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How and when are substrates selected for type III secretion?
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How and when are substrates selected for type III secretion? Phillip Aldridge and Kelly T. Hughes Many Gram-negative bacteria use type III secretion systems to secrete virulence factors as well as the structural components of the flagellum. Some bacterial secretion systems use a secretion signal contained in the amino acid sequence of the secreted substrate. However, substrates of type III systems lack a single, defined secretion signal. There is evidence for the existence of three independent secretion signals – the 5′′ region of the mRNA, the amino terminus of the substrate and the ability of a secretion chaperone to bind the substrate before secretion – that direct substrates for secretion through the type III pathways. One or more of these signals might be used for a given substrate. A recent study of flagellar assembly presented evidence for a role of translation in the type III secretion mechanism. We present a unifying model for type III secretion that can be applied to flagellar assembly, needle assembly and the secretion of virulence factors. The potential role of translation in regulating the timing of substrate secretion is also discussed.
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TTSS and the virulence TTSS is that TTSCs interact with carboxy-terminal amino acids of flagellar substrates and amino-terminal amino acids of virulence substrates. In all cases, the TTSC has been defined by a direct protein–protein interaction between the chaperone and its specific substrate. One secretion model predicts that TTSCs interact directly with their partners to escort the substrate to the base of the TTSS for subsequent secretion (Fig. 1c)6. For some secretion substrates, chaperone binding prevents degradation, whereas other substrates are stable in the cytoplasm in the absence of the chaperone. The identification of TTSCs provided a model to explain the absence of a specific amino acid signal to target proteins for secretion by the TTSSs. The TTSC model predicts that the chaperone interacts with the secretion apparatus, thereby directing secretion of the bound substrate (Fig. 1c). More recently a second model has emerged, which predicts that the 5′ mRNA of the secretion substrate, including both the 5′ untranslated region (5′ UTR) and the mRNA of the amino-terminal coding region, directs substrates for co-translational secretion6. Both models obviate the need for an amino acid secretion signal. However, a common amino acid sequence might be conserved among the chaperones to direct substrate-specific secretion. Chaperones and flagellar assembly
The ability to secrete proteins into the environment is an important part of the bacterial life cycle (Box 1). The type III secretion system (TTSS) is one mechanism utilized by Gram-negative plant and animal pathogens to secrete and translocate virulence determinants into susceptible eukaryotic cells1. Additionally, a TTSS is also employed in bacterial flagellar assembly2. A feature of the TTSS that distinguishes it from other secretion systems is the lack of a defined amino acid secretion signal to direct substrate proteins for secretion. However, in both the flagellar and virulence TTSSs, the amino terminus of many secreted proteins is thought to contain a secretion signal. Work from Schneewind and colleagues3,4 has suggested that an mRNA sequence rather than an amino acid sequence defines the amino-terminal secretion signal in the Yersinia TTSS, and led to a model of co-translational secretion for the TTSSs (Fig. 1a). However, pulse-chase experiments indicate that proteins translated in the cytoplasm can be targeted for secretion, arguing for the existence of an amino acid secretion signal (Fig. 1b). The role of chaperones
Phillip Aldridge Kelly T. Hughes* Dept of Microbiology, University of Washington, Seattle, WA 98195, USA. *e-mail: hughes@ u.washington.edu
Many proteins require accessory proteins for their secretion by a TTSS. These accessory proteins have been termed type III secretion chaperones (TTSCs)5. The loss of TTSCs by mutation resulted in an aberrant secretion pattern of the cognate secretion substrate and the loss of a specific chaperone also resulted in the absence of many of the cognate secretion substrates in the cytoplasm5. A difference between the flagellar
Recent work in the flagellar TTSS of Salmonella has suggested that secretion might be influenced by both chaperone binding and by the mRNA itself. In this system, the FlgN protein acts as a secretion chaperone for two substrates, FlgK and FlgL, which are incorporated into the final flagellar structure7. FlgN also regulates translation of a third secretion substrate, FlgM, which is not incorporated into the flagellar structure8. This led to a unified model for type III secretion in which secretion substrates can be recognized by mRNA signals, chaperone binding and/or amino-terminal amino acid secretion signals. Here, we present a model for the mechanism of type III secretion by the flagellar system of Salmonella typhimurium, and correlate this model to virulence TTSSs (Fig. 2). The closed-gate and open-gate models
Bacteria utilize flagella for motility through liquid environments and on surfaces. The flagellum is composed of two structural components: the hook basal body (HBB, the motor), which spans the membranes and cell wall; and the long, external filament (propeller). The flagellar TTSS can distinguish between secretion substrates at different times during assembly; for example, during HBB assembly, late assembly proteins are not secreted, whereas during filament assembly, HBB substrates are not secreted. Similar to flagellar assembly, it is important for pathogenic bacteria to control the secretion of TTSS structural subunits and virulence factors at the appropriate time and location. As the basic components of the flagellar HBB and virulence TTSS are similar, it
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Box 1. Defining protein secretion in bacteria Mechanisms of protein secretion Type I: secretion is a one-step process across both membranes. The substrates possess an uncleaved signal sequence at the carboxyl terminus. A well known example of a type I secretion substrate is hemolysin. Type II: utilizes the sec-dependent pathway to cross the inner membrane, followed by a second secretion system associated with the outer membrane. Substrates possess a sec-recognition signal sequence at the amino terminus. Examples of secretion substrates include degradative enzymes produced by plant pathogens. Type III: differs from both type I and II in that there is no defined secretion signal that is cleaved during secretion. Secretion occurs across both membranes. The basic type III secretion structure For secretion via a type III system, the structure requires an inner membrane ring that acts as an anchor. Built upon the inner-membrane anchor is a channel that spans the periplasmic space and outer membrane. The channel is held in place by two rings associated with the peptidoglycan layer and the outer membrane. On the outer surface of the bacterial cell is either a needle-like structure or the flagellar filament that is used to allow the release of proteins. The secretion apparatus responsible for driving secretion associates with the cytoplasmic face of the inner-membrane anchor forming another ring.
