Comparative analysis of the secretion capability of early and late flagellar type III secretion substrates

A remarkable feature of the flagellar‐specific type III secretion system (T3SS) is the selective recognition of a few substrate proteins among the many thousand cytoplasmic proteins. Secretion substrates are divided into two specificity classes: early substrates secreted for hook‐basal body (HBB) construction and late substrates secreted after HBB completion. Secretion was reported to require a disordered N‐terminal secretion signal, mRNA secretion signals within the 5′‐untranslated region (5′‐UTR) and for late substrates, piloting proteins known as the T3S chaperones. Here, we utilized translational β‐lactamase fusions to probe the secretion efficacy of the N‐terminal secretion signal of fourteen secreted flagellar substrates in Salmonella enterica. We observed a surprising variety in secretion capability between flagellar proteins of the same secretory class. The peptide secretion signals of the early‐type substrates FlgD, FlgF, FlgE and the late‐type substrate FlgL were analysed in detail. Analysing the role of the 5′‐UTR in secretion of flgB and flgE revealed that the native 5′‐UTR substantially enhanced protein translation and secretion. Based on our data, we propose a multicomponent signal that drives secretion via the flagellar T3SS. Both mRNA and peptide signals are recognized by the export apparatus and together with substrate‐specific chaperones allowing for targeted secretion of flagellar substrates.     

Function of the conserved FHIPEP domain of the flagellar type III export apparatus,protein FlhA

The Type III flagellar protein export apparatus of bacteria consists of five or six membrane proteins, notably FlhA, which controls the export of other proteins and is homologous to the large family of FHIPEP export proteins. FHIPEP proteins contain a highly‐conserved cytoplasmic domain. We mutagenized the cloned Salmonella flhA gene for the 692 amino acid FlhA, changing a single, conserved amino acid in the 68‐amino acid FHIPEP region. Fifty‐two mutations at 30 positions mostly led to loss of motility and total disappearance of microscopically visible flagella, also Western blot protein/protein hybridization showed no detectable export of hook protein and flagellin. There were two exceptions: a D199A mutant strain, which produced short‐stubby flagella; and a V151L mutant strain, which did not produce flagella and excreted mainly un‐polymerized hook protein. The V151L mutant strain also exported a reduced amount of hook‐cap protein FlgD, but when grown with exogenous FlgD it produced polyhooks and polyhook‐filaments. A suppressor mutant in the cytoplasmic domain of the export apparatus membrane protein FlhB rescued export of hook‐length control protein FliK and facilitated growth of full‐length flagella. These results suggested that the FHIPEP region is part of the gate regulating substrate entry into the export apparatus pore.     

Crystal structure of the C-terminal domain of a flagellar hook-capping protein from Xanthomonas campestris

The crystal structure of the C-terminal domain of a hook-capping protein FlgD from the plant pathogen Xanthomonas campestris (Xc) has been determined to a resolution of ca 2.5 Å using X-ray crystallography. The monomer of whole FlgD comprises 221 amino acids with a molecular mass of 22.7 kDa, but the flexible N-terminus is cleaved for up to 75 residues during crystallization. The final structure of the C-terminal domain reveals a novel hybrid comprising a tudor-like domain interdigitated with a fibronectin type III domain. The C-terminal domain of XcFlgD forms three types of dimers in the crystal. In agreement with this, analytical ultracentrifugation and gel filtration experiments reveal that they form a stable dimer in solution. From these results, we propose that the Xc flagellar hook cap protein FlgD comprises two individual domains, a flexible N-terminal domain that cannot be detected in the current study and a stable C-terminal domain that forms a stable dimer.     

Domain structure of Salmonella FlhB, a flagellar export component responsible for substrate specificity switching

