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.     

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 chaperone FlgN regulates flagellar assembly via a negative feedback loop containing its chaperone substrates FlgK and FlgL

The type III secretion (TTS) chaperones are small proteins that act either as cytoplasmic bodyguards, protecting their secretion substrates from degradation and aggregation, facilitators of their cognate substrate secretion or both. FlgN has been previously shown to be a TTS chaperone for the hook-associated proteins FlgK and FlgL (FlgKL), and a translational regulator of the anti-sigma28 factor FlgM. Protein stability assays indicate that a flgN mutation leads to a dramatic decrease in the half-life of intracellular FlgK. However, using gene reporter fusions to flgK we show that a flgN mutation does not affect the translation of a flgK-lacZ fusion. Quantification of FlgM protein levels showed that FlgKL inhibit the positive regulation on flgM translation by FlgN when secretion of FlgKL is inhibited. Suppressors of the motility-defective phenotype of a flgN mutant were isolated and mapped to the clpXP and fliDST loci. Overexpression of flgKL on a plasmid also suppressed the motility defect of a flgN null mutant. These results suggest that FlgN is not required for secretion of FlgKL and that FlgN typifies a class of TTS chaperones that allows for the minimal amount of their substrates expression required in the assembly process by protecting the substrate from proteolysis. Our data leads us to propose a model in which the interaction between FlgN and FlgK or FlgL is a sensing mechanism to determine the stage of flagellar assembly. Furthermore, the interaction between FlgN and FlgK or FlgL inhibits the translational regulation of flgM via FlgN in response to the stage of flagellar assembly.     

Bacterial secretion chaperones

Many Gram-negative bacteria are able to invade hosts by translocation of effectors directly into target cells in processes usually mediated by two very complex secretion systems (SSs), named type III (T3) and type IV (T4) SSs. These syringe-needle injection devices work with intervention of specialized secretion chaperones that, unlike traditional molecular chaperones, do not assist in protein folding and are not energized by ATP. Controversy still surrounds secretion chaperones primary role, but we can say that these chaperones act as: (i) bodyguards to prevent premature aggregation, or as (ii) pilots to direct substrate secretion through the correct secretion system. This family of chaperones does not share primary structure similarity but amazingly equal 3D folds. This mini review has the intent to present updated structural and functional data for several important secretion chaperones, either alone or in complex with their cognate substrates, as well to report on the common features and roles of T3, T4 and flagellar chaperones.     

Yersinia enterocolitica type III secretion: an mRNA signal that couples translation and secretion of YopQ

Pathogenic Yersinia species export Yop proteins via a type III machinery to escape their phagocytic killing during animal infections. Here, we reveal the type III export mechanism of YopQ. In the presence of calcium, when type III secretion was blocked, yopQ mRNA was not translated. The signal of YopQ sufficient for the secretion of translationally fused reporter proteins was contained within the first 10 codons of its open reading frame. Some frameshift mutations that completely altered the peptide sequence specified by this signal did not impair secretion of the reporter protein. Exchanging the upstream untranslated mRNA leader of yopQ for that of E. coli lacZ also did not affect secretion. However, removal of the first 15 codons abolished YopQ export. Pulse-labelled YopE, but not YopQ, could be secreted after the polypeptide had been synthesized within the cytoplasm of Yersinia (post-translational secretion). Thus, YopQ appears to be exported by a mechanism that couples yopQ mRNA translation with the type III secretion of the encoded polypeptide.     

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.     

Type III export: new uses for an old pathway

Gram-negative bacteria use type III secretion (TTS) systems to translocate proteins into the extracellular environment or directly into eukaryotic cells. These complex secretory systems are assembled from over 20 different structural proteins, including 10 that have counterparts in the flagellar export pathway. Secretion substrates are directed to the TTS machinery via mRNA and/or amino acid secretion signals. TTS chaperones bind to select secretion substrates and assist in the export process. Recent progress in the understanding of TTS is reviewed.     

