Visualization of the type III secretion sorting platform of Shigella flexneri

Shigella Minicells as a Model System for Elucidating T3SS Structure.

S. flexneri is an important diarrheal pathogen that uses its Mxi–Spa T3SS to transport effector proteins into human colonocytes, consequently altering host cell signaling to promote bacterial invasion (22). The Shigella T3SS is encoded by ∼25 genes located in the mxi, spa, and ipa operons on a large 230-kb virulence plasmid. Similar to the SpaO–OrgA–OrgB complex in S. enterica (5), homologous proteins Spa33, MxiK, and MxiN in S. flexneri are known to form a high molecular weight complex required for needle formation and substrate secretion (20, 23).

To achieve high-resolution images of intact injectisomes and the cytoplasmic complex, we constructed Shigella strains that produced minicells significantly smaller than normal bacterial cells (Fig. 1A and SI Appendix, Figs. S1 and S2). The purified minicells were able to induce contact hemolysis (SI Appendix, Fig. S3), which is known to be correlated with both phagosomal membrane lysis and tied to the secretion of three Ipa proteins (IpaB, IpaC, and IpaD) (24, 25). Furthermore, our results showed that the purified minicells were able to maintain an intimate association with red blood cells (RBCs) via the T3SS needle in the absence of any added adhesins (Fig. 1B). This is consistent with a previous report that minicells from invasive Shigella strains retained the invasive phenotype (26). A recent study from Salmonella provided further evidence that the T3SS machines in minicells are competent for protein translocation into mammalian cells (27).

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Fig. 1.

Cryo-ET of S. flexneri minicells reveals intact T3SS and its cytoplasmic structure. (A) A cryo-EM image shows tiny S. flexneri minicells with diameters of ∼0.3 μm. Purified S. flexneri minicells are able to interact intimately with an RBC (B). Yellow arrows highlight the five minicells adhering to the RBC, whose membrane is indented at each contact point. A tomographic slice reveals that an injectisome (indicated by a cyan arrow) is directly involved in the interaction with the host cell membrane (C). (D) A central slice and (E) a 3D surface rendering of a tomographic reconstruction of a typical minicell show multiple injectisomes embedded in the cell envelope, including outer membrane (OM) and cytoplasmic membrane (CM). A central section (F) and a 3D surface rendering (G) of the subtomogram average of the intact injectisome show OM, CM, peptidoglycan (PG), basal body, and needle in detail. Importantly, there is a large cytoplasmic complex that is 32 nm in diameter and 24 nm in height (F). Three cross-sections (indicated in F) of the cytoplasmic complex show sixfold symmetric features (H–J). The bottom view (K) and a side view (G) of the injectisome present the apparent discontinuity of the outer ring of six “pod-like” densities. The six pods (colored in red) are linked to the central hub (orange) by radially arranged (spoke-like) linker densities (yellow).

Cryoelectron Tomography of Shigella Minicells Reveals Intact T3SS Machines.

We exploited a newly developed direct electron detector and high-throughput cryoelectron tomography (cryo-ET) to visualize frozen-hydrated minicells derived from WT Shigella. Two different procedures were used to collect and analyze the tomographic data. Initially, low-dose tilt series were collected at 15,500× magnification and 2 × 2 binning, similar to our previous procedure (28). Subsequently, we collected tilt series in dose fractionation mode, which enabled us to correct the motion-induced image blurring (29) and effectively improve the quality of the final reconstructions. A typical 3D reconstruction of a Shigella minicell revealed multiple injectisomes embedded in an intact cell envelope (Fig. 1 D and E and Movie S1). We also observed injectisomes from Shigella minicells directly connected with a host RBC (Fig. 1 B and C and Movie S2), suggesting that the injectisomes in our minicells were intact and could mediate host cell contact.

