter termedmxiH−/mxiH+++, is plated on a Congo Red culture plate containing kanamycin (50 ॖg/ml), ampicillin (100 ॖg/ml), and trimethoprim (5 ॖg/ml) as selecting antibiotics. Within a week of plating the strain, a single Congo Red-positive colony is selected and grown at 37 °C for 8–10 h in 60 ml of trypticase soy broth, plus antibiotics as above. Using this culture as inoculate, it is diluted 1:100 into the main culture volume of trypticase soy broth (plus antibiotics). After induction with 1 mm isopropyl-1-thio-औ-d-galactopyranoside at the time of inoculation, the culture is incubated overnight at 37 °C to obtain maximum bacterial density. 107 weight/volume polyethylene glycol 6000 (catalog number 443915V, BDH Ltd.) is added to the culture to precipitate free needles in the media, and the culture is cooled on ice as soon as the polyethylene glycol has dissolved. Cells (and precipitated needles) are pelleted at 2000 × g (all centrifuge runs at 4 °C) in swing-out buckets. The supernatant is discarded, and the pellet is resuspended in 17 the initial culture volume of phosphate-buffered saline. The cell suspension is transferred to a 40-ml Dounce glass-glass tissue grinder (“tight” pestle, manufactured by Wheaton, Millville, NJ) that shaves needles from the cell surface through the exertion of sheer forces. An examination of the sample in an electron microscope showed that 60 cycles of up and down “grinding” were sufficient to remove the majority of needles from the bacteria while keeping most cells intact. The total volume of the suspension is adjusted with phosphate-buffered saline to 27 the initial culture volume and centrifuged at 2000 × g. The pellet is discarded, and the supernatant is spun at 12,000 × g to remove remaining macromolecular contaminants. Trichloroacetic acid protein precipitation followed by SDS-PAGE analysis and EM confirmed that the supernatant contained MxiH (in the form of long needles) free from major contaminants. Finally, needles are precipitated by adding polyethylene glycol 6000 and NaCl to a final concentration of 107 and 100 mm, respectively. Following 60-min incubation on ice, needles are pelleted at 27,000 × g. After discarding the supernatant, the small opaque needle pellet is resuspended in ∼0.5 ml of buffer (roughly 0.0017 of the initial culture volume) at a physiological pH (10 mm Tris, pH 7.4) when it and at 4 This of MxiH (in the form of needles) to be from a culture of of Shigella grown of TTSS needles were by a of the method of Yamashita I. Vonderviszt F. T. Namba K. J. Mol. Biol. PubMed Scopus Google Scholar, I. Suzuki H. Namba K. J. Mol. Biol. 1998; PubMed Scopus Google Scholar). of needle sample with a concentration is diluted approximately in a buffer at pH and needles by overnight at × a swing-out containing a long using × and The supernatant is and of the pellet are transferred into with an diameter of The are centrifuged at 2000 × g for h in a swing-out to the needles at the of the The of the this effects and sample were to in for at the of a United were in the at of the and were on a using mm, and time needles were diluted in 10 mm Tris, pH An of for 1 on a After the of the with the sample with a of 27 pH The needles were with a of in electron on a at were at a on were on a at a were selected using and analysis using J. M. P. J. M. A. J. Struct. Biol. PubMed Scopus Google Scholar) were using the iterated helical real space reconstruction method 2000; PubMed Scopus Google Scholar). 100 or Å in were from of the mutant and were from needle (9Blocker A. Jouihri N. Larquet E. Gounon P. Ebel F. Parsot C. Sansonetti P. Allaoui A. Mol. Microbiol. 2001; 39: 652-663Crossref PubMed Scopus (289) Google Scholar). The for each of only the of the to the filament This is because the is under the to The of the from each were added to an by two each from of the and the of from these two The of at a of but a significantly more of Å used for the final The for the majority of techniques is a and sample of the structure to be To such of flexneri we used a Shigella mxiH− strain harboring a an promoter This strain can be to the needle subunit The of MxiH a change in the TTSS with the needle from ∼500 Å in the bacteria to Å in the mutant (11Tamano K. Aizawa S. Katayama E. Nonaka T. Imajoh-Ohmi S. Kuwae A. Nagai S. Sasakawa C. EMBO J. 2000; 19: 3876-3887Crossref PubMed Scopus (196) Google Scholar). Using the the needles Å in on be from the mutant bacteria and to than as by SDS-PAGE and electron microscopy analysis fiber diffraction can be a method for helical parameters of as well as for in of helical