Blocker group
Functional and structural analysis of type III secretion systems
Welcome to the website of the Blocker laboratory. Our research focuses on the functional and structural analysis of type III secretion systems, key and widely distributed virulence determinants of Gram-negative bacteria, particularly that of the human bacterial pathogen Shigella flexneri.
Research
Introduction
Figure 1. Scanning electron micrograph of the rod-like Shigella flexneri entering a HeLa cell
Shigella flexneri causes bacillary dysentery by invading the colonic mucosa
The bacterium Shigella flexneriis the etiological agent of the endemic form of bacillary dysentery. Shigellosis is a disease prevalent in many developing countries, with an estimated 20 million new cases per annum, mostly in young malnourished children, causing approximately 600,000 deaths. Transmission of the disease occurs mainly by consumption of water contaminated by feces from infected individuals and it is very efficient, as ingestion of less than 10 bacteria is enough to cause disease. S. flexneri is a Gram-negative enterobacterium closely related to E. coli and causes disease by invading the colonic mucosa (Hale, 1998).After transcytosis across the gut epithelium via M-cells of Peyer's patches, S. flexneri forces its macropinocytic/phagocytic uptake into the basal-lateral face of enterocytes and releases itself into the host cell cytoplasm where it multiplies and spreads to neighboring cells (see Sansonetti, 2001 for a review). The most established animal models for the disease are oral infection of monkeys, ligated rabbit illeal loops, intranasal infection of mice, and the Sereny test (keratoconjunctivitis) in guinea pigs. In vitro tests of bacterial pathogenicity are: entry and dissemination in epithelial cells (Sansonetti et al., 1986; Clerc and Sansonetti, 1987; Bernardini et al., 1989), macrophage apoptosis induction (Zychlinsky et al., 1992) and contact hemolysis (Clerc and Sansonetti, 1986).
Figure 1. Scanning electron micrograph (taken by Roger Wepf, Philippe Sansonetti and Ariel Blocker at the EMBL) of the rod-like Shigella flexneri entering a HeLa cell. The bacterium interacts with the host cell surface, injects (via its type III secreton) its invasins, which act to choreograph a local actin-rich membrane ruffle at the host cell surface (Niebuhr and Sansonetti, 2000). The ruffle engulfs the bacterium and eventually disassembles, internalising the bacterium.
Shigella flexneri uses a type III secretion apparatus and secretes Ipa proteins in order to invade eukaryotic cells
All of the bacterial genes necessary for entry of the bacterium into eukaryotic cells of epithelial origin have been identified and sequenced (Sansonetti et al., 1982; Sansonetti et al., 1983; Maurelli et al., 1985; Parsot, 1994). They are clustered in a 35-kb region of a large virulence plasmid. This region carries two types of genes: the ipa and ipg genes encoding the entry-mediating proteins (IpaA-D and IpgD, known as the invasins) and their individual intrabacterial chaperones, and the mxi/spa genes encoding a type III secretion apparatus (T3SS or secreton) required for secretion of the invasins. T3SSs are essential virulence determinants of many Gram-negative bacteria pathogenic for plants, animals and humans (Cornelis, 2006). They are encoded by approximately 25 genes, which share homology with those encoding flagellar basal bodies (Blocker et al., 2003). Type III secretons serve to secrete, only upon direct physical contact with host cells, and therefore translocate proteins effectors of virulence, the invasins in the case of Shigella, from the bacterial cytoplasm into the host cell cytoplasm.
Our Findings
Overall structure of the Shigella type III secreton and composition of its 'translocation pore': working model for pore insertion/effector translocation
Figure 2. Morphological identification and analysis of the type III secretons of S. flexneri.