To determine 3D structures of intact injectisomes at high resolution, subtomogram averaging was used to analyze 4,631 injectisome subtomograms (SI Appendix, Figs. S4 and S5). We determined the intact injectisome structure at 2.7 nm resolution (SI Appendix, Fig. S6) and observed a previously uncharacterized cytoplasmic complex immediately beneath the cytoplasmic membrane and basal body (Fig. 1 F–K and SI Appendix, Fig. S7). This complex contained six pod-like structures 32 nm in diameter and 24 nm in height (Fig. 1 F–K). The top portion of each pod was associated with the cytoplasmic membrane, whereas the bottom portion was connected to a distal short cylinder by six spoke-like densities (Fig. 1 F and G and SI Appendix, Fig. S7). There are relatively weak densities between the pod and the cytoplasmic membrane, perhaps suggesting that the cytoplasmic complex is loosely attached to the basal body.

The six pods are distinct from the dense, contiguous arrangement of proteins in flagellar C ring structures (15⇓⇓⇓–19). Their connections with the basal body appear to be delicate, potentially explaining why they typically are not retained during purification of basal bodies (9, 10). The pods also were not resolved in a recent in situ structure from whole Yersinia cells (11). Furthermore, our initial effort in determining the injectisome structure from osmotically shocked cells failed to reveal the pod densities (SI Appendix, Figs. S5 D and H and S8), indicating that the basal body is relatively stable, whereas its interactions with the cytoplasmic complex are more delicate and can be disturbed by harsh treatment of the cells (SI Appendix, Fig. S9).

The Presence of MxiN or Spa33 Has a Dramatic Impact on T3SS Machine Structure and Function.

To define the requirements for assembling the complex cytoplasmic structure, we constructed minicell-producing strains of ∆mxiN and ∆spa33 deletion mutants of S. flexneri. Both MxiN and Spa33 are required to assemble functional needles, but not the basal body (20). As expected, we detected basal bodies, but not extracellular needles, in the minicells derived from both deletion mutant strains (Fig. 2). The bottom portion of the needle, which is inserted into the basal body, also was absent (Fig. 2 C and H). These findings indicate that both MxiN and Spa33 are important for translocation of MxiH, the major needle component. The secretion channel appeared to be closed at the base, presumably to prevent leakage (Fig. 2 C and H), reminiscent of findings for the flagellar motor of Borrelia burgdorferi (28).

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Fig. 2.

Injectisome structural differences in S. flexneri minicell mutants lacking either MxiN or Spa33. Analysis of ∆mxiN (A–E) or ∆spa33 (F–J) minicells is shown. Depicted are representative slices of cryo-ET reconstructions of a ∆mxiN minicell (A) or a ∆spa33 minicell (F), followed by the corresponding zoomed-in views (B and G), averaged structures (C and H), and two 3D surface renderings (D, E, and I–J). Both mutants lack the needle (yellow arrows) and the central hub of the cytoplasmic domain (Fig. 1 G and J). The ∆spa33 injectisomes also lack the six outer densities (pods) of the cytoplasmic domain (red arrows and red-colored densities seen in the ∆mxiN injectisomes). The predicted location of the MxiA complex is indicated in purple in the surface renderings.

In the T3SS map derived from ∆mxiN minicells, the six pods and a torus-like structure were detected beneath the basal body (Fig. 2 C–E). The torus-like structure (purple in Fig. 2), which was observed previously (11), has been proposed as a nonameric MxiA ring (13). The presence of the torus-like structure is not affected by the ∆mxiN mutation. In contrast, both the spoke-like densities and the central “hub” are absent in the ∆mxiN mutant (Fig. 2 C and D). Similar to the ATPase FliI in bacterial flagella (17), the hub is likely formed by the Spa47 ATPase. The spoke-like densities probably consist of MxiN, which interconnects the adjacent pods and the central hub. This is consistent with the previous prediction of an indeterminate number of spoke-like linkers comprised of MxiN that connect Spa47 with Spa33 (13). Apparently, the absence of MxiN significantly reduces the colocalization of the Spa47 ATPase with the remainder of the complex, thereby dramatically impacting substrate secretion.