is plausible that the mechanism used to regulate flagellar assembly is the same as that used by other TTSSs. Based on flagellar assembly, we propose there are two pathways by which type III secretion could occur, a closed-gate model and an open-gate model (Fig. 2). In both models, the early stages (Fig. 2, Stages I to IV) occur by self-assembly. Once the basal structure has been assembled and the inner membrane is spanned, a degree of specificity is required to prevent secretion at the wrong time. Recent data from the study of flagellar assembly in Salmonella have shown that substrate secretion requires active translation8. We propose that the specificity of the secretion system could be achieved by gating the cytoplasmic face of the TTSS by translating ribosomes, the closed-gate model (Fig. 2). Alternatively, the ribosomes could associate with the cytoplasmic face, allowing association of translation but not a gating of the channel, an open-gate mechanism. In Salmonella, multiple flagella are present at different stages in development. What mechanism allows flagellin to be secreted only at the flagellumgrowing filament and not at the flagellum-growing hooks? Similarly, in the virulence TTSSs, what mechanism differentiates effector proteins to be secreted through completed needle structures versus incomplete structures that still require secretion of substrates necessary late in needle assembly? Could such a mechanism account for both chaperone-dependent secretion and mRNA co-translational secretion? Secretion systems and sensing
The cytoplasmic-membrane component of the TTSS is constructed during the first stages of assembly (Fig. 2, Stages I and II). This produces a stacked-ring structure of 400 Å in diameter in the cytoplasm and a http://tim.trends.com
TTSS imbedded in the membrane disc sealed off by the MS-ring, the inner membrane anchor, within the cytoplasmic membrane. After this stage all proteins are secreted beyond the cytoplasmic membrane. Rod assembly is followed by hook assembly to complete the HBB structure (Fig. 2, Stage IV). Bonifield et al.9 have shown that the hook protein FlgE can be detected in cellular fractions during Stages I and II of assembly but not during rod assembly (Fig. 2, Stage III). The polymerization of hook subunits requires the transient presence of an FlgD cap to act as a hook scaffold10. Cellular FlgE levels are reduced in flgD mutant strains, suggesting that either flgE translation or FlgE stability is re-established, allowing hook polymerization, only once the basal body is formed and the protruding rod is capped with FlgD (Ref. 10). These results suggest that during the early stages of flagellar assembly, substrate specificity could be determined by actual physical presence. Localized translation of mRNA encoding late secreted proteins could direct flagellin synthesis only at individual flagella undergoing filament elongation. A completed flagellum would require a mechanism that dissociates further translation of late mRNA. It is thought that the sensing of filament completion is accomplished by the capillary action of filament monomers (Fig. 2, Stage VII) and other secreted proteins backing up within the hollow filament until they remain in the cytoplasm. In our model, this could lead to a forced opening of the ribosomal gate, by hindering the placement of ribosomes in the inner C-ring space. Is there a mechanism for direct sensing of this process? Or is an overflow of substrates into the cytoplasm enough? For a pathogenic TTSS, a mechanism similar to the closed-gate model (Fig. 2) could be used to prevent the untimely secretion of effector proteins into an extracellular environment. Secretion of structural components could occur via a co-translational mechanism. On completion of the needle structure, the gate would open and the associated ribosomes could then translate the secretion substrates, allowing chaperone binding and subsequent secretion (similar to the open model). However, our closed model still allows for chaperone-mediated secretion, where the growing polypeptide first associates with the chaperone with immediate donation to the secretion apparatus. The molecular weight of most TTSCs is in the range of 20 kDa (Ref. 5). As these are small proteins even with gating of the C-ring, there could be enough space within the cavity to allow chaperone association and substrate delivery to the secretion apparatus (side view in Fig. 2). Coupling transcription and flagellar assembly
A second regulatory mechanism during flagellar assembly, which ensures that late secretion substrates do not compete with HBB substrates for secretion, is the coupling of transcriptional control to assembly. A flagellar transcription factor, σ28, is required for expression of genes involved in the late stages of
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Fig. 1. Schematic diagram of proposed secretion models for type III systems. The channel spanning both membranes is shown in orange. The cytoplasmic components of the secretion system and the inner membrane anchor are shown in green, whereas the secretion apparatus embedded within the anchor is shown in red. There are three mechanisms by which experimental data suggest a protein can be secreted by a type III secretion system. (a) An mRNA signal-mediated secretion mechanism, which would lead to coupled translation and secretion of the substrate. Substrate-translating ribosomes are brought into association with the cytoplasmic face of the secretion apparatus. (b) An amino-terminal amino acid signal-mediated secretion pathway, where the secretion signal is encoded in the amino-terminus of the secretion substrate (blue lollipops). (c) A chaperone-mediated secretion pathway where a cognate chaperone (light-green squares) binds the secretion substrate (blue circles). On interaction, it is the chaperone that communicates with the secretion apparatus, delivering the substrate for secretion. Unbound substrate is not recognized by the secretion apparatus and is therefore not secreted. Abbreviations: IM, inner membrane; OM, outer membrane; PG, peptidoglycan layer.
flagellar assembly11. The anti-σ28 factor FlgM modulates σ28 activity by direct binding and prevention of the formation of the σ28-RNA polymerase holoenzyme12,13. Both FlgM and σ28 are transcribed with the HBB structural genes14. FlgM binds σ28 and prevents transcription of the late flagellar promoters until the HBB is completed (Fig. 2, Stages I to V). FlgM possesses a late secretion signal and is secreted after HBB assembly, releasing σ28 to interact with RNA polymerase and direct transcription of the late assembly proteins (Fig. 2, Stage V)15,16. In this way, the subunits that make up the external filament are not produced until there is an HBB present for them to polymerize onto17. Both σ28-dependent and -independent promoters drive the transcription of flgM (Ref. 14). Karlinsey et al.17 have recently shown that FlgM secreted from the bacterial cell is predominantly translated from the σ28-dependent transcript and that secretion is dependent on the presence of FlgN and active translation. This suggests FlgM secretion is coupled to flgM translation. In our closed-gate model, this would require FlgN to possibly be within the space between the ribosomal gate and the secretion apparatus. Given that FlgN is a TTSC of small molecular weight, its small size might allow it to fit within such a tight space. This would also enable other forms of chaperone-mediated secretion to occur via the closed-gate pathway.
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(a) mRNA signal
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Substrate specificity of flagellar assembly
The secretion specificity of the flagellar TTSS is partly determined by FliK and FlhB, components of the flagellar TTSS (Refs 18–20). Unlike FlgM, FliK is secreted before hook completion and is retained in the cytoplasm following hook completion (Fig. 2, Stages IV and V). It is believed that, after hook completion, cytoplasmic FliK interacts with FlhB to cause a change in substrate specificity from HBB substrates to late secretion substrates19. Evidence has recently been presented suggesting that, just before hook elongation, hook subunits accumulate within the cytoplasmic space of the C-ring21. Hook elongation would empty this space of hook subunits and allow FliK to interact with FlhB to switch secretion-substrate specificity. It would also allow access of the C-ring space to ribosomes http://tim.trends.com
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to initiate a translation-dependent secretion of mRNA transcripts of late assembly substrates. There is genetic evidence that, during hook elongation, a change in the TTSS apparatus specificity occurs that is independent of the FliK/FlhB substratespecificity-switching mechanism. A novel protein was identifed, Flk, which affects flgM translation and secretion at this stage of assembly. In specific flagellar mutant backgrounds, loss of Flk prevents both flgM translation and allows for premature FlgM secretion22,23. Kutsukake has proposed that Flk will function as a lock on the flagellar TTSS to prevent
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Closed-gate model Side view
Chaperones
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Fig. 2. Schematic diagram of flagellar assembly. The assembly of the flagellum can be divided in to at least seven distinct stages. (I) Site selection and MS-ring formation; (II) assembly of the C-ring and secretion apparatus; (III) rod formation; (IV) assembly of the P and L rings, leading to hook synthesis; (V) hook completion; (VI) a middle to late substrate specificity switch in secretion upon hook completion; and (VII) filament elongation. Upon reaching Stage IV of assembly, the secretion of structural components or secretion substrates can occur by two possible pathways, a closed-gate model or an open-gate model. During the closed-gate pathway, ribosomes associate with Flk bringing them into the vicinity of the C-ring. On induction of the substrate-specificity switch, the ribosomes are brought into the C-ring inner space where they can directly donate growing polypeptide chains to an FliK–FlhB complex, allowing the coupling of translation and secretion. This effectively forms a closed gate, which could prevent the secretion of other substrates at the wrong time. Chaperone-mediated secretion might still take place where the growing polypeptide first associates with the chaperone with immediate donation to an FlhB–FliK complex. A side view of Stage V is shown to demonstrate the possibility that chaperones enter the space between associated ribosomes and the secretion apparatus. An alternative to a closed-gate model would be that the gate is always ‘open’. In the open-gate pathway, Flk brings the ribosomes into the vicinity of the type III secretion apparatus. After translation, the FliK–FlhB complex recognizes the newly synthesized proteins, allowing secretion to occur. On completion of type III secretion, the ribosomal gate would be forced open by the capillary action caused by a build-up of secretion substrates within the secretion channel (Stage VII). On opening, the overflow of secretion substrates into the cytoplasm could be sensed by the secretion chaperones inducing a shutdown of secretion until required. This would leave the secretion apparatus in a primed state ready to repair damage to the type III secretion system or begin to secrete effector proteins when needed. Abbreviations: IM: inner membrane; OM, outer membrane; PG: peptidoglycan layer.