We have investigated the properties of the cytoplasmic domain (FlhB(C)) of the 383-amino-acid Salmonella membrane protein FlhB, a component of the type III flagellar export apparatus. FlhB, along with the hook-length control protein FliK, mediates the switching of export specificity from rod- and hook-type substrates to filament-type substrates during flagellar morphogenesis. Wild-type FlhB(C) was unstable (half-life, ca. 5 min), being specifically cleaved at Pro-270 into two polypeptides, FlhB(CN) and FlhB(CC), which retained the ability to interact with each other after cleavage. Full-length wild-type FlhB was also subject to cleavage. Coproduction of the cleavage products, FlhB(delta CC) (i.e., the N-terminal transmembrane domain FlhB(TM) plus FlhB(CN)) and FlhB(CC), resulted in restoration of both motility and flagellar protein export to an flhB mutant host, indicating that the two polypeptides were capable of productive association. Mutant FlhB proteins that can undergo switching of substrate specificity even in the absence of FliK were much more resistant to cleavage (half-lives, 20 to 60 min). The cleavage products of wild-type FlhB(C), existing as a FlhB(CN)-FlhB(CC) complex on an affinity blot membrane, bound the rod- and hook-type substrate FlgD more strongly than the filament-type substrate FliC. In contrast, the intact form of FlhB(C) (mutant or wild type) or the FlhB(CC) polypeptide alone bound FlgD and FliC to about the same extent. FlhB(CN) by itself did not bind substrates appreciably. We propose that FlhB(C) has two substrate specificity states and that a conformational change, mediated by the interaction between FlhB(CN) and FlhB(CC), is responsible for the specificity switching process. FliK itself is an export substrate; its binding properties for FlhB(C) resemble those of FlgD and do not provide any evidence for a physical interaction beyond that of the export process.     

Substrate specificity classes and the recognition signal for Salmonella type III flagellar export

Most flagellar proteins of Salmonella are exported to their assembly destination via a specialized apparatus. This apparatus is a member of the type III superfamily, which is widely used for secretion of virulence factors by pathogenic bacteria. Extensive studies have been carried out on the export of several of the flagellar proteins, most notably the hook protein (FlgE), the hook-capping protein (FlgD), and the filament protein flagellin (FliC). This has led to the concept of two export specificity classes, the rod/hook type and the filament type. However, little direct experimental evidence has been available on the export properties of the basal-body rod proteins (FlgB, FlgC, FlgF, and FlgG), the putative MS ring-rod junction protein (FliE), or the muramidase and putative rod-capping protein (FlgJ). In this study, we have measured the amounts of these proteins exported before and after hook completion. Their amounts in the culture supernatant from a flgE mutant (which is still at the hook-type specificity stage) were much higher than those from a flgK mutant (which has advanced to the filament-type specificity stage), placing them in the same class as the hook-type proteins. Overproduction of FliE, FlgB, FlgC, FlgF, FlgG, or FlgJ caused inhibition of the motility of wild-type cells and inhibition of the export of the hook-capping protein FlgD. We also examined the question of whether export and translation are linked and found that all substrates tested could be exported after protein synthesis had been blocked by spectinomycin or chloramphenicol. We conclude that the amino acid sequence of these proteins suffices to mediate their recognition and export.     

Role of FliJ in flagellar protein export in Salmonella

We isolated and characterized spontaneous mutants with defects in the 147-amino-acid Salmonella protein FliJ, which is a cytoplasmic component of the type III flagellar export apparatus. These mutants, including ones with null mutations, have the ability to form swarms on motility agar plates after prolonged incubation at 30 degrees C; i.e., they display a leaky motile phenotype. One mutant, SJW277, which formed significantly bigger swarms than the others, encoded only the N-terminal 73 amino acids of FliJ, one-half of the protein. At 30 degrees C, overproduction of this mutant protein improved, to wild-type levels, both motility and the ability to export both rod/hook-type (FlgD; hook capping protein) and filament-type (FliC; flagellin) substrates. At 42 degrees C, however, export was inhibited, indicating that the mutant FliJ protein was temperature sensitive. Taking advantage of this, we performed temperature upshift experiments, which demonstrated that FliJ is directly required for the export of FliC. Co-overproduction of FliJ and either of two export substrates, FliE or FlgG, hindered their aggregation in the cytoplasm. We conclude that FliJ is a general component of the flagellar export apparatus and has a chaperone-like activity for both rod/hook-type and filament-type substrates.     