Spa33 is thought to be a major component of the Shigella cytoplasmic complex, as demonstrated by immunogold EM labeling of Spa33 at the cytoplasmic side of purified basal bodies and biochemical evidence of Spa33 interactions with Spa47, MxiN, and MxiG (20). Our structural studies indicated that the basal bodies assembled in ∆spa33 minicells completely lack this cytoplasmic complex (Fig. 2 H–J). Furthermore, we provide structural evidence that the Spa47 ATPase fails to engage with the MxiA export gate beneath the basal body in the absence of Spa33 (Fig. 2 H–J). We infer from this that Spa33 is an essential component of the pods and provides the docking site for the Spa47 ATPase via the MxiN linkers.

Molecular Model of the T3SS Machine in Situ.

To characterize the overall architecture of the injectisome, we fitted the existing basal body structure of the Shigella T3SS (10) into the intact injectisome map described here (Fig. 3 A and B). The basal body structure matched well with the periplasmic portion of the map (Fig. 3 A and B). Compared with the existing basal body, however, the intact injectisome structure contains extra densities, including outer membrane, cytoplasmic membrane, peptidoglycan, the cytoplasmic complex, and an element associated with the outer membrane (Fig. 3A). The new element may be formed by the pilotin MxiM, which is known to interact with the ring-forming protein MxiD (30). To determine how well molecular structures could dock into our injectisome map, we built a model for MxiG based on the recent pseudoatomic model of the Salmonella PrgH (homolog of MxiG; SI Appendix, Tables S1 and S2) (31). The periplasmic domain fitted well into our map, but an additional shift of the cytoplasmic domain (MxiG_C) was required to position it immediately underneath the cytoplasmic membrane (SI Appendix, Fig. S10). This alteration is apparently needed to facilitate MxiG–Spa33 interactions (20) and is also consistent with the recent model of the Yersinia YscD (homolog of MxiG; SI Appendix, Table S1) (11).

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Fig. 3.

A molecular model of the T3SS injectisome. The isolated Shigella T3SS basal body (10) is fitted onto the intact injectisome map in a central section (A) and a surface rendering (B). Extra densities are apparent, including outer membrane (OM), cytoplasmic membrane (CM), peptidoglycan (PG), a large cytoplasmic complex, and a base element (green arrowhead) where it interacts with the OM. Models of several cytoplasmic proteins (MxiA_C, Spa47, Spa13, Spa33) are fitted in the surface rendering map. Zoomed-in views of the cytoplasmic portion of B are shown from the side (C), top (D), and bottom (E). The Spa47 ATPase hexameric ring (the homolog of V-ATPase, PDB ID code 3J0J, orange) together with Spa13 (the homolog of FliJ, PDB ID code 3AJW, green) is docked into the central hub of the map. Spa13 is long enough to interact with the nonameric ring of MxiA_C (different shades of purple), which fits well into the torus-like structure near the cytoplasmic membrane (C and D). Underneath the MxiG_C ring (the homolog of PrgH_C, PDB ID code 3J1W, dark green), six spa33-encoded complexes form the proposed sorting platform. We place the Spa33 homologs (FliN tetramer, PDB ID code 1YAB, red) into the bottom part of the pod. MxiN forms the spoke-like linker (yellow) that interacts with both the hydrophobic patch (blue) in Spa33 and the C-terminal domain (cyan) of Spa47 as shown in the bottom view (E).

Another key component of the cytoplasmic side of the injectisome is MxiA, which consists of a transmembrane domain and a large cytoplasmic domain (MxiA_C). As MxiA_C has been postulated to form a nonameric ring essential for secretion (13), we docked the crystal structure of this MxiA_C nonameric ring into the torus-like structure, which extends ∼6 nm from the cytoplasmic membrane. The excellent fit supports the proposed juxtapositioning of the MxiA_c nonameric ring beneath the basal body (Fig. 3 B–D and Movie S3) (13). Furthermore, in both ∆mxiN and ∆spa33 mutants, the torus-like structure remained intact (colored purple in Fig. 2), consistent with a direct linkage of the MxiA_C nonameric ring with the MxiA transmembrane domain.