premature FlgM secretion22. Flk has homology to the ribosomal-binding domain of translation-initiation factor S1 and is a membrane-anchored protein. This would provide a mechanism to allow the association of ribosomes with the membrane. However, although both Flk and FlgN affect flgM translation, the effect is seen only in HBB mutant strains8,24. This suggests that redundant translation systems are present, and that http://tim.trends.com
the HBB might also provide a site for association of ribosomes for co-translational secretion. A possible candidate for a second ribosomal-interaction site within the HBB would be the C-ring because of its accessibility to ribosomes within the cytoplasm. At completion of hook synthesis, the FlhB component interacts with FliK to lose affinity for HBB secretion substrates. The C-ring cavity, empty of HBB subunits, would then fill with translating ribosomes containing mRNA that encodes the late secretion substrates (Fig. 2, closed-gate model). The diameter of the ribosome is ~200 Å (Refs 25,26), leading us to predict that the C-ring cavity could accommodate up to three ribosomes. Alternatively, interaction of FlhB and FliK would alter the specificity of FlhB, in that it would only accept translated polypeptides from ribosomes associated with Flk or the C-ring (Fig. 2; open-gate model). The ability of flagellar TTSCs to bind the carboxyl terminus of their respective partners is in contrast to the general trend seen for TTSCs identified from pathogenic TTSSs, which mainly bind an amino-terminal domain5. How can these proteins contribute to flagellar assembly? As they bind the carboxyl termini of flagellar proteins they might prevent degradation or assembly before secretion (Fig. 2, Stages IV to V). They might also act as a system for sensing the build up of secretion substrates in
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the cytoplasm towards the completion of filament elongation (Fig. 2; Stage VII). On filament completion, the substrates for late secretion substrate chaperones would begin to accumulate in the cell, leading to an interaction with their cognate chaperones. On doing so, this could inhibit the ability of the FlgN chaperone to regulate flgM translation or even inhibit FlgM secretion via a direct interaction, allowing a build up of cellular FlgM. This in turn would downregulate the expression of late components via σ28 inactivation. On filament completion, the bacterial cell requires a mechanism by which it can efficiently react to any damage that occurs to the flagella. Damage could automatically lead to a slow induction of substrate recognition by the secretion apparatus resulting in a reduction in substrates bound to their respective chaperones. For FlgN, this would lead to an increase in FlgM secretion, allowing an upregulation of the necessary promoters of the flagellar regulon until the flagellum has been resynthesized. Conclusions
Can our model also be true for general type III secretion of virulence factors in pathogenic bacteria? The majority of effector proteins have a partner chaperone that binds to an amino-terminal region instead of the carboxyl terminus like FlgN. This would allow an association of translation, reducing the time taken for secretion but also allowing for a measure of variation in the transcripts brought to TTSSassociated ribosomes. Many of the chaperones identified so far show a degree of selectivity, in that their respective partners can be secreted by the type III system but these are only correctly translocated to eukaryotic cells, from completed needles, when the chaperone is present6. A recent report by Wolf-Watz and colleagues27 has suggested that, contrary to earlier work, a secreted virulence factor in Yersinia does not possess an mRNA signal but the secretion is References 1 Hueck, C.J. (1998) Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol. Mol. Biol. Rev. 62, 379–433 2 Macnab, R.M. (1999) The bacterial flagellum: reversible rotary propellor and type III export apparatus. J. Bacteriol. 181, 7149–7153 3 Anderson, D.M. and Schneewind, O. (1999) Yersinia enterocolitica type III secretion: an mRNA signal that couples translation and secretion of YopQ. Mol. Microbiol. 31, 1139–1148 4 Anderson, D.M. and Schneewind, O. (1997) A mRNA signal for the type III secretion of Yop proteins by Yersinia enterocolitica. Science 278, 1140–1143 5 Bennett, J.C. and Hughes, C. (2000) From flagellum assembly to virulence: the extended family of type III export chaperones. Trends Microbiol. 8, 202–204 6 Cheng, L.W. and Schneewind, O. (2000) Type III machines of Gram-negative bacteria: delivering the goods. Trends Microbiol. 8, 214–220 7 Fraser, G.M. et al. (1999) Substrate-specific binding of hook-associated proteins by FlgN and FliT, putative chaperones for flagellum assembly. Mol. Microbiol. 32, 569–580 http://tim.trends.com
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Questions for future research • •
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Are ribosomes directly associated with the type III secretion apparatus? Is translation of secretion substrates associated with the type III secretion apparatus? Is there an FlgM secretion chaperone? Is σ28 or FlgN the FlgM chaperone? Do type III secretion chaperones act as sensors for completion of the secretion apparatus? If so, by what mechanism? Are there direct interactions between the different chaperones and the secretion apparatus? If so, what is the mechanism of these interactions?
exclusively determined by the amino terminus of the protein. A pertinent question here would be: is the translation of this protein still localized before chaperone binding and subsequent secretion? Completion of needle structures would also lead to an accumulation of distal structural subunits within the cell. Allowing secretion chaperones to act as a sensor, for completion of the type III machine, would allow for an induction of a specificity switch in secretion substrates. This would lead to the TTSS being left in a primed state ready to begin immediate delivery of effector proteins on contact with eukaryotic cells. In general, these observations could be the reason why much research in flagellar assembly and type III virulence factor secretion has identified a requirement for a signal in both the mRNA and in the secreted proteins: the mRNA signal allows the translation of the prospective secretion substrates to occur near the secretion apparatus, and the protein signal is there to allow for selectivity and control when deciding whether a protein should or should not be secreted.