The type III flagellar export specificity switch is dependent on FliK ruler and a molecular clock

Salmonella flagellar hook length is controlled at the level of export substrate specificity of the FlhB component of the type III flagellar export apparatus. FliK is believed to be the hook length sensor and interacts with FlhB to change its export specificity upon hook completion. To find properties of FliK expected of such a molecular ruler, we assayed binding of FliK to the hook and found that the N-terminal domain of FliK (FliK(N)) bound to the hook-capping protein FlgD with high affinity and to the hook protein FlgE with low affinity. To investigate a possible role of FlgE in hook length control, flgE mutants with partially impaired motility were isolated and analyzed. Eight flgE mutants obtained all formed flagellar filaments. The mutants produced significantly shorter hooks while the hook-type substrates such as FlgE, FliK and FlgD were secreted in large amounts, suggesting defective hook assembly with the mutant FlgE proteins. Upon overexpression, mutant FlgEs produced hooks of normal length and wild-type FlgE produced longer hooks. These results suggest that hook length is dependent on the hook polymerization rate and that the start of hook polymerization initiates a "time countdown" for the specificity switch to occur or for significant slow down of rod/hook-type export after hook length reaches around 55 nm for later infrequent FliK(C)-FlhB(C) interaction. We propose that FliK(N) acts as a flexible tape measure, but that hook length is also dependent on the hook elongation rate and a switch timing mechanism.     

Components of the Salmonella flagellar export apparatus and classification of export substrates

Until now, identification of components of the flagellar protein export apparatus has been indirect. We have now identified these components directly by establishing whether mutants defective in putative export components could translocate export substrates across the cytoplasmic membrane into the periplasmic space. Hook-type proteins could be exported to the periplasm of rod mutants, indicating that rod protein export does not have to precede hook-type protein export and therefore that both types of proteins belong to a single export class, the rod/hook-type class, which is distinct from the filament-type class. Hook-capping protein (FlgD) and hook protein (FlgE) required FlhA, FlhB, FliH, FliI, FliO, FliP, FliQ, and FliR for their export to the periplasm. In the case of flagellin as an export substrate, because of the phenomenon of hook-to-filament switching of export specificity, it was necessary to use temperature-sensitive mutants and establish whether flagellin could be exported to the cell exterior following a shift from the permissive to the restrictive temperature. Again, FlhA, FlhB, FliH, FliI, and FliO were required for its export. No suitable temperature-sensitive fliQ or fliR mutants were available. FliP appeared not to be required for flagellin export, but we suspect that the temperature-sensitive FliP protein continued to function at the restrictive temperature if incorporated at the permissive temperature. Thus, we conclude that these eight proteins are general components of the flagellar export pathway. FliJ was necessary for export of hook-type proteins (FlgD and FlgE); we were unable to test whether FliJ is needed for export of filament-type proteins. We suspect that FliJ may be a cytoplasmic chaperone for the hook-type proteins and possibly also for FliE and the rod proteins. FlgJ was not required for the export of the hook-type proteins; again, because of lack of a suitable temperature-sensitive mutant, we were unable to test whether it was required for export of filament-type proteins. Finally, it was established that there is an interaction between the processes of outer ring assembly and of penetration of the outer membrane by the rod and nascent hook, the latter process being of course necessary for passage of export substrates into the external medium. During the brief transition stage from completion of rod assembly and initiation of hook assembly, the L ring and perhaps the capping protein FlgD can be regarded as bona fide export components, with the L ring being in a formal sense the equivalent of the outer membrane secretin structure of type III virulence factor export systems.     

Targeting early proximal-rod component substrate FlgB to FlhB for flagellar-type III secretion in Salmonella

The Salmonella flagellar secretion apparatus is a member of the type III secretion (T3S) family of export systems in bacteria. After completion of the flagellar motor structure, the hook-basal body (HBB), the flagellar T3S system undergoes a switch from early to late substrate secretion, which results in the expression and assembly of the external, filament propeller-like structure. In order to characterize early substrate secretion-signals in the flagellar T3S system, the FlgB, and FlgC components of the flagellar rod, which acts as the drive-shaft within the HBB, were subject to deletion mutagenesis to identify regions of these proteins that were important for secretion. The β-lactamase protein lacking its Sec-dependent secretion signal (Bla) was fused to the C-terminus of FlgB and FlgC and used as a reporter to select for and quantify the secretion of FlgB and FlgC into the periplasm. Secretion of Bla into the periplasm confers resistance to ampicillin. In-frame deletions of amino acids 9 through 18 and amino acids 39 through 58 of FlgB decreased FlgB secretion levels while deleting amino acid 6 through 14 diminished FlgC secretion levels. Further PCR-directed mutagenesis indicated that amino acid F45 of FlgB was critical for secretion. Single amino acid mutagenesis revealed that all amino acid substitutions at F45 of FlgB position impaired rod assembly, which was due to a defect of FlgB secretion. An equivalent F49 position in FlgC was essential for assembly but not for secretion. This study also revealed that a hydrophobic patch in the cleaved C-terminal domain of FlhB is critical for recognition of FlgB at F45.     