The main component of the hub, Spa47, energizes secretion of effector proteins and shares significant amino acid similarity with FliI and V-ATPases (SI Appendix, Table S3). By analogy with a FliI hexamer, which is positioned within the in situ flagellar motor (17), a Spa47 hexamer likely accounts for density present ∼10 nm beneath the MxiA_C ring (Fig. 3A). Spa47 is also known to interact with MxiN and Spa13 (the homolog of FliJ; SI Appendix, Table S4) (20, 32, 33). Together, we built a model of the Spa47–Spa13–MxiN complex (Movie S3). The C-terminal domains of Spa47 are connected to the MxiA_C ring via Spa13, whereas the N-terminal domains of Spa47 interact with MxiN (Fig. 3 B–E).

Spa33 is likely a main constituent of the pods. The C-terminal region of Spa33 is homologous to the flagellar protein FliN (SI Appendix, Table S5), which has been proposed to form a homotetramer (34, 35) at the bottom of the flagellar C ring. Recent studies showed that the Spa33 ortholog YscQ from Yersinia exists as two translation products: intact YscQ and a C-terminal fragment (36). Similar to FliN, the C-terminal domain structure of YscQ reveals an intertwined homodimer (36). Spa33 may also form a multimeric complex. The tetramer model of FliN fills well into the bottom portion of each pod density, suggesting that the C-terminal fragment of Spa33 likely forms a stable tetramer and an intact Spa33 and another protein MxiK could account for the remainder of the density of the pods (Fig. 3D), as they are known to form a high molecular weight complex (20). It remains to be resolved how these proteins are elegantly organized within the pods.

The discovery of the spa33-encoded hexameric complex and its physical connections with the ATPase highlights the similarities and differences between an injectisome and a bacterial flagellum (Fig. 4). Both systems contain an export gate powered by the proton motive force and a cytoplasmic ATPase complex (21, 37, 38). A similar integrated network and molecular mechanism are likely used for the recognition and secretion of specific substrates. Nevertheless, the six discrete pods observed in the injectisome are distinct from the flagellar C ring, which is not only indispensable for substrate secretion but also essential for flagellar rotation and switching (Fig. 4).

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Fig. 4.

Comparative structural models of injectisomes and bacterial flagella. The cytoplasmic hexamer of the Spa33 complex positions the Spa47 ATPase (via MxiN) directly beneath and in line with the export gate, beginning with the MxiA cytoplasmic domain. In contrast, the flagellar C ring contains multiple copies of FliG, FliM, and FliN that are involved in secretion, rotation, and switching. FliH, the homolog of MxiN, is likely sufficiently flexible to interact with different FliN proteins at the bottom of the C ring. The outer membrane (OM), cytoplasmic membrane (CM), peptidoglycan (PG), a large cytoplasmic complex, and the basal body are colored consistently as those in previous figures.

The conserved cytoplasmic complex in S. enterica T3SS provides a sorting platform that determines the order of protein secretion (5). We propose that the hub–spoke–pod complex functions specifically as the sorting platform. The Spa47 ATPase within the complex is aligned with the central channel of the MxiA gate, suggesting that they function together to engage and secrete needle components and effector proteins. The multiple contacts we identified among components of the cytoplasmic complex are likely to be important for the integrity of the sorting platform and its functions in needle formation and subsequent substrate secretion (Fig. 4). We postulate that this platform functions in the recruitment of needle components and effectors either without or within a complex with dedicated chaperones. The sorting platform delivers these substrates to the Spa47 ATPase for chaperone dissociation and substrate unfolding, whereupon the substrates are delivered to MxiA and the export gate for secretion (Fig. 4 and Movie S3). Although many questions remain regarding recognition of chaperone/substrate complexes and the temporal control of substrate selection, our architectural definition of the Shigella T3SS sorting platform provides a structural basis for further dissecting the mechanisms underlying the T3SS-mediated secretion and pathogenesis.