8 Karlinsey, J.E. et al. (2000) Translation/secretion coupling by type III secretion systems. Cell 102, 487–497 9 Bonifield, H.R. et al. (2000) The flagellar hook protein, FlgE, of Salmonella enterica serovar typhimurium is posttranscriptionally regulated in response to the stage of flagellar assembly. J. Bacteriol. 182, 4044–4050 10 Ohnishi, K. et al. (1994) FlgD is a scaffolding protein needed for flagellar hook assembly in Salmonella typhimurium. J. Bacteriol. 176, 2272–2281 11 Ohnishi, K. et al. (1990) Gene fliA encodes an alternative sigma factor specific for flagellar operons in Salmonella typhimurium. Mol. Gen. Genet. 221, 139–147 12 Chadsey, M.S. et al. (1998) The flagellar antisigma factor FlgM actively dissociates Salmonella typhimurium σ28 RNA polymerase holoenzyme. Genes Dev. 12, 3123–3136 13 Ohnishi, K. et al. (1992) A novel transcriptional regulation mechanism in the flagellar regulon of Salmonella typhimurium: an antisigma factor inhibits the activity of the flagellum-specific σ factor, σF. Mol. Microbiol. 6, 3149–3157
14 Gillen, K.L. and Hughes, K.T. (1993) Transcription from two promoters and autoregulation contribute to the control of expression of the Salmonella typhimurium flagellar regulatory gene flgM. J. Bacteriol. 175, 7006–7015 15 Kutsukake, K. and Iino, T. (1994) Role of the FliA–FlgM regulatory system on the transcriptional control of the flagellar regulon and flagellar formation in Salmonella typhimurium. J. Bacteriol. 176, 3598–3605 16 Hughes, K.T. et al. (1993) Sensing structural intermediates in bacterial flagellar assembly by export of a negative regulator. Science 262, 1277–1280 17 Karlinsey, J.E. et al. (2000) Completion of the hookbasal body complex of the Salmonella typhimurium flagellum is coupled to FlgM secretion and fliC transcription. Mol. Microbiol. 37, 1220–1231 18 Minamino, T. et al. (1999) FliK, the protein responsible for flagellar hook length control in Salmonella, is exported during hook assembly. Mol. Microbiol. 34, 295–304 19 Minamino, T. and Macnab, R.M. (2000) Domain structure of Salmonella FlhB, a flagellar
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export component responsible for substrate specificity switching. J. Bacteriol. 182, 4906–4914 20 Williams, A.W. et al. (1996) Mutations in fliK and flhB affecting flagellar hook and filament assembly in Salmonella typhimurium. J. Bacteriol. 178, 2960–2970 21 Makishima, S. et al. Length of the flagellar hook and the capacity of the type III export apparatus. Science (in press)
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22 Kutsukake, K. (1997) Hook-length control of the export-switching machinery involves a double-locked gate in Salmonella typhimurium flagellar morphogenesis. J. Bacteriol. 179, 1268–1273 23 Karlinsey, J.E. et al. (1998) Flk couples flgM translation to flagellar ring assembly in Salmonella typhimurium. J. Bacteriol. 180, 5384–5397 24 Karlinsey, J.E. et al. (1997) The flk gene of Salmonella typhimurium couples flagellar P- and
Intimin and the host cell – is it bound to end in Tir(s)? Gad Frankel, Alan D. Phillips, Luiz R. Trabulsi, Stuart Knutton, Gordon Dougan and Stephen Matthews Intimate bacterial adhesion to the intestinal epithelium is a pathogenic mechanism shared by several human and animal enteric pathogens, including enteropathogenic and enterohaemorrhagic Escherichia coli. Two bacterial protein partners involved in this intimate association have been identified, intimin and Tir. Some key remaining questions include whether intimin specifically interacts with one or more host-cell-encoded molecules and whether these contacts are a prerequisite for the subsequent intimate intimin–Tir association. Recent data support the hypothesis that the formation of a stable intimin–Tir relationship is the consequence of intimin protein interactions involving both host and bacterial components.
Intimin is the name given to an outer-membrane adhesion molecule expressed by a group of pathogenic Escherichia coli that can cause disease in both humans and animals through formation of attaching and effacing (A/E) intestinal lesions1. These lesions are characterized by effacement of brush-border microvilli in the vicinity of adhering bacteria and intimin-mediated bacterial attachment to the plasma membrane of infected cells. The intimate nature of this interaction is revealed in electron micrographs of infected intestinal epithelia, which show a uniform ~10 nm gap between bacterial and host cell membranes (Fig. 1). A/E lesions were first described for strains of enteropathogenic E. coli (EPEC)2,3, an established etiological agent of human infantile diarrhoea in the developing world4. Similar intestinal lesions were later associated with other enteric mucosal pathogens including enterohaemorrhagic E. coli (EHEC)5,6, an emerging microbial pathogen associated with food poisoning that often has lifethreatening complications owing to the production of shiga toxins (verocytotoxins)4; rabbit diarrhoeagenic
L-ring assembly to flagellar morphogenesis. J. Bacteriol. 179, 2389–2400 25 Ban, N. et al. (2000) The complete atomic structure of the large ribosomal subunit at 2.4 Å resolution. Science 289, 905–920 26 Wimberly, B.T. et al. (2000) Structure of the 30S ribosomal subunit. Nature 407, 327–339 27 Lloyd, S.A. et al. (2001) Yersinia YopE is targeted for type III secretion by N-terminal, not mRNA, signals. Mol. Microbiol. 39, 520–532
E. coli (REPEC)7; and the mouse pathogen Citrobacter rodentium8. Using animal and ex vivo infection models, intimin was shown, in most cases, to be essential for colonization of the intestinal mucosa and in all cases for A/E lesion formation and pathogenicity9–13.
‘...the formation of a stable intimin–Tir relationship is the consequence of intimin protein interactions involving both host and bacterial components.’ Receptor-binding activities of intimin – early studies
In 1990, intimin, a 94-kDa outer membrane protein, was the first gene product reported to be associated with A/E lesion formation in EPEC (Ref. 14); an intimin homologue was subsequently discovered in EHEC O157:H7 (Ref. 15). Although no specific function was assigned to intimin at this stage, the realization that the protein had significant homology, at least among the amino-terminal 650 amino acids, to Yersinia invasin was very significant for subsequent studies. Invasin had been shown to be an outer membrane Yersinia protein that mediated bacterial internalization into mammalian cells by binding to β1 integrins; the integrin-binding activity of invasin had been localized to the carboxyl terminus of the polypeptide16, a region with only limited similarity to intimin. It was on this basis that, in 1993, we decided to investigate the possibility that the carboxyl terminus of intimin contained receptorbinding activity. By fusing the carboxy-terminal 280 amino acids of intimin (Int280) to maltosebinding protein (MBP), we demonstrated that soluble and latex-bead-coated MBP–Int280 from both EPEC and EHEC could directly bind to HEp-2 cells17. Independently however, Rosenshine et al.18 were unable to demonstrate significant MBP–Int280 binding to uninfected epithelial cells, but were able to demonstrate MBP–Int280 binding to cells previously infected with an intimin-negative EPEC mutant, thus indicating that a bacterial ‘signal’ is required for intimin binding. Importantly, both observations (i.e. binding of intimin to
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How and when are substrates selected for type III secretion? Phillip Aldridge and Kelly T. Hughes Many Gram-negative bacteria use type III secretion systems to secrete virulence factors as well as the structural components of the flagellum. Some bacterial secretion systems use a secretion signal contained in the amino acid sequence of the secreted substrate. However, substrates of type III systems lack a single, defined secretion signal. There is evidence for the existence of three independent secretion signals – the 5′′ region of the mRNA, the amino terminus of the substrate and the ability of a secretion chaperone to bind the substrate before secretion – that direct substrates for secretion through the type III pathways. One or more of these signals might be used for a given substrate. A recent study of flagellar assembly presented evidence for a role of translation in the type III secretion mechanism. We present a unifying model for type III secretion that can be applied to flagellar assembly, needle assembly and the secretion of virulence factors. The potential role of translation in regulating the timing of substrate secretion is also discussed.