When in Prof. Sansonetti's laboratory at the Institute Pasteur, Paris, France, Dr. Blocker identified the type III secreton of Shigella flexneri she showed that during intimate contact with eukaryotic cells it inserts, with the help of IpaD, IpaB and IpaC to form a pore within the host cell membrane, through which some of the remaining invasins may become translocated (Blocker et al., 1999). The secreton morphologically resembles bacterial flagellar hook-basal body complexes. It composed of: a 10x60 nm external needle inserted within a 30 nm in diameter cylinder traversing both bacterial membranes and the peptidoglycan and of a large proximal bulb, 45x25 nm, located on the cytoplasmic face of the inner membrane.
Figure 2. Morphological identification and analysis of the type III secretons of S. flexneri. (A) Osmotically shocked and negatively stained wild-type Shigella which had been induced to secrete with Congo red were visualized by electron microscopy. Arrows show the position of the secreton at the margin of bacteria. The neck and bulb are inside the body of the bacterium while the needle is protruding outside the outer membrane. Inset shows the periphery of partially lysed bacteria. Only the protruding needle is clearly visible. The neck is poorly distinguished and the bulb is masked inside the body of the bacterium. With the method used here one can not assume that the two lines at the margin of the bacterium are its inner and outer membranes. (B) Image of the secreton bulb resulting from alignment and averaging of the 22 best preserved secretons found in A and deduced projection density map of the averaged image at 2.8 nm resolution. (C) Typical secretons found in wild-type, ipaB, ipaC and ipaD strains. Bars: (A) 200 nm; (B) 50 nm. Taken from Blocker et al., 1999.
The first two parts of the secreton strongly resemble the "needle complex" previously discovered by Kubori et al. (1998), as the type III secretion machinery of Salmonella typhimurium. Protein translocation appears to be a one-step process (Blocker et al., 1999) directly from the bacterial cytoplasm through a channel in the secreton and its needle (Blocker et al., 2001) into the bacterially inserted pore in the host membrane (Blocker et al., 1999). Understanding this complex protein translocation step is the main aim of the ongoing work in my laboratory (Blocker et al., 2003; Blocker et al., 2008).
Figure 3. Initial structural analysis of the NC by electron microscopy
Figure 3. Initial structural analysis of the NC by electron microscopy. A. Negative staining of isolated NCs; arrows point to incomplete NCs, lacking the base. B. CryoEM of isolated NCs; three typical and enlarged NCs are shown at the top. The bar is 100 nm. C. Results of hierarchical ascendant classification with Ward's criterion. The diagram is read from bottom to top. Each node-connecting branch represents a merged class. For instance, the classes 4 (n = 267) and 1 (n = 74) are merged into a new class with n = 341. The vertical height of the horizontal lines (in arbitrary units, a.u.) is a measure of the gain in intraclass variance obtained through the merging; if that gain is small, then the two classes are very similar. For each class, the average (above) and the variance (below) image is shown. The asterisk marks the major class for which the variance is the lowest and the particle number is the highest (n = 432). D. Enlargement of the average image of the major particle class obtained in (C). E. Surface representation of the NC volume obtained assuming cylindrical symmetry. F. Central axial section of the volume obtained in (E). G. Presentation of half of the volume obtained in (E). H. The same as (F) but with an interpretative model representation of the three parts of the NC; a = base; b = upper ring doublet; c = needle. The parts of the full type III secreton defined by Blocker et al. (1999) are represented for reference; 1 = needle, 2 = transmembrane neck domain, 3 = bulb. Taken from Blocker et al. 2001.
This protein transport process can be broken down into several steps:
- Assembly of a mature T3SS
- Activation of the type III secreton by direct physical contact with host cells
- Intrabacterial energisation of secretion
- Secretion/assembly of the bacterial translocation pore within the host plasma membrane
- Secretion of the effector proteins to be translocated into the host cytoplasm
- Activation of the transcription/translation of later effector proteins destined for secretion after the bacterium has entered the host cell
Although we have been working on all of these aspects, our progress in recent year has been concentrated mainly on points 1, 2 and 4 (Blocker et al., 2008).
