The Locus of Enterocyte Effacement Type III Secretion Specificity Switch: the Devil's in the Data for a Common Mechanism

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The locus of enterocyte effacement (LEE) is a pathogenicity island present in enteropathogenic Escherichia coli (EPEC), atypical enteropathogenic E. coli (ATEC), and enterohemorrhagic E. coli (EHEC) (for a recent review, see reference 17). They are all nasty pathogens. Like all virulence-associated type III secretion (T3S) systems, the LEE system comes with an array of effector proteins, each interesting in its own right, that enter host cells and manipulate their physiology to facilitate pathogenic processes. One aspect of the LEE system that is not well characterized is that of LEE injectisome structure and assembly. This is not surprising, given the extensive characterization of virulence-associated injectisome structure and assembly for other animal pathogens, in particular, the Salmonella, Shigella, and Yersinia systems, and the related flagellar structure and assembly with its associated T3S system.

T3S systems all regulate the assembly of the injectisome structure and protein secretion. The T3S protein family that performs this common function includes the Yersinia YscP protein and the flagellar FliK protein. YscP and FliK control the length of the needle of the Yersinia injectisome and that of the flagellar hook, respectively (1, 6). Length control works in coordination with a secretion specificity switch through an interaction between YscP/FliK and an integral membrane component of their respective T3S systems. YscP and FliK are not homologous in amino acid sequence, which makes them difficult to identify by standard homology searches. In an accompanying paper in this issue, Monjarás Feria et al. identify the Orf16 protein of the LEE T3S system of EPEC and provide a thorough set of experiments that allow them to rename Orf16 as EscP, the LEE T3S system member of the YscP/FliK protein family (13). Monjarás Feria et al. do a terrific job of characterizing the role of EscP in aspects of LEE injectisome assembly related to needle length control, substrate secretion, and the secretion specificity switch mechanism (13). The assembly of the LEE injectisome, based on the assembly pathway reported for the Yersinia T3S system, is diagrammed in Fig. 1 (2). The homologous proteins for a subset of T3S systems are listed in Table 1.

Fig 1.

Diagram of the assembly of the LEE injectisome based on the assembly pathway reported by Diepold et al. for the Yersinia T3S system (2). They propose that outer membrane (OM)-associated EscC (secretin) and the inner membrane (IM) EscD cytoplasmic-ring structure (M ring) assemble first (A) and then core membrane-associated (MA) components EscR, -S, -T, and -V enter the M ring (B). However, the evidence for this is not very strong. This is followed by the addition of an inner membrane structure composed of EscJ subunits and the addition of EscU to the MA core secretion components to complete the T3S system (C). The EscQ C ring provides an affinity site for the EscN-EscL ATPase complex to pilot secretion substrates to the cytoplasmic base of the T3S system (D). Inner rod and needle structures are assembled onto the basal structure (E). Upon needle completion, EscP catalyzes a secretion specificity switch to allow filament and translocon assembly on the cell-distal portion of the injectisome (F).

Table 1.

Members of a subset of the type III injectisome protein family and the E. coli/Salmonella flagellar biogenesis system^a

Type III system	Secretin	M ring	MA core	M/C core	IM ring	M/C core	C ring	Inner rod	Length control	Needle/hook	ATPase complex
Escherichia LEE	EscC	EscD	EscRST	EscV	EscJ	EscU	EscQ	EscI	EscP	EscF	EscLN
Yersinia	YscC	YscD	YscRST	YscV	YscJ	YscU	YscQ	YscI	YscP	YscF	YscKLN
Salmonella Spi1	InvG	PrgH	SpaPQR	InvA	PrgK	SpaS	SpaO	PrgJ	InvJ	PrgI	OrgB, InvCI
Salmonella Spi2	SsaC	SsaD	SsaRST	SsaV	SsaJ	SsaU	SsaQ	SsaI	SsaP	SsaG	SsaKNO
Shigella	MxiD	MxiG	SpaPQR	MxiA	MxiJ	SpaS	SpaO	SpaM	Spa32	MxiH	MxiK, Spa47
Flagella	FlgHI	FliG	FliPQR	FlhA	FliF	FlhB	FliN	FlgBCFG	FliK	FlgE	FliHIJ

^aThe EscC family includes the secretin component that forms in the outer membrane (Fig. 1). The EscD family forms a ring in the inner membrane (M ring). Inner membrane-associated core (MA core) components include the EscRST family of proteins, while the EscV family inner membrane core (M/C core) component contains a large cytoplasmic domain at its C terminus. The EscJ family contains an inner membrane ring (IM ring) component that is homologous to the flagellar MS ring component (FliF). Secretion specificity is determined by the EscU family inner membrane core (M/C core) component. EscU undergoes autocleavage to release a cleaved C-terminal cytoplasmic domain that remains associated with the core T3S components. The EscQ family includes a cytoplasmic ring (C ring)-forming protein required for secretion substrate localization by ATPase complex (EscLN family)-bound secretion substrates. The EscI family subunits form the inner rod; EscF family subunits polymerize into the extracellular needle structure, whose length is determined by EscP family proteins, which catalyze a conformational change in EscU upon completion of the needle.