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TTSS and the virulence TTSS is that TTSCs interact with carboxy-terminal amino acids of flagellar substrates and amino-terminal amino acids of virulence substrates. In all cases, the TTSC has been defined by a direct protein–protein interaction between the chaperone and its specific substrate. One secretion model predicts that TTSCs interact directly with their partners to escort the substrate to the base of the TTSS for subsequent secretion (Fig. 1c)6. For some secretion substrates, chaperone binding prevents degradation, whereas other substrates are stable in the cytoplasm in the absence of the chaperone. The identification of TTSCs provided a model to explain the absence of a specific amino acid signal to target proteins for secretion by the TTSSs. The TTSC model predicts that the chaperone interacts with the secretion apparatus, thereby directing secretion of the bound substrate (Fig. 1c). More recently a second model has emerged, which predicts that the 5′ mRNA of the secretion substrate, including both the 5′ untranslated region (5′ UTR) and the mRNA of the amino-terminal coding region, directs substrates for co-translational secretion6. Both models obviate the need for an amino acid secretion signal. However, a common amino acid sequence might be conserved among the chaperones to direct substrate-specific secretion. Chaperones and flagellar assembly
The ability to secrete proteins into the environment is an important part of the bacterial life cycle (Box 1). The type III secretion system (TTSS) is one mechanism utilized by Gram-negative plant and animal pathogens to secrete and translocate virulence determinants into susceptible eukaryotic cells1. Additionally, a TTSS is also employed in bacterial flagellar assembly2. A feature of the TTSS that distinguishes it from other secretion systems is the lack of a defined amino acid secretion signal to direct substrate proteins for secretion. However, in both the flagellar and virulence TTSSs, the amino terminus of many secreted proteins is thought to contain a secretion signal. Work from Schneewind and colleagues3,4 has suggested that an mRNA sequence rather than an amino acid sequence defines the amino-terminal secretion signal in the Yersinia TTSS, and led to a model of co-translational secretion for the TTSSs (Fig. 1a). However, pulse-chase experiments indicate that proteins translated in the cytoplasm can be targeted for secretion, arguing for the existence of an amino acid secretion signal (Fig. 1b). The role of chaperones
Phillip Aldridge Kelly T. Hughes* Dept of Microbiology, University of Washington, Seattle, WA 98195, USA. *e-mail: hughes@ u.washington.edu
Many proteins require accessory proteins for their secretion by a TTSS. These accessory proteins have been termed type III secretion chaperones (TTSCs)5. The loss of TTSCs by mutation resulted in an aberrant secretion pattern of the cognate secretion substrate and the loss of a specific chaperone also resulted in the absence of many of the cognate secretion substrates in the cytoplasm5. A difference between the flagellar
Recent work in the flagellar TTSS of Salmonella has suggested that secretion might be influenced by both chaperone binding and by the mRNA itself. In this system, the FlgN protein acts as a secretion chaperone for two substrates, FlgK and FlgL, which are incorporated into the final flagellar structure7. FlgN also regulates translation of a third secretion substrate, FlgM, which is not incorporated into the flagellar structure8. This led to a unified model for type III secretion in which secretion substrates can be recognized by mRNA signals, chaperone binding and/or amino-terminal amino acid secretion signals. Here, we present a model for the mechanism of type III secretion by the flagellar system of Salmonella typhimurium, and correlate this model to virulence TTSSs (Fig. 2). The closed-gate and open-gate models
Bacteria utilize flagella for motility through liquid environments and on surfaces. The flagellum is composed of two structural components: the hook basal body (HBB, the motor), which spans the membranes and cell wall; and the long, external filament (propeller). The flagellar TTSS can distinguish between secretion substrates at different times during assembly; for example, during HBB assembly, late assembly proteins are not secreted, whereas during filament assembly, HBB substrates are not secreted. Similar to flagellar assembly, it is important for pathogenic bacteria to control the secretion of TTSS structural subunits and virulence factors at the appropriate time and location. As the basic components of the flagellar HBB and virulence TTSS are similar, it
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Box 1. Defining protein secretion in bacteria Mechanisms of protein secretion Type I: secretion is a one-step process across both membranes. The substrates possess an uncleaved signal sequence at the carboxyl terminus. A well known example of a type I secretion substrate is hemolysin. Type II: utilizes the sec-dependent pathway to cross the inner membrane, followed by a second secretion system associated with the outer membrane. Substrates possess a sec-recognition signal sequence at the amino terminus. Examples of secretion substrates include degradative enzymes produced by plant pathogens. Type III: differs from both type I and II in that there is no defined secretion signal that is cleaved during secretion. Secretion occurs across both membranes. The basic type III secretion structure For secretion via a type III system, the structure requires an inner membrane ring that acts as an anchor. Built upon the inner-membrane anchor is a channel that spans the periplasmic space and outer membrane. The channel is held in place by two rings associated with the peptidoglycan layer and the outer membrane. On the outer surface of the bacterial cell is either a needle-like structure or the flagellar filament that is used to allow the release of proteins. The secretion apparatus responsible for driving secretion associates with the cytoplasmic face of the inner-membrane anchor forming another ring.