Substrate specificity switching of the flagellum-specific export apparatus during flagellar morphogenesis in Salmonella typhimurium.

During flagellar morphogenesis in Salmonella typhimurium, the flagellum-specific anti-sigma factor FlgM is exported out of the cells only after completion of hook assembly. In this study, we examined the export of the flagellar proteins, FlgD (hook capping protein), FlgE (hook protein), FlgK and FlgL (hook-filament junction proteins), FliD (filament capping protein), and FliC (flagellin), before and after completion of hook assembly. Like the FlgM protein, the FlgK, FlgL, FliD, and FliC proteins are exported efficiently only after completion of hook assembly. On the other hand, the FlgD and FlgE proteins are exported efficiently before, but poorly after, hook completion. These results indicate that the export properties are different between these two groups and that their export order exactly parallels the assembly order of the hook-filament structure. We propose that the substrate specificity switching occurs in the flagellum-specific export apparatus upon completion of hook assembly.     

Amino‐terminal residues dictate the export efficiency of the Campylobacter jejuni filament proteins via the flagellum

Bacterial flagella play an essential role in the pathogenesis of numerous enteric pathogens. The flagellum is required for motility, colonization, and in some instances, for the secretion of effector proteins. In contrast to the intensively studied flagella of Escherichia coli and Salmonella typhimurium, the flagella of Campylobacter jejuni, Helicobacter pylori and Vibrio cholerae are less well characterized and composed of multiple flagellin subunits. This study was performed to gain a better understanding of flagellin export from the flagellar type III secretion apparatus of C. jejuni. The flagellar filament of C. jejuni is comprised of two flagellins termed FlaA and FlaB. We demonstrate that the amino‐termini of FlaA and FlaB determine the length of the flagellum and motility of C. jejuni. We also demonstrate that protein‐specific residues in the amino‐terminus of FlaA and FlaB dictate export efficiency from the flagellar type III secretion system (T3SS) of Yersinia enterocolitica. These findings demonstrate that key residues within the amino‐termini of two nearly identical proteins influence protein export efficiency, and that the mechanism governing the efficiency of protein export is conserved among two pathogens belonging to distinct bacterial classes. These findings are of additional interest because C. jejuni utilizes the flagellum to export virulence proteins.     

Interactions among components of the Salmonella flagellar export apparatus and its substrates

We have examined the cytoplasmic components (FliH, FliI and FliJ) of the type III flagellar protein export apparatus, plus the cytoplasmic domains (FlhAC and FlhBC) of two of its six membrane components. FliH, FlhAC and FliJ, when overproduced, caused inhibition of motility of wild-type cells and inhibition of the export of substrates such as the hook protein FlgE. Co-overproduction of FliH and FliI substantially relieved the inhibition caused by FliH, suggesting that it is excess free FliH that is inhibitory and that FliH and FliI form a complex. We purified His-FLAG-tagged versions of: (i) export components FliH, FliI, FliJ, FlhAC and FlhBC; (ii) rod/hook-type export substrates FlgB (rod protein), FlgE (hook protein), FlgD (hook capping protein) and FliE (basal body protein); and (iii) filament-type export substrates FlgK and FlgL (hook-filament junction proteins) and FliC (flagellin). We tested for protein-protein interactions by affinity blotting. In many cases, a given protein interacted with more than one other component, indicating that there are likely to be multiple dynamic interactions or interactions that involve more than two components. Interactions of FlhBC with rod/hook-type substrates were strong, whereas those with filament-type substrates were very weak; this may reflect the role of FlhB in substrate specificity switching. We propose a model for the flagellar export apparatus in which FlhA and FlhB and the other four integral membrane proteins of the apparatus form a complex at the base of the flagellar motor. A soluble complex of at least three proteins (FliH, FliI and FliJ) bind the protein to be exported and then interact with the complex at the motor to deliver the protein, which is then exported in an ATP-dependent process mediated by FliI.     

