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

Scanning electron micrograph of the rod-like Shigella flexneri entering a HeLa cell
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 flexneri is 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

Morphological identification and analysis of the type III secretons of S. flexneri
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.

Larger version of figure 2

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 cell-contact mediated protein translocation step is the main aim of the ongoing work in the laboratory (Blocker et al., 2003; Blocker et al., 2008).

Initial structural analysis of the NC by electron microscopy
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.

Larger version of figure 3

This protein transport process can be broken down into several steps:

      1. Assembly of a mature T3SS
      2. Activation of the type III secreton by direct physical contact with host cells
      3. Intrabacterial energisation of secretion
      4. Secretion/assembly of the bacterial translocation pore within the host plasma membrane
      5. Secretion of the effector proteins to be translocated into the host cytoplasm
      6. 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 years has 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.

Three-dimensional reconstruction of needle complexes with C12 symmetry
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.

Larger version of figure 4

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 Prof. 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 (Yonekura et al., 2000).

Helical three-dimensional (3D) reconstruction of the needle using electron microscopy (verified also by X-ray fiber diffraction)
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.

Larger version of figure 5

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).

MxiH mutagenesis findings

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 resolution electron density map of the needle

More recently, in collaboration with Prof. Keiichi Namba’s laboratory in Osaka, we provided a higher resolution three-dimensional reconstruction of the needle, derived from images collected using electron cryomicroscopy (Fujii et al., 2012). This map has recently been used by others, who combined it with solid-state NMR, to propose an atomic model of the T3SS needle (Loquet et al., 2012; Demers et al., 2013; Demers et al., 2014). It is now beyond doubt that the N-terminus of the needle subunit faces the outside of the polymer. However, because the method used to derive this model is entirely novel, it remains to be validated by independent means. Puzzlingly for instance, this atomic model does not readily explain the phenotypes of the mutants above.

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 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).  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 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.

Analysis of Ipa proteins associated with purified needles derived from MxiH point mutants
Figure 6. Analysis of Ipa proteins associated with purified needles derived from MxiH point mutants.

Figure 6. 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).

Model for needle tip-mediated host cell sensing and translocon insertion
Figure 7. Model for needle tip-mediated host cell sensing and translocon insertion

Figure 7. 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.

Three-dimensional electron microscopy reconstruction of the tip complex (TC)

In the last few years, we sought to test this model directly using cysteine-mediated crosslinking and three-dimensional reconstruction from electron micrographs (Cheung et al., 2014). We showed that TCs from a ΔipaB strain contain five IpaD subunits while the TCs from wild-type can also contain one IpaB and four IpaD subunits. Electron microscopy followed by single particle and helical image analysis was used to reconstruct three-dimensional images of TCs at ∼20 Å resolution. Docking of the IpaD crystal structure, constrained by the crosslinks observed, reveals that TC organisation is different from that of all previously proposed models (see below). Our findings suggest new mechanisms for TC assembly and function. The TC is the only site within these secretion systems targeted by disease-protecting antibodies. By suggesting how these act, our work will allow improvement of prophylactic and therapeutic strategies.

Figure 8: Three-dimensional reconstruction of the TC and needle from the wild-type strain.

Figure 8: Three-dimensional reconstruction of the TC and needle from the wild-type strain. (A) 24 Å resolution electron density map of the TC and needle from wild-type Shigella reconstructed using negative-stain EM, displaying view from the side (left) and top (right, top). Slices through the TC, TC/needle junction and needle portions are displayed of the lower right panels. (B) Electron density map of needle reconstructed using cryoEM (Fujii et al., 2012) (EMD-5352) docked into the proposed needle portion of our TC-needle reconstruction with protofilament P1, containing the lowest MxiH subunit atop the needle helix below the highest and largest subunit in the TC. (C) Isolated density of the TC following subtraction of the density corresponding to the needle. Four copies of a partial IpaD crystal structure (derived from PDB ID 2j0o(Johnson et al., 2007), molecule A as outlined in the main text) were docked into the individual subunit densities, their location colour coded as in Fig. 1A (right), i.e. the lightest subunit corresponding to position β. Black arrows indicate upper bulges assumed to correspond to the C-terminal globular domains of IpaD, while white arrows indicate lower bulges assumed to correspond to IpaD N-terminal domains (not found in the modified 2j0o structure). (D) Isolated view of the wild-type TC with either IpaDβ (top) or IpaDε (bottom) docked in, to show larger size of subunit in position α. The new map is displayed throughout at a contour level of 0.0688 in Chimera. Scale bar, 70 Å.

Regulation of type III secretion from the bacterial cytoplasm

Finally we have been studying the regulation of T3SS from within the bacterial cytoplasm. Within the T3SS base lies the protein export apparatus (EA), formed of inner membrane and peripheral/cytoplasmic proteins, which energise the secretion process. T3SSs are activated by physical contact with host cells and then allow rapid, hierarchical secretion of effectors. Recent evidence suggests this is mediated by the interplay of conserved, T3SS-secreted, negative regulators acting externally, at the needle tip, and also on the EA, within the bacterial cytoplasm (Martinez-Argudo and Blocker, 2010). Surprisingly, IpaD, the main tip complex component acts both internally and externally to regulate the T3SS (Roehrich et al., 2013), alongside MxiC which acts internally. Given recent work on the Pseudomonas T3SS, it may well be that removal of IpaD from the cytoplasm activates the rate of export of the apparatus, whilst subsequent secretion of MxiC regulates its substrate selectivity (Lee et al., 2014). But, how is not yet understood.

Figure 9: Working model for the regulation of T3SS activation in Shigella.

Figure 9: Working model for the regulation of T3SS activation in Shigella. Modified from Martinez-Argudo and Blocker (2010); early effectors are in light pink, regulators in dark pink & purples. The TC may prevent premature effector secretion by allosterically constraining the T3SS in a secretion “off” conformation without blocking the secretion channel (Cheung et al., 2014). Upon physical contact of the TC with host cells (Roehrich et al., 2013), a signal, termed Signal 1, is transmitted via the TC (Roehrich et al., 2013) and needle (Kenjale et al., 2005) to the cytoplasm where it triggers secretion (Martinez-Argudo and Blocker, 2010). Next, translocators are secreted to form the pore in the host cell membrane. Successful pore formation at the needle tip generates Signal 2, also transmitted via the needle (Kenjale et al., 2005; Martinez-Argudo and Blocker, 2010), that allows inactivation or T3S-mediated removal of a conserved cytoplasmic regulatory protein, MxiC in Shigella. Third, early effector proteins are secreted and translocated into the host cell (Martinez-Argudo and Blocker, 2010) and late effector expression is activated

Our next research directions

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 and biophysical approaches to probe the detailed 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', by cryo-electron microscopy and single particle analysis and by electron cryotomography. We hope that once we can visualise how these elements all exactly fit together in wild type and 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.

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