is plausible that the mechanism used to regulate flagellar assembly is the same as that used by other TTSSs. Based on flagellar assembly, we propose there are two pathways by which type III secretion could occur, a closed-gate model and an open-gate model (Fig. 2). In both models, the early stages (Fig. 2, Stages I to IV) occur by self-assembly. Once the basal structure has been assembled and the inner membrane is spanned, a degree of specificity is required to prevent secretion at the wrong time. Recent data from the study of flagellar assembly in Salmonella have shown that substrate secretion requires active translation8. We propose that the specificity of the secretion system could be achieved by gating the cytoplasmic face of the TTSS by translating ribosomes, the closed-gate model (Fig. 2). Alternatively, the ribosomes could associate with the cytoplasmic face, allowing association of translation but not a gating of the channel, an open-gate mechanism. In Salmonella, multiple flagella are present at different stages in development. What mechanism allows flagellin to be secreted only at the flagellumgrowing filament and not at the flagellum-growing hooks? Similarly, in the virulence TTSSs, what mechanism differentiates effector proteins to be secreted through completed needle structures versus incomplete structures that still require secretion of substrates necessary late in needle assembly? Could such a mechanism account for both chaperone-dependent secretion and mRNA co-translational secretion? Secretion systems and sensing
The cytoplasmic-membrane component of the TTSS is constructed during the first stages of assembly (Fig. 2, Stages I and II). This produces a stacked-ring structure of 400 Å in diameter in the cytoplasm and a http://tim.trends.com
TTSS imbedded in the membrane disc sealed off by the MS-ring, the inner membrane anchor, within the cytoplasmic membrane. After this stage all proteins are secreted beyond the cytoplasmic membrane. Rod assembly is followed by hook assembly to complete the HBB structure (Fig. 2, Stage IV). Bonifield et al.9 have shown that the hook protein FlgE can be detected in cellular fractions during Stages I and II of assembly but not during rod assembly (Fig. 2, Stage III). The polymerization of hook subunits requires the transient presence of an FlgD cap to act as a hook scaffold10. Cellular FlgE levels are reduced in flgD mutant strains, suggesting that either flgE translation or FlgE stability is re-established, allowing hook polymerization, only once the basal body is formed and the protruding rod is capped with FlgD (Ref. 10). These results suggest that during the early stages of flagellar assembly, substrate specificity could be determined by actual physical presence. Localized translation of mRNA encoding late secreted proteins could direct flagellin synthesis only at individual flagella undergoing filament elongation. A completed flagellum would require a mechanism that dissociates further translation of late mRNA. It is thought that the sensing of filament completion is accomplished by the capillary action of filament monomers (Fig. 2, Stage VII) and other secreted proteins backing up within the hollow filament until they remain in the cytoplasm. In our model, this could lead to a forced opening of the ribosomal gate, by hindering the placement of ribosomes in the inner C-ring space. Is there a mechanism for direct sensing of this process? Or is an overflow of substrates into the cytoplasm enough? For a pathogenic TTSS, a mechanism similar to the closed-gate model (Fig. 2) could be used to prevent the untimely secretion of effector proteins into an extracellular environment. Secretion of structural components could occur via a co-translational mechanism. On completion of the needle structure, the gate would open and the associated ribosomes could then translate the secretion substrates, allowing chaperone binding and subsequent secretion (similar to the open model). However, our closed model still allows for chaperone-mediated secretion, where the growing polypeptide first associates with the chaperone with immediate donation to the secretion apparatus. The molecular weight of most TTSCs is in the range of 20 kDa (Ref. 5). As these are small proteins even with gating of the C-ring, there could be enough space within the cavity to allow chaperone association and substrate delivery to the secretion apparatus (side view in Fig. 2). Coupling transcription and flagellar assembly
A second regulatory mechanism during flagellar assembly, which ensures that late secretion substrates do not compete with HBB substrates for secretion, is the coupling of transcriptional control to assembly. A flagellar transcription factor, σ28, is required for expression of genes involved in the late stages of
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Fig. 1. Schematic diagram of proposed secretion models for type III systems. The channel spanning both membranes is shown in orange. The cytoplasmic components of the secretion system and the inner membrane anchor are shown in green, whereas the secretion apparatus embedded within the anchor is shown in red. There are three mechanisms by which experimental data suggest a protein can be secreted by a type III secretion system. (a) An mRNA signal-mediated secretion mechanism, which would lead to coupled translation and secretion of the substrate. Substrate-translating ribosomes are brought into association with the cytoplasmic face of the secretion apparatus. (b) An amino-terminal amino acid signal-mediated secretion pathway, where the secretion signal is encoded in the amino-terminus of the secretion substrate (blue lollipops). (c) A chaperone-mediated secretion pathway where a cognate chaperone (light-green squares) binds the secretion substrate (blue circles). On interaction, it is the chaperone that communicates with the secretion apparatus, delivering the substrate for secretion. Unbound substrate is not recognized by the secretion apparatus and is therefore not secreted. Abbreviations: IM, inner membrane; OM, outer membrane; PG, peptidoglycan layer.
flagellar assembly11. The anti-σ28 factor FlgM modulates σ28 activity by direct binding and prevention of the formation of the σ28-RNA polymerase holoenzyme12,13. Both FlgM and σ28 are transcribed with the HBB structural genes14. FlgM binds σ28 and prevents transcription of the late flagellar promoters until the HBB is completed (Fig. 2, Stages I to V). FlgM possesses a late secretion signal and is secreted after HBB assembly, releasing σ28 to interact with RNA polymerase and direct transcription of the late assembly proteins (Fig. 2, Stage V)15,16. In this way, the subunits that make up the external filament are not produced until there is an HBB present for them to polymerize onto17. Both σ28-dependent and -independent promoters drive the transcription of flgM (Ref. 14). Karlinsey et al.17 have recently shown that FlgM secreted from the bacterial cell is predominantly translated from the σ28-dependent transcript and that secretion is dependent on the presence of FlgN and active translation. This suggests FlgM secretion is coupled to flgM translation. In our closed-gate model, this would require FlgN to possibly be within the space between the ribosomal gate and the secretion apparatus. Given that FlgN is a TTSC of small molecular weight, its small size might allow it to fit within such a tight space. This would also enable other forms of chaperone-mediated secretion to occur via the closed-gate pathway.
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Substrate specificity of flagellar assembly
The secretion specificity of the flagellar TTSS is partly determined by FliK and FlhB, components of the flagellar TTSS (Refs 18–20). Unlike FlgM, FliK is secreted before hook completion and is retained in the cytoplasm following hook completion (Fig. 2, Stages IV and V). It is believed that, after hook completion, cytoplasmic FliK interacts with FlhB to cause a change in substrate specificity from HBB substrates to late secretion substrates19. Evidence has recently been presented suggesting that, just before hook elongation, hook subunits accumulate within the cytoplasmic space of the C-ring21. Hook elongation would empty this space of hook subunits and allow FliK to interact with FlhB to switch secretion-substrate specificity. It would also allow access of the C-ring space to ribosomes http://tim.trends.com
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to initiate a translation-dependent secretion of mRNA transcripts of late assembly substrates. There is genetic evidence that, during hook elongation, a change in the TTSS apparatus specificity occurs that is independent of the FliK/FlhB substratespecificity-switching mechanism. A novel protein was identifed, Flk, which affects flgM translation and secretion at this stage of assembly. In specific flagellar mutant backgrounds, loss of Flk prevents both flgM translation and allows for premature FlgM secretion22,23. Kutsukake has proposed that Flk will function as a lock on the flagellar TTSS to prevent
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Closed-gate model Side view
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Fig. 2. Schematic diagram of flagellar assembly. The assembly of the flagellum can be divided in to at least seven distinct stages. (I) Site selection and MS-ring formation; (II) assembly of the C-ring and secretion apparatus; (III) rod formation; (IV) assembly of the P and L rings, leading to hook synthesis; (V) hook completion; (VI) a middle to late substrate specificity switch in secretion upon hook completion; and (VII) filament elongation. Upon reaching Stage IV of assembly, the secretion of structural components or secretion substrates can occur by two possible pathways, a closed-gate model or an open-gate model. During the closed-gate pathway, ribosomes associate with Flk bringing them into the vicinity of the C-ring. On induction of the substrate-specificity switch, the ribosomes are brought into the C-ring inner space where they can directly donate growing polypeptide chains to an FliK–FlhB complex, allowing the coupling of translation and secretion. This effectively forms a closed gate, which could prevent the secretion of other substrates at the wrong time. Chaperone-mediated secretion might still take place where the growing polypeptide first associates with the chaperone with immediate donation to an FlhB–FliK complex. A side view of Stage V is shown to demonstrate the possibility that chaperones enter the space between associated ribosomes and the secretion apparatus. An alternative to a closed-gate model would be that the gate is always ‘open’. In the open-gate pathway, Flk brings the ribosomes into the vicinity of the type III secretion apparatus. After translation, the FliK–FlhB complex recognizes the newly synthesized proteins, allowing secretion to occur. On completion of type III secretion, the ribosomal gate would be forced open by the capillary action caused by a build-up of secretion substrates within the secretion channel (Stage VII). On opening, the overflow of secretion substrates into the cytoplasm could be sensed by the secretion chaperones inducing a shutdown of secretion until required. This would leave the secretion apparatus in a primed state ready to repair damage to the type III secretion system or begin to secrete effector proteins when needed. Abbreviations: IM: inner membrane; OM, outer membrane; PG: peptidoglycan layer.
