Type III secretion systems and bacterial flagella: Insights into their function from structural similarities

The flagellar chaperone FlgN.

How is the choice made between signals operating at different levels for proteins that have both export routes? The chaperones may regulate the transcription and translation of two different mRNAs encoding such effectors. Flagellar FlgN binds two proteins, FlgK and FlgL, exported upon hook completion (50). FlgN is also required for translation of the anti-σ factor-encoding flgM class III transcript but not its earlier transcribed class II transcript (51). FlgM translated from the class II transcript is retained in the cytoplasm, perhaps because FlgN is then binding FlgK and FlgL. However, when it is translated from the class III transcript, FlgM is secreted cotranslationally. Upon secretion of FlgM, transcription from class III promoters initiates via a specific σ factor.

Evidence for regulatory chaperones from virulence TTSSs.

We propose that the equivalent of FlgN in TTSSs is the chaperone of the translocator complex IpgC (binds IpaC and IpaB of Shigella, Fig. 2A; see Supporting Text). Increasing the level of free IpgC (Fig. 2B) leads to premature expression of genes encoding putative late effectors and even to secretion of their products (IpaHs and Ops, Fig. 2D, point 10). Free IpgC interacts with the transcription factor MxiE to activate transcription from these late gene promoters (ref. 45; Fig. 2D, point 8). Mutants in Shigella ipaD or ipaB and flagellar flgK and flgL demonstrate constitutive secretion of class III products (43, 52). Thus, cotranslational secretion of effectors produced from class III mRNAs is contact-independent (Fig. 2, point 10). However, contact-dependent secretion of effectors may remain possible because some return to storage before secretion (Fig. 2, points 5 and 6). Termination of secretion may occur by mRNA degradation when the effector becomes abundant enough to compete for chaperone binding with the mRNA encoding it or proteins secreted downstream in the pathway (ref. 53; Fig. 2D, point 7).

Hence, cotranslational secretion (Fig. 2 C and D) may involve a chaperone/transcription factor complex activating the relevant DNA region(s) and later binding the mRNAs and/or any proteins derived from them to the secreton. If and how chaperone-protein/mRNA complexes become localized at the C-ring is unknown (54). No TTSS equivalent of FlgM has been identified (Fig. 2 B and C, points 2 and 3). In assembly of flagella and TTSSs, the regulatory circuit involves a morphogenic switch from exporting hook/needle to filament/effector components (refs. 55 and 56 and Supporting Text). Flagellum assembly is a continuum, whereas TTSSs arrest at needle completion.

Are effector motifs recognized by the secreton?

Recently determined structures of effectors alone or in complex with their chaperone suggest that (i) the binding of the chaperone partially unfolds the underlying effector region, preventing the N-terminal amphipathic helix from folding against the rest of the protein, and (ii) the part of the effector beyond the chaperonebinding domain is folded during storage (see Supporting Text). If and how part(s) of the secreton recognize the opened helix is unknown. How a motif as common as a loose amphipathic helix serves as a selective secretion signal is unclear. The hypothetical localization provided by chaperones to the C-ring may be key to specificity.

In what state do the substrate proteins travel down the central channel?

The channels through flagella and TTSSs could accommodate several α-helices or a β-sheet domain, short or unfolded into hairpins. The structure of flagellin (11, 12) shows that it could readily unfold into subdomains <30 Å in diameter, connected by linearized regions. The mode of unfolding of tightly folded TTSS effector domains (60–63) is less obvious. Some proteins have high rates of conformational breathing, which is key for their import into mitochondria. Experiments with chimeric proteins show that the ability to readily unfold is required for TTSS export (64) and used to exclude cytoplasmic proteins from export.

What are the energizers of posttranslational and cotranslational secretion?

The flagellar ATPase FliI is required for export of all flagellar proteins except the outer membrane components (5). Without it only the inner membrane and cytoplasmic components are assembled. Mutants in homologous TTSS ATPases display analogous phenotypes (65). Does cotranslational secretion occur by docking of the ribosome to the cytoplasmic part of the TTS machine like cotranslational export across the membrane of the endoplasmic reticulum? An empty flagellar C-ring could only accommodate two ribosomes, the protein channels of which could not directly dock to the HBB without a gap being left. Therefore, cotranslational secretion is probably also driven by the ATPase and hence indirect.

How is energy transduced by the export motor during secretion?

The ATPases interact with cytoplasmic components of TTSSs or flagella but the function(s) of these interactions are mostly unidentified (66, 67). The biological cycle of the enzyme is unknown and its localization is debated [cytosolic or membrane-bound (69)?]. How might these ATPases catalyze processive protein export? Spa47 (the Shigella FliI homolog) shares 33% amino acid identity with the β-subunit of F1-ATPase. Proteins with >30% sequence identity have a high probability of sharing similar structures (69). Active F1-ATPase is a heterohexamer consisting of alternating α- and β-subunits with a γ-subunit inserted in a central channel where it rotates during the catalytic cycle (70). No equivalent of the α-subunit of F1-ATPases is found within flagellar or TTSS-encoding operons, so we assume that the type III export motor is a homohexamer. When modeled on the F1 structure, Spa47 fits at the inner membrane base of our NC structure (Fig. 3). It would contain a central channel aligned with the one found within the NC and of similar diameter to it, through which the proteins could be secreted (see Supporting Text).

Evidence for a pore.

The insertion of a bacterial pore into the host cell membrane (Fig. 4) is central to translocation of effectors. Yersinia, Shigella, and enteropathogenic Escherichia coli TTSSs form 1.5- to 3-nm (in diameter) pores in eukaryotic cells (73) by using at least two bacterial proteins, YopB/IpaB/EspD and YopD/IpaC/EspB, sharing disposition of hydrophobic α-helical regions. IpaB/IpaC and EspD/EspB are inserted into eukaroytic cell membranes during this process (8, 72, 73). Association of YopB, IpaB, IpaC, and SipB individually with artificial membranes does not generate pores. Incubating secreting Yersinia with liposomes leads to association of YopB and YopD with liposomes. This is necessary and sufficient for detection of ionically aselective channels with 105 pS of conductance (74). Pore protein insertion is mysterious. Whether eukaryotic cell receptors are used by TTSSs to initiate activation and/or pore insertion is also unclear (details and species-specific modifications are discussed in Supporting Text).

A model for pore function.

Might the pore “passively” translocate effectors like the flagellar cap FliD allows insertion of flagellin at the flagellar tip (14)? FliD is a pentamer, whereas the flagellar filament contains 5.5 subunits per helical turn. There is a symmetry mismatch that may cause FliD to rotate by 6.5° per step as it catalyzes insertion of new flagellin subunits by using the energy provided by the cytoplasmic ATPase during substrate unfolding and stored in the extended flagellin during export. IpaC may be equivalent to FliD, whereas IpaD and IpaB may be analogous to FlgK and FlgL (Fig. 1). This would explain why pore formation by most TTSSs does not cause host cell lysis: the pore is closed from the outside and inside (Fig. 4).

Continuity between the secreton needle and the translocation pore is necessary for probable energetic coupling of secretion, needle growth, pore insertion, and effector translocation. Continuity is provided in the flagellum by packing of the components into a hollow helical superstructure. In TTSSs, evidence for continuity is still circumstantial. For instance, continuity between needle and pore is suggested by the similarity of the inner diameters of the pore and of the NC channel (refs. 8 and 10; Supporting Text).