Translocated intimin receptor and its chaperone interact with ATPase of the type III secretion apparatus of enteropathogenic Escherichia coli

Few interactions have been reported between effectors and components of the type III secretion apparatus, although many interactions have been demonstrated between type III effectors and their cognate chaperones. It is thought that chaperones may play a role in directing effectors to the type III secretion apparatus. The ATPase FliI in the flagellar assembly apparatus plays a pivotal role in interacting with other components of the apparatus and with substrates of the flagellar system. We performed experiments to determine if there were any interactions between the effector Tir and its chaperone CesT and the type III secretion apparatus of enteropathogenic Escherichia coli (EPEC). Specifically, based on analogies with the flagella system, we examined Tir-CesT interactions with the putative ATPase EscN. We showed by affinity chromatography that EscN and Tir bind CesT specifically. Tir is not necessary for CesT and EscN interactions, and EscN binds Tir specifically without its chaperone CesT. Moreover, Tir directly binds EscN, as shown via gel overlay and enzyme-linked immunosorbent assay, and coimmunoprecipitation experiments revealed that Tir interacts with EscN inside EPEC. These data provide evidence for direct interactions between a chaperone, effector, and type III component in the pathogenic type III secretion system and suggest a model for Tir translocation whereby its chaperone, CesT, brings Tir to the type III secretion apparatus by specifically interacting with the type III ATPase EscN.     

Identification of potential type III secretion proteins via heterologous expression of Vibrio parahaemolyticus DNA

We employed a heterologous secretion assay to identify proteins potentially secreted by type III secretion systems (T3SSs) in Vibrio parahaemolyticus. N-terminal sequences from 32 proteins within T3SS genomic islands and seven proteins from elsewhere in the chromosome included proteins that were recognized for export by the Yersinia enterocolitica flagellar T3SS.     

Expanded roles for multicargo and class 1B effector chaperones in type III secretion

Bacterial type III secretion systems (T3SS) are complex protein assemblies that mediate the secretion of protein substrates outside the cell. Type III secretion chaperones (T3SC) are always found associated with T3SS, and they serve in multiple roles to ensure that protein substrates are efficiently targeted for secretion. Bacterial pathogens with T3SS express T3SC proteins that bind effectors, a process important for effector protein delivery into eukaryotic cells during infection. In this minireview, we focus on multicargo and class 1B T3SC that associate with effectors within significant pathogens of animals and plants. As a primary role, multicargo and class 1B T3SC form homodimers and specifically bind different effectors within the cytoplasm, maintaining the effectors in a secretion-competent state. This role makes T3SC initial and central contributors to effector-mediated pathogenesis. Recent findings have greatly expanded our understanding of cellular events linked to multicargo T3SC function. New binding interactions with T3SS components have been reported in different systems, thereby implicating multicargo T3SC in critical roles beyond effector binding. Three notable interactions with the YscN, YscV, and YscQ family members are well represented in the literature. Similar T3SC interactions are reported in the putative related flagellar T3SS, suggesting that secretion mechanisms may be more similar than previously thought. The evidence implicates multicargo and class 1B T3SC in effector binding and stabilization, in addition to T3SS recruitment and docking events.     

Functional analysis of the enteropathogenic Escherichia coli type III secretion system chaperone CesT identifies domains that mediate substrate interactions

In many Gram-negative bacteria, a key indicator of pathogenic potential is the possession of a specialized type III secretion system, which is utilized to deliver virulence effector proteins directly into the host cell cytosol. Many of the proteins secreted from such systems require small cytosolic chaperones to maintain the secreted substrates in a secretion-competent state. One such protein, CesT, serves a chaperone function for the enteropathogenic Escherichia coli (EPEC) translocated intimin receptor (Tir) protein, which confers upon EPEC the ability to alter host cell morphology following intimate bacterial attachment. Using a combination of complementary biochemical approaches, functional domains of CesT that mediate intermolecular interactions, involved in both chaperone-chaperone and chaperone-substrate associations, were determined. The CesT N-terminal is implicated in chaperone dimerization, whereas the amphipathic alpha-helical region of the C-terminal, is intimately involved in substrate binding. By functional complementation of chaperone domains using the Salmonella SicA chaperone to generate chaperone chimeras, we show that CesT-Tir interaction proceeds by a mechanism potentially common to other type III secretion system chaperones.     

