Function of FlhB,a Membrane Protein Implicated in the Bacterial Flagellar Type III Secretion System

The membrane protein FlhB is a highly conserved component of the flagellar secretion system, and it plays an active role in the regulation of protein export. In this study conserved properties of FlhB that are important for its function were investigated. Replacing the flhB gene (or part of the gene) in Salmonella typhimurium with the flhB gene of the distantly related bacterium Aquifex aeolicus greatly reduces motility. However, motility can be restored to some extent by spontaneous mutations in the part of flhB gene coding for the cytoplasmic domain of Aquifex FlhB. Structural analysis suggests that these mutations destabilize the structure. The secondary structure and stability of the mutated cytoplasmic fragments of FlhB have been studied by circular dichroism spectroscopy. The results suggest that conformational flexibility could be important for FlhB function. An extragenic suppressor mutation in the fliS gene, which decreases the affinity of FliS to FliC, partially restores motility of the FlhB substitution mutants.     

The Structure of the Salmonella typhimurium Type III Secretion System Needle Shows Divergence from the Flagellar System

The type III secretion system (T3SS) is essential for the infectivity of many pathogenic Gram-negative bacteria. The T3SS contains proteins that form a channel in the inner and outer bacterial membranes, as well as an extracellular needle that is used for transporting and injecting effector proteins into a host cell. The homology between the T3SS and the bacterial flagellar system has been firmly established, based upon both sequence similarities between respective proteins in the two systems and the structural homology of higher-order assemblies. It has previously been shown that the Shigella flexneri needle has a helical symmetry of ∼ 5.6 subunits/turn, which is quite similar to that of the most intensively studied flagellar filament (from Salmonella typhimurium), which has ∼ 5.5 subunits/turn. We now show that the Sa. typhimurium needle, expected by homology arguments to be more similar to the Sa. typhimurium flagellar filament than is the needle from Shigella, actually has ∼ 6.3 subunits/turn. It is not currently understood how host cell contact, made at the tip of the needle, is communicated to the secretory system at the base. In contrast to the Sa. typhimurium flagellar filament, which shows a nearly crystalline order, the Sa. typhimurium needle has a highly variable symmetry, which could be used to transmit information about host cell contact.     

Functional Activation of the Flagellar Type III Secretion Export Apparatus

Flagella are assembled sequentially from the inside-out with morphogenetic checkpoints that enforce the temporal order of subunit addition. Here we show that flagellar basal bodies fail to proceed to hook assembly at high frequency in the absence of the monotopic protein SwrB of Bacillus subtilis. Genetic suppressor analysis indicates that SwrB activates the flagellar type III secretion export apparatus by the membrane protein FliP. Furthermore, mutants defective in the flagellar C-ring phenocopy the absence of SwrB for reduced hook frequency and C-ring defects may be bypassed either by SwrB overexpression or by a gain-of-function allele in the polymerization domain of FliG. We conclude that SwrB enhances the probability that the flagellar basal body adopts a conformation proficient for secretion to ensure that rod and hook subunits are not secreted in the absence of a suitable platform on which to polymerize.     

An Arabidopsis Secondary Metabolite Directly Targets Expression of the Bacterial Type III Secretion System to Inhibit Bacterial Virulence

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Flagellar Formation in C-Ring-Defective Mutants by Overproduction of FliI,the ATPase Specific for Flagellar Type III Secretion