Two parts of the T3S4 domain of the hook-length control protein FliK are essential for the substrate specificity switching of the flagellar type III export apparatus

The switch in export specificity of the type III flagellar protein export apparatus from rod/hook type to filament type is believed to occur upon completion of hook assembly by way of an interaction of the type III secretion substrate specificity switch (T3S4) domain of the hook-length control protein FliK, with the integral membrane export apparatus component FlhB. The T3S4 domain of FliK (FliKT3S4) consisting of amino acid residues 265-405 has an unstable and flexible conformation in its last 35 residues (FliKCT). To investigate the role of FliKT3S4 in substrate specificity switching, we studied the effect of deletions and point mutations within this domain and characterized suppressor mutations. Deletions of ten amino acid residues within the region of residues 301-350 and five amino acids of residues 401-405 abolished switching of export specificity. Site directed mutagenesis showed that highly conserved residues, Val302, Ile304, Leu335, Val401 and Ala405, are essential, and that the five C terminal residues (401-405) are restricted in conformation for the switching process. Suppressor mutant analysis of the fliK(S319Y) mutant, which produces extended hooks with filaments attached due to delayed switching, suggested that FliKT3S4 interacts with the C terminal half of the cytoplasmic domain of FlhB (FlhBC). We propose a two step binding model of FliKT3S4 and FlhBC, in which residues 301-350 of FliK bind to FlhBC upon hook assembly completion at about 55 nm, and then unfolded FliKCT binds to FlhBC to trigger the switch in substrate specificity.     

Similar modes of polypeptide recognition by export chaperones in flagellar biosynthesis and type III secretion

Assembly of the bacterial flagellum and type III secretion in pathogenic bacteria require cytosolic export chaperones that interact with mobile components to facilitate their secretion. Although their amino acid sequences are not conserved, the structures of several type III secretion chaperones revealed striking similarities between their folds and modes of substrate recognition. Here, we report the first crystallographic structure of a flagellar export chaperone, Aquifex aeolicus FliS. FliS adopts a novel fold that is clearly distinct from those of the type III secretion chaperones, indicating that they do not share a common evolutionary origin. However, the structure of FliS in complex with a fragment of FliC (flagellin) reveals that, like the type III secretion chaperones, flagellar export chaperones bind their target proteins in extended conformation and suggests that this mode of recognition may be widely used in bacteria.     

An amino-terminal secretion signal is required for YplA export by the Ysa, Ysc, and flagellar type III secretion systems of Yersinia enterocolitica biovar 1B

Yersinia enterocolitica biovar 1B maintains three distinct type III secretion (TTS) systems, which independently operate to target proteins to extracellular sites. The Ysa and Ysc systems are prototypical contact-dependent TTS systems that translocate toxic effectors to the cytosols of targeted eukaryotic host cells during infection. The flagellar TTS system is utilized during the assembly of the flagellum and is required for secretion of the virulence-associated phospholipase YplA to the bacterial milieu. When ectopically produced, YplA is also a secretion substrate for the Ysa and Ysc TTS systems. In this study, we define elements that allow YplA recognition and export by the Ysa, Ysc, and flagellar TTS systems. Fusion of various amino-terminal regions of YplA to Escherichia coli alkaline phosphatase (PhoA) lacking its native secretion signal demonstrated that the first 20 amino acids or corresponding mRNA codons of YplA were sufficient for export of YplA-PhoA chimeras by each TTS system. Export of native YplA by each of the three TTS systems was also found to depend on the integrity of its amino terminus. Introduction of a frameshift mutation or deletion of yplA sequences encoding the amino-terminal 20 residues negatively impacted YplA secretion. Deletion of other yplA regions was tolerated, including that resulting in the removal of amino acid residues 30 through 40 of the polypeptide and removal of the 5' untranslated region of the mRNA. This work supports a model in which independent and distantly related TTS systems of Y. enterocolitica recognize protein substrates by a similar mechanism.     

Yersinia enterocolitica type III secretion. On the role of SycE in targeting YopE into HeLa cells.

Yersinia enterocolitica inject toxic proteins (effector Yops) into the cytosol of eukaryotic cells by a mechanism requiring the type III machinery. Previous work mapped a signal sufficient for the targeting of fused reporter proteins to amino acids 1-100 of YopE. Targeting requires the binding of SycE to YopE residues 15-100 in the bacterial cytoplasm. We asked whether SycE functions only to stabilize YopE in the bacterial cytoplasm, or whether the secretion chaperone itself contributes to substrate recognition by the type III machinery. Fusions of glutathione S-transferase to either the N or C terminus of SycE resulted in hybrid proteins that bound YopE but prevented targeting of the export substrate into HeLa cells. As compared with wild-type SycE, glutathione S-transferase-SycE bound and stabilized YopE in the bacterial cytoplasm but failed to release the polypeptide for export by the type III machinery. Thus, it appears that SycE functions to deliver YopE to the type III secretion machinery. A model is presented that accounts for substrate recognition of effector Yops, a group of proteins that do not share amino acid sequence or physical similarities.     