when only the structure of a is known D. W. W. Nature. PubMed Scopus Google Scholar). no structure yet for However, has been used on the needles to that is to that by electron microscopy The key to is the of a sample of the macromolecular complex to be the of the biological in the form of a We used a on the method of Yamashita I. Vonderviszt F. T. Namba K. J. Mol. Biol. PubMed Scopus Google I. Suzuki H. Namba K. J. Mol. Biol. 1998; PubMed Scopus Google Scholar), which and of the in a to needle The of these to for the of such as that in of the showed the diffraction of a helical assembly, that MxiH in a helical fashion to The parameters to the helical are the the filament subunit and the the filament The subunit to a in the that is on the and the at a of Å were also at a of and Å. The of this from using electron To obtain more detailed structural the needle, we a of electron from The to of helical analysis and three-dimensional reconstruction D.J. A. Nature. PubMed Scopus Google Scholar) were not in because of the very by these not be that showed the for a single used 2000; PubMed Scopus Google Scholar) on the of from the each long for the mutant and long in for the The derived from these is the of the because the diffraction have been added from that have been only to have the filament in the with of and were seen in both the and in the The that were that it from a P. M. J. PubMed Scopus Google Scholar) of these not to a these The to be and to a of The at An analysis of this showed that it also and that it most to a of the at to be and most to a of The only the at and from with the and the at from a helix with the We are to determine the of this structure from these and have a on of the flagellar hook and major filament helix and helix (14Morgan D.G. Macnab R.M. Francis N.R. Derosier D.J. J. Mol. Biol. 1993; 229: 79-84Crossref PubMed Scopus (41) Google Y. Yamashita I. Murata K. Fujiyoshi Y. Yonekura K. Toyoshima C. Namba K. J. Mol. Biol. 1995; 249: 69-87Crossref PubMed Scopus (118) Google Scholar). three-dimensional reconstruction by from the and this used as an initial for the method 2000; PubMed Scopus Google Scholar). This method for the of helical during the of cycles where the is to change in this to a structure with of a 24-Å helix from the of this reconstruction that the of the from the of the structure to both the and the either the or the involved no this is a strong on the The of very different to the either the cycles to with the when the subunit than with or to a three-dimensional reconstruction not the or with 3 shows the helical parameters as a function of number during the of from The parameters to approximately the when the is with the results of the method an subunit of well the of the EM This is the the of by in the of the EM seen in the as by the to this This can that have or of the 24-Å an of or respectively. were made from long needles and and also from needles from of needle as by (9Blocker A. Jouihri N. Larquet E. Gounon P. Ebel F. Parsot C. Sansonetti P. Allaoui A. Mol. Microbiol. 2001; 39: 652-663Crossref PubMed Scopus (289) Google not The helical parameters both to similar indicating that the architecture of the needle is by the of We have the of the reconstruction to be ∼16 a from this shows a at Å that is in with the at this in the The needle is to be a of an outer diameter of Å traversed by a central of Å in The three-dimensional shows subunits by in the that each subunit to a single of we can determine an molecular volume a specific volume of protein of The reconstruction is when a is that this a is that a much smaller or the reconstruction is no is the volume in the is and that there is a single of MxiH parameters from the Shigella needle and with those for the flagellar filament I. Hasegawa K. Suzuki H. Vonderviszt F. Mimori-Kiyosue Y. Namba K. Nat. Struct. Biol. 1998; 5: 125-132Crossref PubMed Scopus (108) Google Scholar) and hook (14Morgan D.G. Macnab R.M. Francis N.R. Derosier D.J. J. Mol. Biol. 1993; 229: 79-84Crossref PubMed Scopus (41) Google needles in helical filament I. Hasegawa K. Suzuki H. Vonderviszt F. Mimori-Kiyosue Y. Namba K. Nat. Struct. Biol. 1998; 5: 125-132Crossref PubMed Scopus (108) Google (14Morgan D.G. Macnab R.M. Francis N.R. Derosier D.J. J. Mol. Biol. 1993; 229: 79-84Crossref PubMed Scopus (41) Google in a Common ancestry of the and flagellar systems to the (10Blocker A. Komoriya K. Aizawa S. Proc. Natl. Acad. Sci. U. S. A. 2003; (in press)PubMed Google Scholar) that there be TTSS homologues of either the hook proteins. show that the needle has the architecture (∼5.6 subunits/turn, helical as the flagellar filament (12Samatey F.A. Imada K. Nagashima S. Vonderviszt F. Kumasaka T. Yamamoto M. Namba K. Nature. 