premature FlgM secretion22. Flk has homology to the ribosomal-binding domain of translation-initiation factor S1 and is a membrane-anchored protein. This would provide a mechanism to allow the association of ribosomes with the membrane. However, although both Flk and FlgN affect flgM translation, the effect is seen only in HBB mutant strains8,24. This suggests that redundant translation systems are present, and that http://tim.trends.com
the HBB might also provide a site for association of ribosomes for co-translational secretion. A possible candidate for a second ribosomal-interaction site within the HBB would be the C-ring because of its accessibility to ribosomes within the cytoplasm. At completion of hook synthesis, the FlhB component interacts with FliK to lose affinity for HBB secretion substrates. The C-ring cavity, empty of HBB subunits, would then fill with translating ribosomes containing mRNA that encodes the late secretion substrates (Fig. 2, closed-gate model). The diameter of the ribosome is ~200 Å (Refs 25,26), leading us to predict that the C-ring cavity could accommodate up to three ribosomes. Alternatively, interaction of FlhB and FliK would alter the specificity of FlhB, in that it would only accept translated polypeptides from ribosomes associated with Flk or the C-ring (Fig. 2; open-gate model). The ability of flagellar TTSCs to bind the carboxyl terminus of their respective partners is in contrast to the general trend seen for TTSCs identified from pathogenic TTSSs, which mainly bind an amino-terminal domain5. How can these proteins contribute to flagellar assembly? As they bind the carboxyl termini of flagellar proteins they might prevent degradation or assembly before secretion (Fig. 2, Stages IV to V). They might also act as a system for sensing the build up of secretion substrates in
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the cytoplasm towards the completion of filament elongation (Fig. 2; Stage VII). On filament completion, the substrates for late secretion substrate chaperones would begin to accumulate in the cell, leading to an interaction with their cognate chaperones. On doing so, this could inhibit the ability of the FlgN chaperone to regulate flgM translation or even inhibit FlgM secretion via a direct interaction, allowing a build up of cellular FlgM. This in turn would downregulate the expression of late components via σ28 inactivation. On filament completion, the bacterial cell requires a mechanism by which it can efficiently react to any damage that occurs to the flagella. Damage could automatically lead to a slow induction of substrate recognition by the secretion apparatus resulting in a reduction in substrates bound to their respective chaperones. For FlgN, this would lead to an increase in FlgM secretion, allowing an upregulation of the necessary promoters of the flagellar regulon until the flagellum has been resynthesized. Conclusions
Can our model also be true for general type III secretion of virulence factors in pathogenic bacteria? The majority of effector proteins have a partner chaperone that binds to an amino-terminal region instead of the carboxyl terminus like FlgN. This would allow an association of translation, reducing the time taken for secretion but also allowing for a measure of variation in the transcripts brought to TTSSassociated ribosomes. Many of the chaperones identified so far show a degree of selectivity, in that their respective partners can be secreted by the type III system but these are only correctly translocated to eukaryotic cells, from completed needles, when the chaperone is present6. A recent report by Wolf-Watz and colleagues27 has suggested that, contrary to earlier work, a secreted virulence factor in Yersinia does not possess an mRNA signal but the secretion is References 1 Hueck, C.J. (1998) Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol. Mol. Biol. Rev. 62, 379–433 2 Macnab, R.M. (1999) The bacterial flagellum: reversible rotary propellor and type III export apparatus. J. Bacteriol. 181, 7149–7153 3 Anderson, D.M. and Schneewind, O. (1999) Yersinia enterocolitica type III secretion: an mRNA signal that couples translation and secretion of YopQ. Mol. Microbiol. 31, 1139–1148 4 Anderson, D.M. and Schneewind, O. (1997) A mRNA signal for the type III secretion of Yop proteins by Yersinia enterocolitica. Science 278, 1140–1143 5 Bennett, J.C. and Hughes, C. (2000) From flagellum assembly to virulence: the extended family of type III export chaperones. Trends Microbiol. 8, 202–204 6 Cheng, L.W. and Schneewind, O. (2000) Type III machines of Gram-negative bacteria: delivering the goods. Trends Microbiol. 8, 214–220 7 Fraser, G.M. et al. (1999) Substrate-specific binding of hook-associated proteins by FlgN and FliT, putative chaperones for flagellum assembly. Mol. Microbiol. 32, 569–580 http://tim.trends.com
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• •
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Are ribosomes directly associated with the type III secretion apparatus? Is translation of secretion substrates associated with the type III secretion apparatus? Is there an FlgM secretion chaperone? Is σ28 or FlgN the FlgM chaperone? Do type III secretion chaperones act as sensors for completion of the secretion apparatus? If so, by what mechanism? Are there direct interactions between the different chaperones and the secretion apparatus? If so, what is the mechanism of these interactions?
exclusively determined by the amino terminus of the protein. A pertinent question here would be: is the translation of this protein still localized before chaperone binding and subsequent secretion? Completion of needle structures would also lead to an accumulation of distal structural subunits within the cell. Allowing secretion chaperones to act as a sensor, for completion of the type III machine, would allow for an induction of a specificity switch in secretion substrates. This would lead to the TTSS being left in a primed state ready to begin immediate delivery of effector proteins on contact with eukaryotic cells. In general, these observations could be the reason why much research in flagellar assembly and type III virulence factor secretion has identified a requirement for a signal in both the mRNA and in the secreted proteins: the mRNA signal allows the translation of the prospective secretion substrates to occur near the secretion apparatus, and the protein signal is there to allow for selectivity and control when deciding whether a protein should or should not be secreted.