The flagellar cytoplasmic ring (C ring), which consists of three proteins, FliG, FliM, and FliN, is located on the cytoplasmic side of the flagellum. The C ring is a multifunctional structure necessary for flagellar protein secretion, torque generation, and switching of the rotational direction of the motor. The deletion of any one of the fliG, fliM, and fliN genes results in a Fla⁻ phenotype. Here, we show that the overproduction of the flagellum-specific ATPase FliI overcomes the inability of basal bodies with partial C-ring structures to produce complete flagella. Flagella made upon FliI overproduction were paralyzed, indicating that an intact C ring is essential for motor function. In FliN- or FliM-deficient mutants, flagellum production was about 10% of the wild-type level, while it was only a few percent in FliG-deficient mutants, suggesting that the size of partial C rings affects the extent of flagellation. For flagella made in C-ring mutants, the hook length varied considerably, with many being markedly shorter or longer than that of the wild type. The broad distribution of hook lengths suggests that defective C rings cannot control the hook length as tightly as the wild type even though FliK and FlhB are both intact.The flagellum is the ultrastructure for motility in many bacterial species (1). Flagellar assembly requires about 50 genes, among which about 20 gene products are incorporated in the complete flagellum (12). Most structural proteins and others necessary for assembly are exported through a flagellum-specific type III secretion apparatus housed within the basal body. The apparatus consists of at least six integral membrane proteins: FlhA, FlhB, FliP, FliQ, FliR, and FliO (for salmonellae and other species) (1, 12). Other proteins are also involved. FliI is the only known ATPase among flagellar proteins (2). FliI interacts with FliJ, which is of unknown function, and with a dimer of FliH, an inhibitor of FliI. The apparatus can be visualized by quick-freeze electron microscopy and has been termed the C (cytoplasmic) rod by virtue of its appearance and membrane-proximal location inside the C ring (7). The C ring is composed of three component proteins: FliG, FliM, and FliN (3). Mutations or deletions of any of these proteins cause a nonflagellate (Fla⁻) phenotype, strongly suggesting that the C ring is necessary for flagellar protein export (6, 22, 26). The trimer FliH₂-FliI specifically binds FliN (4, 15), suggesting that FliI docks at the periphery of the C ring through interactions with FliN-bound FliH, standing ready to escort export substrates to the secretion gate that is probably composed by FlhA, FlhB, and others (15).The C ring has long been studied with respect to motor function rather than export function. It has been proposed that FliG plays a major role in torque generation in concert with MotAB complexes, leaving the other two proteins, FliM and FliN, in minor and supporting roles (10, 11). However, as mentioned above, all three components are required for flagellar protein export (6, 22, 26). Together with the C ring, FliI pushes export substrates into the gate using the energy of ATP hydrolysis. Just recently, it was shown that FliI ATPase activity is not absolutely necessary for protein export and that increasing proton motive force (PMF) or reversion mutations in FlhA and FlhB can compensate for its absence (17, 21).In order to elucidate the roles that FliG, FliM, and FliN play in export, we employed C-ring-defective mutants. Here, we show that the overproduction of FliI allows flagellar formation in C-ring-defective mutants. We closely examined flagella formed in those mutants by electron microscopy, noting percentages of flagellation in each population, analyzing partially formed structures, and measuring hook length.     

Role of the C-Terminal Cytoplasmic Domain of FlhA in Bacterial Flagellar Type III Protein Export