Three-dimensional reconstruction of the Shigella T3SS transmembrane regions reveals 12-fold symmetry and novel features throughout
An incomplete understanding of the structure of the needle complex has hampered studies of T3SS function. To estimate the stoichiometry of its components, we measured the mass of its subdomains by scanning transmission electron microscopy (STEM). We then determined subunit symmetries by analysis of top and side views within negatively stained samples in low-dose transmission electron microscopy (TEM). Application of 12-fold symmetry allowed generation of a 21–25-Å resolution, three-dimensional reconstruction of the needle complex "base" or transmembrane region, revealing many new features and permitting tentative docking of the crystal structure of EscJ, an inner membrane component (Hodgkinson et al., 2009; reviewed in Waksman, 2009). Our task for the future is to obtain higher-resolution reconstructions of wild-type and mutant (Kenjale et al., 2005) needle complexes from cryo-EM images without imposition of symmetry, where existing (Yip et al., 2005; Deane et al., 2006; Johnson et al., 2007; Zavirach et al., 2008; Spreter et al., 2009) and future needle complex component structures can be precisely fitted.
Figure 4. 3D reconstruction of needle complexes with C12 symmetry
Figure 4. Three-dimensional reconstruction of needle complexes with C12 symmetry (a) Surface representation. Labels on the left-hand side define structural regions in the map and numbers on the right-hand side define points at which slices were taken for b.Conn., connector; Le, legs. The bacterial (cytoplasmic) side of the needle complex is defined as the base of the complex. (b) Slices through the three-dimensional reconstructions at the linker (1), IMR shoulder (2), shoulder-connector transition (3), connector (4), connector-OMR3 transition (5), OMR2 (6), OMR2-OMR1 transition (7) and OMR1 (8). Arrow in (2) shows radial density spokes extending inward from between each pair of outer IMR subunits. (3) shows the top of the IMR (large radius ring indicated) and start of the connector (smaller radius ring indicated). (3) and (5) show azimuthal tilts of connector subunits. Twelve-fold symmetry is seen throughout the OMR rings (6)–(8). More slices are shown in Supplementary Figure 6 of Hodgkinson et al. (2009) online. (c) Longitudinal cutaway of map reveals details of internal components with a distinct socket ring (SR, blue arrow), with strong density modulations and 12-fold connections to the socket cup (S/C) directly below it. Twelve spokes connect the socket cup to the IMR (S). Two slender linkers (Li) connect the legs to the underside of the IMR shoulder. The top of the connector shows 12-fold connectivity with the underside of OMR3. N indicates the needle. The hollow arrow on the left side of connector indicates the point at which the structure is cut to give the view in d (where the black ring is the cut-through volume). (d) The top view surface cut back to the socket ring shows its 12-fold symmetry and domains with azimuthal tilt (blue arrow). The spokes connecting the outer IMR to the socket cup are seen below this ring. (e) View of the needle complex base from the cytoplasmic side, showing 12-fold symmetry of the socket cup (black arrow). The 12-fold spokes show azimuthal tilting. Scale bars in a, c, d and e represent 100 Å. Scale bar in b represents 100 Å.
The hollow needle is a helical polymer of MxiH and is directly involved in sensing host cells and regulation of secretion
While at the Sir William Dunn School of Pathology at the University of Oxford, we showed in collaboration with Dr. Susan M. Lea's laboratory, that the external needle is formed by polymerisation of a single 8 kDa protein, MxiH. The hollow helical structure displays structural parameters almost identical to those of all axial components of bacterial flagella (Cordes et al., 2003). We therefore assume that the needle polymerises in a similar fashion, by addition of new subunits at its distal end via the catalytic action of a rotating assembly cap (Yonekura et al., 2000), which remains to be identified.
Figure 5. Helical 3D reconstruction of the needle using electron microscopy.
Figure 5. Helical three-dimensional (3D) reconstruction of the needle using electron microscopy (verified also by X-ray fiber diffraction). All images produced using AESOP (M.E.M. Noble, unpublished program). A and B show a top view and side view (respectively) of the 3D reconstruction of the Shigella flexneri needles. The electron density is thresholded to generate the solid surface. C is a composite image giving a model for the structure of the needle complex in the bacterial membranes using the needle reconstruction presented here in combination with the earlier reconstruction of the basal body of the needle complex (Blocker et al., 2001). Taken from Cordes et al., 2003.