8 Karlinsey, J.E. et al. (2000) Translation/secretion coupling by type III secretion systems. Cell 102, 487–497 9 Bonifield, H.R. et al. (2000) The flagellar hook protein, FlgE, of Salmonella enterica serovar typhimurium is posttranscriptionally regulated in response to the stage of flagellar assembly. J. Bacteriol. 182, 4044–4050 10 Ohnishi, K. et al. (1994) FlgD is a scaffolding protein needed for flagellar hook assembly in Salmonella typhimurium. J. Bacteriol. 176, 2272–2281 11 Ohnishi, K. et al. (1990) Gene fliA encodes an alternative sigma factor specific for flagellar operons in Salmonella typhimurium. Mol. Gen. Genet. 221, 139–147 12 Chadsey, M.S. et al. (1998) The flagellar antisigma factor FlgM actively dissociates Salmonella typhimurium σ28 RNA polymerase holoenzyme. Genes Dev. 12, 3123–3136 13 Ohnishi, K. et al. (1992) A novel transcriptional regulation mechanism in the flagellar regulon of Salmonella typhimurium: an antisigma factor inhibits the activity of the flagellum-specific σ factor, σF. Mol. Microbiol. 6, 3149–3157
14 Gillen, K.L. and Hughes, K.T. (1993) Transcription from two promoters and autoregulation contribute to the control of expression of the Salmonella typhimurium flagellar regulatory gene flgM. J. Bacteriol. 175, 7006–7015 15 Kutsukake, K. and Iino, T. (1994) Role of the FliA–FlgM regulatory system on the transcriptional control of the flagellar regulon and flagellar formation in Salmonella typhimurium. J. Bacteriol. 176, 3598–3605 16 Hughes, K.T. et al. (1993) Sensing structural intermediates in bacterial flagellar assembly by export of a negative regulator. Science 262, 1277–1280 17 Karlinsey, J.E. et al. (2000) Completion of the hookbasal body complex of the Salmonella typhimurium flagellum is coupled to FlgM secretion and fliC transcription. Mol. Microbiol. 37, 1220–1231 18 Minamino, T. et al. (1999) FliK, the protein responsible for flagellar hook length control in Salmonella, is exported during hook assembly. Mol. Microbiol. 34, 295–304 19 Minamino, T. and Macnab, R.M. (2000) Domain structure of Salmonella FlhB, a flagellar
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export component responsible for substrate specificity switching. J. Bacteriol. 182, 4906–4914 20 Williams, A.W. et al. (1996) Mutations in fliK and flhB affecting flagellar hook and filament assembly in Salmonella typhimurium. J. Bacteriol. 178, 2960–2970 21 Makishima, S. et al. Length of the flagellar hook and the capacity of the type III export apparatus. Science (in press)
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22 Kutsukake, K. (1997) Hook-length control of the export-switching machinery involves a double-locked gate in Salmonella typhimurium flagellar morphogenesis. J. Bacteriol. 179, 1268–1273 23 Karlinsey, J.E. et al. (1998) Flk couples flgM translation to flagellar ring assembly in Salmonella typhimurium. J. Bacteriol. 180, 5384–5397 24 Karlinsey, J.E. et al. (1997) The flk gene of Salmonella typhimurium couples flagellar P- and
Intimin and the host cell – is it bound to end in Tir(s)? Gad Frankel, Alan D. Phillips, Luiz R. Trabulsi, Stuart Knutton, Gordon Dougan and Stephen Matthews Intimate bacterial adhesion to the intestinal epithelium is a pathogenic mechanism shared by several human and animal enteric pathogens, including enteropathogenic and enterohaemorrhagic Escherichia coli. Two bacterial protein partners involved in this intimate association have been identified, intimin and Tir. Some key remaining questions include whether intimin specifically interacts with one or more host-cell-encoded molecules and whether these contacts are a prerequisite for the subsequent intimate intimin–Tir association. Recent data support the hypothesis that the formation of a stable intimin–Tir relationship is the consequence of intimin protein interactions involving both host and bacterial components.
Intimin is the name given to an outer-membrane adhesion molecule expressed by a group of pathogenic Escherichia coli that can cause disease in both humans and animals through formation of attaching and effacing (A/E) intestinal lesions1. These lesions are characterized by effacement of brush-border microvilli in the vicinity of adhering bacteria and intimin-mediated bacterial attachment to the plasma membrane of infected cells. The intimate nature of this interaction is revealed in electron micrographs of infected intestinal epithelia, which show a uniform ~10 nm gap between bacterial and host cell membranes (Fig. 1). A/E lesions were first described for strains of enteropathogenic E. coli (EPEC)2,3, an established etiological agent of human infantile diarrhoea in the developing world4. Similar intestinal lesions were later associated with other enteric mucosal pathogens including enterohaemorrhagic E. coli (EHEC)5,6, an emerging microbial pathogen associated with food poisoning that often has lifethreatening complications owing to the production of shiga toxins (verocytotoxins)4; rabbit diarrhoeagenic
L-ring assembly to flagellar morphogenesis. J. Bacteriol. 179, 2389–2400 25 Ban, N. et al. (2000) The complete atomic structure of the large ribosomal subunit at 2.4 Å resolution. Science 289, 905–920 26 Wimberly, B.T. et al. (2000) Structure of the 30S ribosomal subunit. Nature 407, 327–339 27 Lloyd, S.A. et al. (2001) Yersinia YopE is targeted for type III secretion by N-terminal, not mRNA, signals. Mol. Microbiol. 39, 520–532
E. coli (REPEC)7; and the mouse pathogen Citrobacter rodentium8. Using animal and ex vivo infection models, intimin was shown, in most cases, to be essential for colonization of the intestinal mucosa and in all cases for A/E lesion formation and pathogenicity9–13.
‘...the formation of a stable intimin–Tir relationship is the consequence of intimin protein interactions involving both host and bacterial components.’ Receptor-binding activities of intimin – early studies
In 1990, intimin, a 94-kDa outer membrane protein, was the first gene product reported to be associated with A/E lesion formation in EPEC (Ref. 14); an intimin homologue was subsequently discovered in EHEC O157:H7 (Ref. 15). Although no specific function was assigned to intimin at this stage, the realization that the protein had significant homology, at least among the amino-terminal 650 amino acids, to Yersinia invasin was very significant for subsequent studies. Invasin had been shown to be an outer membrane Yersinia protein that mediated bacterial internalization into mammalian cells by binding to β1 integrins; the integrin-binding activity of invasin had been localized to the carboxyl terminus of the polypeptide16, a region with only limited similarity to intimin. It was on this basis that, in 1993, we decided to investigate the possibility that the carboxyl terminus of intimin contained receptorbinding activity. By fusing the carboxy-terminal 280 amino acids of intimin (Int280) to maltosebinding protein (MBP), we demonstrated that soluble and latex-bead-coated MBP–Int280 from both EPEC and EHEC could directly bind to HEp-2 cells17. Independently however, Rosenshine et al.18 were unable to demonstrate significant MBP–Int280 binding to uninfected epithelial cells, but were able to demonstrate MBP–Int280 binding to cells previously infected with an intimin-negative EPEC mutant, thus indicating that a bacterial ‘signal’ is required for intimin binding. Importantly, both observations (i.e. binding of intimin to
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