For construction of the bacterial flagellum, many of the flagellar proteins are exported into the central channel of the flagellar structure by the flagellar type III protein export apparatus. FlhA and FlhB, which are integral membrane proteins of the export apparatus, form a docking platform for the soluble components of the export apparatus, FliH, FliI, and FliJ. The C-terminal cytoplasmic domain of FlhA (FlhA_C) is required for protein export, but it is not clear how it works. Here, we analyzed a temperature-sensitive Salmonella enterica mutant, the flhA(G368C) mutant, which has a mutation in the sequence encoding FlhA_C. The G368C mutation did not eliminate the interactions with FliH, FliI, FliJ, and the C-terminal cytoplasmic domain of FlhB, suggesting that the mutation blocks the export process after the FliH-FliI-FliJ-export substrate complex binds to the FlhA-FlhB platform. Limited proteolysis showed that FlhA_C consists of at least three subdomains, a flexible linker, FlhA_CN, and FlhA_CC, and that FlhA_CN becomes sensitive to proteolysis by the G368C mutation. Intragenic suppressor mutations were identified in these subdomains and restored flagellar protein export to a considerable degree. However, none of these suppressor mutations suppressed the protease sensitivity. We suggest that FlhA_C not only forms part of the docking platform for the FliH-FliI-FliJ-export substrate complex but also is directly involved in the translocation of the export substrate into the central channel of the growing flagellar structure.The bacterial flagellum, which is responsible for motility, is a supramolecular complex of about 30 different proteins, and it consists of at least three substructures: the basal body, the hook, and the filament. Flagellar assembly begins with the basal body, followed by the hook and finally the filament. Many of the flagellar component proteins are translocated into the central channel of the growing flagellar structure and then to the distal end of the structure for self-assembly by the flagellar type III protein export apparatus (11, 16, 22). This export apparatus consists of six integral membrane proteins, FlhA, FlhB, FliO, FliP, FliQ, and FliR, and three soluble proteins, FliH, FliI, and FliJ (18, 21). These protein components show significant sequence and functional similarities to those of the type III secretion systems of pathogenic bacteria, which directly inject virulence factors into their host cells (11, 16).FliI is an ATPase (4) and forms an FliH₂-FliI complex with its regulator, FliH, in the cytoplasm (20). FliI self-assembles into a homo-hexamer and hence exhibits full ATPase activity (1, 8, 17). FliH and FliI, together with FliJ and the export substrate, bind to the export core complex, which is composed of the six integral membrane proteins, to recruit export substrates from the cytoplasm to the core complex (14) and facilitate the initial entry of export substrates into the export gate (23). FliJ not only prevents premature aggregation of export substrates in the cytoplasm (13) but also plays an important role in the escort mechanism for cycling export chaperones during flagellar assembly (3). The export core complex is believed to be located in the central pore of the basal body MS ring (11, 16, 22). In fact, it has been found that FlhA, FliP, and FliR are associated with the MS ring (5, 9). The FliR-FlhB fusion protein is partially functional, suggesting that FliR and FlhB interact with each other within the MS ring (29). The export core complex utilizes a proton motive force across the cytoplasmic membrane as the energy source to drive the successive unfolding of export substrates and their translocation into the central channel of the growing flagellum (23, 27). Here we refer to the export core complex as the “export gate,” as we have previously (8, 16, 23, 24).FlhA is a 692-amino-acid protein consisting of two regions: a hydrophobic N-terminal transmembrane region with eight predicted α-helical transmembrane spans (FlhA_TM) and a hydrophilic C-terminal cytoplasmic region (FlhA_C) (12, 15). FlhA_TM is responsible for the association with the MS ring (9). FlhA_C interacts with FliH, FliI, FliJ, and the C-terminal cytoplasmic domain of FlhB (6, 12, 21, 24) and plays a role in the initial export process with these proteins (28). It has been shown that the V404M mutation in FlhA_C increases not only the probability of FliI binding to the export gate in the absence of FliH (14) but also the efficiency of substrate translocation through the export gate in the absence of FliH and FliI (23). Recently, it has been shown that FlhA_C is also required for substrate recognition (7). These observations suggest that an interaction between FlhA_C and FliI is coupled with substrate entry, although it is not clear how.In order to understand the mechanism of substrate entry into the export gate, we characterized a temperature-sensitive Salmonella enterica mutant, the flhA(G368C) mutant, whose mutation blocks the flagellar protein export process at 42°C (28). We show here that this mutation severely inhibits translocation of flagellar proteins through the export gate after the FliH-FliI-FliJ complex binds to the FlhA-FlhB platform of the gate and that the impaired ability of the flhA(G368C) mutant to export flagellar proteins is restored almost to wild-type levels by intragenic second-site mutations that may alter the interactions between subdomains of FlhA_C for possible rearrangement for the export function.     