Tetratricopeptide repeats are essential for PcrH chaperone function in Pseudomonas aeruginosa type III secretion

The type III secretion system (T3SS) is a specialized apparatus evolved by Gram-negative bacteria to deliver effector proteins into host cells, thus facilitating the establishment of an infection. Effector translocation across the target cell plasma membrane is believed to occur via pores formed by at least two secreted translocator proteins, the functions of which are dependent upon customized class II T3SS chaperones. Recently, three internal tetratricopeptide repeats (TPRs) were identified in this class of chaperones. Here, defined mutagenesis of the class II chaperone PcrH of Pseudomonas aeruginosa revealed these TPRs to be essential for chaperone activity towards the translocator proteins PopB and PopD and subsequently for the translocation of exoenzymes into host cells.     

Salmonella type III secretion-associated chaperones confer secretion-pathway specificity

Type III protein secretion systems (TTSSs) are ancestrally related to the flagellar export system and are essential for the virulence of many bacteria pathogenic for humans, animals and plants. Most proteins destined to travel the TTSS pathway possess at least two domains that specifically target them to the secretion apparatus. One of the domains is located within the amino terminal first approximately 20 amino acids and the second domain, located within the first approximately 140 amino acids, serves as a binding site for specific chaperones. It has been previously proposed that these two secretion signals are capable of operating independently of one another to facilitate secretion into the extracellular environment. We have found that in the absence of their chaperone-binding domains, the Salmonella typhimurium TTSS-secreted proteins SptP and SopE are no longer targeted for secretion through their cognate TTSS and, instead, are secreted through the flagellar export pathway. These results indicate the existence of an 'ancestral' flagellar secretion signal within TTSS-exported proteins that is revealed in the absence of the chaperone-binding domain. Furthermore, we found that secretion into culture supernatants as well as translocation into host cells by the cognate TTSS require both, the amino terminal and chaperone-binding domains. We conclude from these studies that a critical function for the TTSS-associated chaperones is to confer secretion-pathway specificity to their cognate secreted proteins.     

Soluble components of the flagellar export apparatus,FliI, FliJ,and FliH,do not deliver flagellin,the major filament protein,from the cytosol to the export gate

Flagella, the locomotion organelles of bacteria, extend from the cytoplasm to the cell exterior. External flagellar proteins are synthesized in the cytoplasm and exported by the flagellar type III secretion system. Soluble components of the flagellar export apparatus, FliI, FliH, and FliJ, have been implicated to carry late export substrates in complex with their cognate chaperones from the cytoplasm to the export gate. The importance of the soluble components in the delivery of the three minor late substrates FlgK, FlgL (hook–filament junction) and FliD (filament-cap) has been convincingly demonstrated, but their role in the transport of the major filament component flagellin (FliC) is still unclear.     

Spa15 of Shigella flexneri, a third type of chaperone in the type III secretion pathway

The type III secretion (TTS) pathway is used by numerous Gram-negative pathogens to inject virulence factors into eukaryotic cells. In addition to a functional TTS apparatus, secretion of effector proteins depends upon specific chaperones. Using a two-hybrid screen in yeast and a co-purification assay in Shigella flexneri, we demonstrated that Spa15, which is encoded by an operon for components of the TTS apparatus, is associated in the cytoplasm with three proteins that are secreted by the TTS pathway, IpaA, IpgB1 and OspC3. Spa15 was found to be necessary for stability of IpgB1 but not IpaA, and for secretion of IpaA molecules that were stored in the cytoplasm but not those that were synthesized while the secretion apparatus was active. The ability of Spa15 to associate with several non-homologous secreted proteins, the presence of Spa15 homologues in other TTS systems and the location of the corresponding genes within operons for components of the TTS apparatus suggest that Spa15 belongs to a new class of TTS chaperones.     

Substrate recognition by the Yersinia type III protein secretion machinery

Type III secretion is the designation given to those protein secretion pathways, primarily in pathogenic Gram-negative bacteria, whose secretion machinery components share an amino acid sequence homology to components of the flagellar basal body. In Yersinia spp., these secretion machineries inject virulence proteins called Yops into the cytosol of target macrophages in an effort to evade phagocytic killing. To date, a clear mechanism by which Yops are recognized by the type III secretion machinery has not been elucidated. Unlike most, if not all, previously characterized protein sorting pathways, the information that identifies Yops as substrates for secretion seems not to be wholly encoded within the Yop peptide sequence. In fact, it appears that at least some of this information is contained within yop mRNAs. This review summarizes recent observations that have been made in this unusual field and proposes models by which proteins may be initiated into this pathway.