We next showed by site-directed mutagenesis of MxiH and functional assays that the needle is directly involved in sensing host cells and regulating secretion (Kenjale et al., 2005), although we found no evidence that this occurs through helical transitions within the needle (Cordes et al., 2005).
Summary of MxiH mutagenesis findings. Congo red is a small amphipathic dye molecule known to be an artificial inducer of Ipa protein secretion in Shigella. See Kenjale et al., 2005.
A pseudo-atomic model of the needle reveals the location of amino acids important for MxiH function
More recently, still in collaboration with the Lea laboratory in Oxford, we solved the crystal structure of the MxiH monomer (Deane et al., 2006a and b). This revealed two short α-helices held in a hairpin shape by a tiny double proline loop. When the crystal structure of MxiH was used to build a pseudo-atomic model of the needle, it was found that the amino acids identified by mutagenesis as key for MxiH function are located within the needle head (P44 and Q51), and thus at the distal tip of the needle, or at subunit interfaces (D73) within a single protofilament. Although the molecular means of signal transduction through the needle remains unknown, these findings do suggest some sort of "tip-to-toe" transmission path (Blocker et al., 2008).
Figure 6a. Crystal structure of an MxiH monomer
Figure 6b. Construction of a pseudo-atomic model of the needle
Figure 6a. Crystal structure of an MxiH monomer. Ribbon diagram of MxiH (molecule A) coloured 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 labelled. Taken from Deane et al., 2006b.
Figure 6b. Construction of a pseudo-atomic model of the needle. The structure of MxiH (molecule A) was repositioned into our 16 Å resolution electron microscopy reconstruction of the needle by using both the helical parameters of the needle we had determined previously (Cordes et al., 2003) and the shape of the reconstructed volume as a guide. Each subunit was rotated 64.3º about long axis and translated 4.31 Å along long axis. See Deane et al., 2006b.
MxiH needle mutants have altered distal tip complex compositions
Although we are still searching for a signal transmission path through or along the needle, further studies of the MxiH mutants revealed that they have fascinating defects in the composition of their needle distal tip complexes. The major component of this complex was first identified in the T3SS of Yersinia spp. by Mueller et al. (2005) as the LcrV protein, also known as the only protective antigen against plague. We had proposed some years previously that IpaD, which we had identified as required for the insertion of IpaB/IpaC translocon (Blocker et al., 1999), was the functional homolog of LcrV (Blocker et al., 2003; Picking et al., 2005). While searching for the needle assembly cap, we then serendipitously discovered a way to further purify polymerised needles (Cordes et al., 2003) that allowed identification of specifically attached minor components (Veenendaal et al., 2007). These components revealed themselves to be not the assembly cap as we had initially hypothesised, but instead IpaD and IpaB, in a stoichiometric ratio of approximately 1:10. We showed by immuno-electron microscopy that these proteins where located only at the distal tip of needles. When we examined the distal tip of MxiH mutant needles we found that these had strikingly altered needle tip complex compositions, which grossly correlated with the ability of these mutants to sense host cells, regulate secretion and insert the translocation pore. From this work it hence appears that the interaction between IpaB and IpaD at needle tips is key to host cell sensing, orchestration of IpaC secretion and its subsequent assembly at needle tips. We thus proposed that this is the translocon assembly pathway that allows insertion into the host cell membrane of a translocation pore that is continuous with the needle.
Figure 7. Analysis of Ipa proteins associated with purified needles derived from MxiH point mutants.