Interaction between FliJ and FlhA,Components of the Bacterial Flagellar Type III Export Apparatus

A soluble protein, FliJ, along with a membrane protein, FlhA, plays a role in the energy coupling mechanism for bacterial flagellar protein export. The water-soluble FliH_X-FliI₆ ATPase ring complex allows FliJ to efficiently interact with FlhA. However, the FlhA binding site of FliJ remains unknown. Here, we carried out genetic analysis of a region formed by well-conserved residues—Gln38, Leu42, Tyr45, Tyr49, Phe72, Leu76, Ala79, and His83—of FliJ. A structural model of the FliI₆-FliJ ring complex suggests that they extend out of the FliI₆ ring. Glutathione S-transferase (GST)-FliJ inhibited the motility of and flagellar protein export by both wild-type cells and a fliH-fliI flhB(P28T) bypass mutant. Pulldown assays revealed that the reduced export activity of the export apparatus results from the binding of GST-FliJ to FlhA. The F72A and L76A mutations of FliJ significantly reduced the binding affinity of FliJ for FlhA, thereby suppressing the inhibitory effect of GST-FliJ on the protein export. The F72A and L76A mutations were tolerated in the presence of FliH and FliI but considerably reduced motility in their absence. These two mutations affected neither the interaction with FliI nor the FliI ATPase activity. These results suggest that FliJ(F72A) and FliJ(L76A) require the support of FliH and FliI to exert their export function. Therefore, we propose that the well-conserved surface of FliJ is involved in the interaction with FlhA.     

FlgM as a Secretion Moiety for the Development of an Inducible Type III Secretion System

Regulation and assembly of the flagellar type III secretion system is one of the most investigated and best understood regulational cascades in molecular biology. Depending on the host organism, flagellar morphogenesis requires the interplay of more than 50 genes. Direct secretion of heterologous proteins to the supernatant is appealing due to protection against cellular proteases and simplified downstream processing. As Escherichia coli currently remains the predominant host organism used for recombinant prokaryotic protein expression, the generation of a strain that exhibits inducible flagellar secretion would be highly desirable for biotechnological applications.Here, we report the first engineered Escherichia coli mutant strain featuring flagellar morphogenesis upon addition of an external inducer. Using FlgM as a sensor for direct secretion in combination with this novel strain may represent a potent tool for significant improvements in future engineering of an inducible type III secretion for heterologous proteins.     

Effective Identification of Bacterial Type III Secretion Signals Using Joint Element Features

Type III secretion system (T3SS) plays important roles in bacteria and host cell interactions by specifically translocating type III effectors into the cytoplasm of the host cells. The N-terminal amino acid sequences of the bacterial type III effectors determine their specific secretion via type III secretion conduits. It is still unclear as to how the N-terminal sequences guide this specificity. In this work, the amino acid composition, secondary structure, and solvent accessibility in the N-termini of type III and non-type III secreted proteins were compared and contrasted. A high-efficacy mathematical model based on these joint features was developed to distinguish the type III proteins from the non-type III ones. The results indicate that secondary structure and solvent accessibility may make important contribution to the specific recognition of type III secretion signals. Analysis also showed that the joint feature of the N-terminal 6^th–10^th amino acids are especially important for guiding specific type III secretion. Furthermore, a genome-wide screening was performed to predict Salmonella type III secreted proteins, and 8 new candidates were experimentally validated. Interestingly, type III secretion signals were also predicted in gram-positive bacteria and yeasts. Experimental validation showed that two candidates from yeast can indeed be secreted through Salmonella type III secretion conduit. This research provides the first line of direct evidence that secondary structure and solvent accessibility contain important features for guiding specific type III secretion. The new software based on these joint features ensures a high accuracy (general cross-validation sensitivity of ∼96% at a specificity of ∼98%) in silico identification of new type III secreted proteins, which may facilitate our understanding about the specificity of type III secretion and the evolution of type III secreted proteins.     