How and when are substrates selected for type III secretion?

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.     

The type III secretion determinants of the flagellar anti-transcription factor, FlgM, extend from the amino-terminus into the anti-σ28 domain

The flagellar-specific anti-sigma factor, FlgM, inhibits the expression of late flagellar genes until the hook–basal body structure is assembled and competent for export of the flagellins and hook-associated proteins (flagellar late proteins). FlgM monitors this assembly checkpoint by being a substrate for export via the hook–basal body structure, which includes a type III protein secretion complex. Amino acid sequence alignment of late-secreted flagellar proteins identified a region of homology present in the amino-terminus of FlgM and the other late flagellar proteins, but not in flagellar proteins secreted earlier during flagellar biosynthesis. Single amino acid substitutions at specific positions within this motif decreased the export of FlgM. Deletion of this region (S3-P11) resulted in lower intracellular FlgM levels, but did not prevent recognition and export by the flagellar-specific secretion system. Mutations were isolated in a second region of FlgM spanning residues K27 to A65 that exhibited increased anti-σ²⁸ activity. These FlgM 'hyperinhibitor' mutants were secreted less than wild-type FlgM. Mutations that interfere with the secretion of FlgM without abolishing anti-σ²⁸ activity have a negative effect upon the secretion of a His-tagged FlgM mutant that lacks anti-σ²⁸ activity. Models are proposed to explain the dominant negative phenotype of the FlgM secretion mutants reported in this study.     

Flipping the switch: bringing order to flagellar assembly

The bacterial flagellum is a complex self-assembling nanomachine that contains its own type III protein export apparatus. Upon completion of early flagellar structure, this apparatus switches substrate specificity to export late structural subunits, thereby coupling sequential flagellar gene expression with flagellar assembly. The switch is achieved by a conformational change of the export apparatus component FlhB driven by the flagellar hook-length control protein FliK. Two basic models of FliK- and FlhB-based switching are currently being pursued, together with the investigation of another factor, Flk, which prevents premature export of late substrates. Here, we review in detail each of these three export switch components and present the current understanding of how they work in concert in the making of a flagellum.     

The chaperone binding domain of SopE inhibits transport via flagellar and SPI-1 TTSS in the absence of InvB

Type III secretion systems (TTSS) are used by many Gram-negative pathogens for transporting effector proteins into eukaryotic host cells. Two modes of type III effector protein transport can be distinguished: transport into the surrounding medium (secretion) and cell-contact induced injection of effector proteins directly into the host cell cytosol (translocation). Two domains within the N-terminal regions of effector proteins determine the mode of transport. The amino terminal approximately 20 amino acids (N-terminal secretion signal, NSS) mediate secretion. In contrast, translocation generally requires the NSS, the adjacent approximately 100 amino acids (chaperone binding domain, CBD) and binding of the cognate chaperone to this CBD. TTSS are phylogenetically related to flagellar systems. Because both systems are expressed in Salmonella Typhimurium, correct effector protein transport involves at least two decisions: transport via the Salmonella pathogenicity island 1 (SPI-1) but not the flagellar TTSS (= specificity) and translocation into the host cell instead of secretion into the surrounding media (= transport mode). The mechanisms guiding these decisions are poorly understood. We have studied the S. Typhimurium effector protein SopE, which is specifically transported via the SPI-1 TTSS. Secretion and translocation strictly require the cognate chaperone InvB. Alanine replacement of amino acids 30-42 (and to some extent 44-54) abolished tight InvB binding, abolished translocation into the host cell and led to secretion of SopE via both, the flagellar and the SPI-1 TTSS. In clear contrast to wild-type SopE, secretion of SopE(Ala30-42) and SopE(Ala44-54) via the SPI-1 and the flagellar export system did not require InvB. These data reveal a novel function of the CBD: the CBD inhibits secretion of wild-type SopE via the flagellar and the SPI-1 TTSS in the absence of the chaperone InvB. Our data provide new insights into mechanisms ensuring specific effector protein transport by TTSS.