Figure 7. Analysis of Ipa proteins associated with purified needles derived from MxiH point mutants. Needles were purified from wild-type Shigella overexpressing MxiH and mxiH- strains overexpressing the indicated MxiH mutants. The secretion phenotypes of the different mutants is indicated in red (top panel): WT, wild-type; CS, constitutive secreter; NI, non-inducible. The samples were normalised for the amount of MxiH (arrowheads) by Silver stained SDS-PAGE (bottom panels) and analysed by Western blotting. The antibodies used for the blots are indicated on the left. The high signal obtained for IpaC in P44A+Q51A actually corresponds to stoichiometric amounts of IpaC relative to IpaD and IpaB when visualized by Silver stain. Please note also that this latter mutant retained the ability to insert wild-type like translocons into red blood cells during contact hemolysis, even though it was non-invasive when assayed on HeLa cells. Found in, and further details available, from Veenendaal et al., 2007.
A working model for the assembly of the tip complex and translocon
In parallel, the crystal structure of IpaD was solved in collaboration with the Lea laboratory at Oxford (Johnson et al., 2007). Although it could not directly be docked onto our needle model, a pentamer of LcrV -its functional homologue from Yersinia- could and this generated model of the tip complex that grossly resembled that observed by Mueller et al. 2005, suggesting that any completely formed tip complex assembly contained maximally 5 such molecules (Deane et al., 2006; Blocker et al., 2008). Taken in combination with our own biochemical data, this lead to the model for tip complex assembly, host cell sensing and translocon insertion shown immediately below (Veenendaal et al., 2007; Blocker et al., 2008).
Figure 8. Model for needle tip-mediated host cell sensing and translocon insertion
Figure 8. Model for needle tip-mediated host cell sensing and translocon insertion. Left, assembly of the tip complex on a resting needle prior to host cell contact. B, C and D stand for IpaB, IpaC and IpaD, respectively. Middle, contact of IpaB with the host cell leads to its membrane insertion and a conformational change relayed by IpaD and the needle to the base of the secreton. Right, activation of secretion inside the bacterium leads to IpaC secretion, its association with the needle tip and insertion into the host cell membrane. This finalises translocon formation. Top, cross-sections through the middle of the needle. Bottom, top views from the distal end of the needle.
Our next research directions
We are investigating the biological means by which the needle and its tip complex become assembled and the mechanisms, which regulate these events.
We are seeking, within the mature secreton, the original and physiological secretion-inducing event, through mutagenesis and cell biological studies.
We are also taking a number of different biochemical approaches to probe the detailed assembly/function of the tip complex and the process of translocon assembly during the initial activation phase.
Finally, we are working to generate higher resolution views of the type III secreton base or 'needle complex' of the needle itself (Fujii et al., in preparation) and of its distal tip complex, by cryo-electron microscopy and single particle analysis. We hope that once we can visualise how these elements all exactly fit together in wild type and needle/tip mutant structures, it will be easier to understand how they function in a coordinated fashion in sensing and transmitting the host cell-contact activation signal.
It is our long term aim, in the coming years, to try to reconstitute the process of T3SS-mediate protein export across inverted inner membrane vesicles in vitro, so as to be able to dissect the mechanics and energetics of the export process in molecular detail. We would also like to understand whether structural and/or biochemical changes in the secretion machinery are involved in leading to timely, ordered secretion of different effectors during the Shigella life cycle and how these proteins function to mediate specific aspects of the cell biology of this infection.
Last but not least, we have begun to translate our recent work into prophylactic vaccine development against shigellosis and novel therapeutic drugs against Gram-negative pathogens carrying T3SSs.
Conclusion
Thus, this project seeks to provide novel and fundamental insight into the workings of type III secretons. This knowledge should enrich our understanding of the diverse mechanisms of protein targeting in prokaryotes and eukaryotes. Type III secreton are highly complex but self-contained devices and as such they are a splendid example of a "molecular machine", the assembly and function of which can truly be studied in an integrated approach, rather than simply an analytical one. Such studies should lead to technical developments of general interest within the biochemical and structural protocols taken. Importantly, what we learn could be used to design specific inhibitors of these systems, which might become important and broad-range new anti-bacterial agents at a time when antibiotic resistance is increasing.