Complete Type III Secretion System of a Mesophilic Aeromonas hydrophila Strain

We have investigated the existence and genetic organization of a functional type III secretion system (TTSS) in a mesophilic Aeromonas strain by initially using the Aeromonas hydrophila strain AH-3. We report for the first time the complete TTSS DNA sequence of an Aeromonas strain that comprises 35 genes organized in a similar disposition as that in Pseudomonas aeruginosa. Using several gene probes, we also determined the presence of a TTSS in clinical or environmental strains of different Aeromonas species: A. hydrophila, A. veronii, and A. caviae. By using one of the TTSS genes (ascV), we were able to obtain a defined insertion mutant in strain AH-3 (AH-3AscV), which showed reduced toxicity and virulence in comparison with the wild-type strain. Complementation of the mutant strain with a plasmid vector carrying ascV was fully able to restore the wild-type toxicity and virulence.     

Structural Analysis of a Specialized Type III Secretion System Peptidoglycan-cleaving Enzyme

The Gram-negative bacterium enteropathogenic Escherichia coli uses a syringe-like type III secretion system (T3SS) to inject virulence or “effector” proteins into the cytoplasm of host intestinal epithelial cells. To assemble, the T3SS must traverse both bacterial membranes, as well as the peptidoglycan layer. Peptidoglycan is made of repeating N-acetylmuramic acid and N-acetylglucosamine disaccharides cross-linked by pentapeptides to form a tight mesh barrier. Assembly of many macromolecular machines requires a dedicated peptidoglycan lytic enzyme (PG-lytic enzyme) to locally clear peptidoglycan. Here we have solved the first structure of a T3SS-associated PG-lytic enzyme, EtgA from enteropathogenic E. coli. Unexpectedly, the active site of EtgA has features in common with both lytic transglycosylases and hen egg white lysozyme. Most notably, the β-hairpin region resembles that of lysozyme and contains an aspartate that aligns with lysozyme Asp-52 (a residue critical for catalysis), a conservation not observed in other previously characterized lytic transglycosylase families to which the conserved T3SS enzymes had been presumed to belong. Mutation of the EtgA catalytic glutamate, Glu-42, conserved across lytic transglycosylases and hen egg white lysozyme, and this differentiating aspartate diminishes type III secretion in vivo, supporting its essential role in clearing the peptidoglycan for T3SS assembly. Finally, we show that EtgA forms a 1:1 complex with the building block of the polymerized T3SS inner rod component, EscI, and that this interaction enhances PG-lytic activity of EtgA in vitro, collectively providing the necessary strict localization and regulation of the lytic activity to prevent overall cell lysis.     

Identification of Novel Protein-Protein Interactions of Yersinia pestis Type III Secretion System by Yeast Two Hybrid System

Type III secretion system (T3SS) of the plague bacterium Y. pestis encodes a syringe-like structure consisting of more than 20 proteins, which can inject virulence effectors into host cells to modulate the cellular functions. Here in this report, interactions among the possible components in T3SS of Yersinia pestis were identified using yeast mating technique. A total of 57 genes, including all the pCD1-encoded genes except those involved in plasmid replication and partition, pseudogenes, and the putative transposase genes, were subjected to yeast mating analysis. 21 pairs of interaction proteins were identified, among which 9 pairs had been previously reported and 12 novel pairs were identified in this study. Six of them were tested by GST pull down assay, and interaction pairs of YscG-SycD, YscG-TyeA, YscI-YscF, and YopN-YpCD1.09c were successfully validated, suggesting that these interactions might play potential roles in function of Yersinia T3SS. Several potential new interactions among T3SS components could help to understand the assembly and regulation of Yersinia T3SS.     