T3S systems have the ability to change their repertoire of secretion substrates. This provides a mechanism to time the secretion of substrates to when they are needed—either for the assembly of the injectisome or the flagellum or for the secretion of effectors after completion of the structure. In the injectisome system, the secretion and assembly of translocator proteins, which form the translocon at the tip of the injectisome to allow injection of effector proteins into host cells, follow the completion of the extracellular needle structure. The secretion specificity switch results in translocator protein secretion after needle completion. All T3S structures require the assembly of a membrane-embedded complex that recognize substrates through an N-terminal secretion signal and/or an associated secretion chaperone. An ATPase complex of two or three proteins that bind and target substrates facilitates secretion and aids in their delivery to the membrane-associated base of the cognate structure. ATP hydrolysis is believed to release bound substrate and facilitate the presentation of an unfolded N-terminal peptide to initiate the secretion process (8). The actual secretion process is driven by energy derived from the proton motive force (12, 15).

For the virulence-associated T3S systems, the secretion specificity switch occurs through a conformational change in one of the membrane-embedded core secretion complex proteins, the EscU family of homologous proteins, i.e., EscU, YscU, SsaU, SpaS, and HrcU, which are all homologs of the FlhB component of the flagellar T3S system. All of the EscU family proteins that have been characterized to date undergo autocleavage, which produces a cleaved C-terminal cytoplasmic (CC) domain (5, 7, 11, 21). The cleaved CC domain remains associated with the membrane-embedded core T3S complex proteins, and its conformational state determines substrate specificity. Initially, the CC domain of the EscU family protein (EscU_CC) adopts a conformation that confers secretion specificity for components that make up the injectisome or flagellar hook-basal body (HBB) structures. Interaction of EscU_CC with the C terminus of an EscP family protein during EscP secretion catalyzes a change in the conformation of EscU_CC to allow the secretion of translocon or flagellar filament proteins later in the assembly process.

Thus, a productive interaction with the C-terminal domain of the secreted EscP family protein changes the secretion specificity from injectisome-type or HBB-type secretion substrates to substrates needed after injectisome or HBB assembly, such as translocator proteins (EspA filament- and pore-forming proteins) or flagellar filament subunits. That interaction occurs at a point during needle or hook polymerization that is dependent on the length of the EscP protein, resulting in the cessation of needle or hook growth that correlates with EscP length. In effect, the EscP family proteins act as molecular rulers to terminate needle or hook growth once the needle or hook has achieved a minimal length. The report by Monjarás Feria et al. provides evidence that EscP is the secreted molecular ruler in the LEE T3S system that interacts with EscU_CC to catalyze the secretion specificity switch and control needle length (13).

In Yersinia, loss of the EscP family protein YscP results in reduced secretion of effector proteins called Yops (3). In Shigella, loss of the EscP family protein Spa32 results in reduced secretion of Ipa effector proteins and IpgD (9). In the Salmonella Spi1 system, loss of the EscP family protein InvJ results in reduced secretion of effector protein SipB (16). In the Salmonella/E. coli flagellar system, loss of the EscP family protein FliK results in no secretion of the FliC filament protein (14, 18). In the study of Monjarás Feria et al., removal of EscP reduced translocator protein secretion but effector protein secretion was increased. This suggests that the secretion specificity switch in the LEE system acts to broaden the repertoire of secreted substrates rather than change from early-type to translocator-type substrate secretion.

As with all members of the EscP protein family, loss of EscP results in needles or hooks of uncontrolled length. All EscP family proteins lack significant amino acid similarity; however, the C terminus of the EscP family proteins contains a region of moderate similarity, termed the T3S4 (type III secretion and substrate specificity switch) domain, which interacts with the CC domain of the cognate EscU family protein (EscU_CC). The T3S4 domain is essential to trigger the secretion specificity conformational switch in EscU_CC. As predicted, Monjarás Feria et al. demonstrate that EscP interacts with EscU_CC. EscP also interacts with the substrates EscI and EscF, which make up the inner rod and needle structures, respectively (13). This situation is similar to the flagellar T3S system, in which the FliK protein interacts with the hook-cap and hook subunit proteins FlgE and FlgD, respectively. The hook-cap and hook are assembled after rod completion.

In both the Salmonella Spi1 and Yersinia type III injectisome systems, completion of the inner rod component is coupled to the T3S specificity switch. Similarly, in flagellar mutants that are blocked in assembly after rod completion, the T3S specificity switch can occur, but only in the absence of proteins that prevent the premature switch in secretion specificity. These results suggest that the mechanism of the T3S specificity switch is common to all T3S systems.