2001; 410: 331-337Crossref PubMed Scopus (384) Google Scholar, Y. Yamashita I. Murata K. Fujiyoshi Y. Yonekura K. Toyoshima C. Namba K. J. Mol. Biol. 1995; 249: 69-87Crossref PubMed Scopus (118) Google Scholar) and hook (14Morgan D.G. Macnab R.M. Francis N.R. Derosier D.J. J. Mol. Biol. 1993; 229: 79-84Crossref PubMed Scopus (41) Google Scholar), that MxiH and the flagellar hook protein and be structural but not sequence homologues of one This is the in the of these two proteins is is and the hook protein However, the three-dimensional reconstruction of the flagellar filament I. Hasegawa K. Suzuki H. Vonderviszt F. Mimori-Kiyosue Y. Namba K. Nat. Struct. Biol. 1998; 5: 125-132Crossref PubMed Scopus (108) Google Scholar, Y. Yamashita I. Murata K. Fujiyoshi Y. Yonekura K. Toyoshima C. Namba K. J. Mol. Biol. 1995; 249: 69-87Crossref PubMed Scopus (118) Google Scholar) shows homologies than of helical parameters to needle structure. The flagellar filament structure can be into a of different an inner of Å in diameter with a central channel of the outer that and has an outer of and and that from the helical The of an x-ray structure for into this EM reconstruction Yamashita I. Hasegawa K. Suzuki H. Vonderviszt F. Mimori-Kiyosue Y. Namba K. Nat. Struct. Biol. 1998; 5: 125-132Crossref PubMed Scopus (108) Google Scholar) to determine that the made up of the and of flagellin, which were not present in the x-ray as they to be to the protein from The and helical architecture of the inner is very similar to the structure we present here for the Shigella the as to the MxiH shares any homology with the of Although we any sequence homology, there is of homology at the of predicted structure such as for Scholar, C. Struct. 19: PubMed Scopus Google Scholar, C. J. Mol. Biol. 1993; PubMed Scopus Google Scholar) and M. 1998; PubMed Scopus Google Scholar)) because we find that the and of MxiH and as well as those of the and filament proteins strong for at that the needle the to a helix of this with this the subunit a inserted between the and that is not for helix but to of flagellar the homology with the flagellar system, it that the assembly of MxiH into the Shigella needle require the of a at the needle tip (10Blocker A. Komoriya K. Aizawa S. Proc. Natl. Acad. Sci. U. S. A. 2003; (in press)PubMed Google Scholar). at the of the the by subunits into the the helix K. S. Namba K. J. Struct. Biol. 2001; PubMed Scopus Google Scholar). This protein be present in the needle complex at such a to MxiH (and is to be of a similar that it is not that we not it in will be to this The hypothesis of common ancestry is in providing a by which secretion be on by the tip of needle a host cell (10Blocker A. Komoriya K. Aizawa S. Proc. Natl. Acad. Sci. U. S. A. 2003; (in press)PubMed Google Scholar). The by which a signal is from the needle tip to the at the of the basal body at an is difficult to understand on a molecular The flagellar filament is able to switch helical forms on the of or in to the Nature. PubMed Scopus Google Scholar). is to be to small changes in the of the to changes in the filament as a (12Samatey F.A. Imada K. Nagashima S. Vonderviszt F. Kumasaka T. Yamamoto M. Namba K. Nature. 2001; 410: 331-337Crossref PubMed Scopus (384) Google Scholar). This in leads to changes in the of the the flagellum and thus the change in to the The that this helical architecture be to switch between different that the of contact of the needle with the host cell be to switch the helical of the needle, thus a signal that is transmitted to the basal of the needle and the proteins with the basal Although this functional hypothesis remains to be structure in that the Shigella needle to at the of the flagellar filament strong support for the hypothesis that secretion is by changes in the architecture of the needle This remains to be confirmed by of activated these are to be difficult because the of of activated needles that will to be We can of the structural needle to present the for TTSS in This the assembly of the TTSS and the structural to the flagellar hook-basal body questions on how are and the of the between the basal body and the needle and the of the between the needle and its which a signal to be to an We Aizawa Japan) for and with the needle on in bacterial We Namba for on the structure of bacterial flagellar and on EM and x-ray fiber diffraction DeRosier for that we a single and M. E. M. United for to to the of these