Process of Protein Transport by the Type III Secretion System

The type III secretion system (TTSS) of gram-negative bacteria is responsible for delivering bacterial proteins, termed effectors, from the bacterial cytosol directly into the interior of host cells. The TTSS is expressed predominantly by pathogenic bacteria and is usually used to introduce deleterious effectors into host cells. While biochemical activities of effectors vary widely, the TTSS apparatus used to deliver these effectors is conserved and shows functional complementarity for secretion and translocation. This review focuses on proteins that constitute the TTSS apparatus and on mechanisms that guide effectors to the TTSS apparatus for transport. The TTSS apparatus includes predicted integral inner membrane proteins that are conserved widely across TTSSs and in the basal body of the bacterial flagellum. It also includes proteins that are specific to the TTSS and contribute to ring-like structures in the inner membrane and includes secretin family members that form ring-like structures in the outer membrane. Most prominently situated on these coaxial, membrane-embedded rings is a needle-like or pilus-like structure that is implicated as a conduit for effector translocation into host cells. A short region of mRNA sequence or protein sequence in effectors acts as a signal sequence, directing proteins for transport through the TTSS. Additionally, a number of effectors require the action of specific TTSS chaperones for efficient and physiologically meaningful translocation into host cells. Numerous models explaining how effectors are transported into host cells have been proposed, but understanding of this process is incomplete and this topic remains an active area of inquiry.     

T346Hunter: A Novel Web-Based Tool for the Prediction of Type III,Type IV and Type VI Secretion Systems in Bacterial Genomes

T346Hunter (Type Three, Four and Six secretion system Hunter) is a web-based tool for the identification and localisation of type III, type IV and type VI secretion systems (T3SS, T4SS and T6SS, respectively) clusters in bacterial genomes. Non-flagellar T3SS (NF-T3SS) and T6SS are complex molecular machines that deliver effector proteins from bacterial cells into the environment or into other eukaryotic or prokaryotic cells, with significant implications for pathogenesis of the strains encoding them. Meanwhile, T4SS is a more functionally diverse system, which is involved in not only effector translocation but also conjugation and DNA uptake/release. Development of control strategies against bacterial-mediated diseases requires genomic identification of the virulence arsenal of pathogenic bacteria, with T3SS, T4SS and T6SS being major determinants in this regard. Therefore, computational methods for systematic identification of these specialised machines are of particular interest. With the aim of facilitating this task, T346Hunter provides a user-friendly web-based tool for the prediction of T3SS, T4SS and T6SS clusters in newly sequenced bacterial genomes. After inspection of the available scientific literature, we constructed a database of hidden Markov model (HMM) protein profiles and sequences representing the various components of T3SS, T4SS and T6SS. T346Hunter performs searches of such a database against user-supplied bacterial sequences and localises enriched regions in any of these three types of secretion systems. Moreover, through the T346Hunter server, users can visualise the predicted clusters obtained for approximately 1700 bacterial chromosomes and plasmids. T346Hunter offers great help to researchers in advancing their understanding of the biological mechanisms in which these sophisticated molecular machines are involved. T346Hunter is freely available at http://bacterial-virulence-factors.cbgp.upm.es/T346Hunter.     

Weak Interactions between Salmonella enterica FlhB and Other Flagellar Export Apparatus Proteins Govern Type III Secretion Dynamics

The bacterial flagellum contains its own type III secretion apparatus that coordinates protein export with assembly at the distal end. While many interactions among export apparatus proteins have been reported, few have been examined with respect to the differential affinities and dynamic relationships that must govern the mechanism of export. FlhB, an integral membrane protein, plays critical roles in both export and the substrate specificity switching that occurs upon hook completion. Reported herein is the quantitative characterization of interactions between the cytoplasmic domain of FlhB (FlhB_C) and other export apparatus proteins including FliK, FlhA_C and FliI. FliK and FlhA_C bound with micromolar affinity. K_D for FliI binding in the absence of ATP was 84 nM. ATP-induced oligomerization of FliI induced kinetic changes, stimulating fast-on, fast-off binding and lowering affinity. Full length FlhB purified under solubilizing, nondenaturing conditions formed a stable dimer via its transmembrane domain and stably bound FliH. Together, the present results support the previously hypothesized central role of FlhB and elucidate the dynamics of protein-protein interactions in type III secretion.