A remaining issue in the T3S field is how inner rod completion is coupled to needle length control and the secretion specificity switch. The answer may lie in the rate of secretion of the secreted molecular ruler protein. In the flagellar system, completion of the hook is determined by a secreted molecular ruler, FliK, which, when secreted through a hook with a minimal length (42 nm or greater), causes a conformational change in the membrane-embedded EscU family core secretion complex protein FlhB. The FliK-dependent conformational change in FlhB results in a change from the secretion of hook-type subunits of the HBB to so-called late secretion substrates, which include the subunits of the long external rotating helical filament that can extend about 10 μm from the cell surface. An interaction between the C terminus of FliK and the FlhB_CC domain during FliK secretion catalyzes a change in the conformation of FlhB_CC to allow the secretion of flagellar filament proteins after HBB completion. FliK is secreted throughout HBB assembly. In the absence of the hook, the secretion of FliK occurs at a rate that is at least 10 times higher than the rate of secretion when hooks are present (4). Presumably, the low rate of secretion allows time for a productive interaction between the C terminus of FliK, during the act of secretion, and the FlhB_CC domain to catalyze the secretion specificity switch. The mechanism that controls the rate of FliK secretion is not fully understood. However, it has been shown that the N terminus of FliK interacts with the hook protein FlgE and the hook-cap protein FlgD. These interactions could account for the decreased rate of FliK secretion through longer hook structures. The longer the hook, the more FlgE subunits are present to interact with the N terminus of secreted FliK, resulting in a lower rate of secretion.

Monjarás Feria et al. demonstrate that, similar to FliK binding to hook and hook-cap proteins, EscP interacts with the inner rod and needle subunit proteins EscI and EscF (13). It has been shown in both Salmonella Spi1 and Yersinia injectisome T3S systems that the secretion specificity switch and needle length control require the presence of the inner rod protein (10, 20). In fact, the inner rod of the Salmonella Spi1 system, which is composed of PrgJ subunits, does not form in the Spi1 system in the absence of the needle length control protein InvJ. This suggests that InvJ might act as the secretion chaperone for PrgJ and would account for the lack of inner rod formation in the absence of InvJ. The current work of Monjarás Feria et al. and earlier work on the flagellar system predict that interaction between needle length control proteins and inner rod and needle subunit proteins might be used as a diagnostic tool in the characterization of these proteins in other T3S systems.

On the basis of the work of Monjarás Feria et al., we can propose a model of needle length control and the secretion specificity switch that incorporates data from all T3S systems (Fig. 2). During the assembly of the injectisome basal structure, EscP is secreted at a rate that is too high to allow an interaction between EscP_C and EscU_CC to flip the secretion specificity switch (Fig. 2A). Inner rod subunits are secreted but fail to assemble (Fig. 2B) until they are able to interact with EscP_N. Interaction with EscP_N during EscP secretion and with inner rod subunits of EscI in the periplasm results in inner rod formation (Fig. 2C). During needle assembly after inner rod formation, the rate of EscP secretion is slow, and once the rod reaches a minimal length, EscP_C can interact with EscU_CC during EscP secretion to flip the secretion specificity switch and allow translocon substrate secretion (Fig. 2D).

Fig 2.

Model of the LEE injectisome T3S specificity switch. After assembly of the membrane-embedded basal body structure, inner membrane-associated EscU is in EscP, -F, and -I secretion mode. Secretion of the needle length control protein EscP occurs at a secretion rate that is too high to allow an interaction between EscP_C and Esc_CC to flip the secretion specificity switch (A). Inner rod subunits (EscI) are secreted into the periplasm (B). Interaction of periplasmic EscI subunits with secreted EscP_N in the periplasm results in inner rod formation (C). After inner rod assembly and during needle assembly, the rate of EscP secretion is low and once the rod reaches a minimal length, EscP_C interacts with EscU_CC to flip the secretion specificity switch and allow translocon-type substrate secretion, resulting in a structure that is competent for effector secretion into host cells (D).

A major point of controversy for injectisome T3S systems is how assembly of the inner rod (EscI) is dependent on EscP. The role of EscP homologs in inner rod assembly remains an unsolved mystery in injectisome assembly. In the Salmonella Spi1 system, removal of the EscP homolog prevents inner rod assembly but not secretion of the inner rod subunit protein PrgJ (19). Yersinia inner rod protein YscI mutants were isolated that would not form a needle on the injectisome unless YscP was removed (20). How do the YscI mutants prevent YscF secretion in the presence of YscP? The fact that they are poorly secreted suggests that they might jam the secretion apparatus. EscI is secreted in the absence of EscP but does not assemble into an inner rod structure. I propose that EscP_N acts as a scaffold for EscI assembly in the periplasm (Fig. 2C). EscI assembly into higher-ordered structures in the presence of EscP_N is consistent with this model. However, if either the periplasmic environment or other periplasmic components were essential for inner rod assembly, such a mechanism would be difficult to test.