FlhA Undergoes Cyclic Open-close Domain Motions During Flagellar Protein Export in Salmonella

The flagellar type III secretion system (fT3SS) transports flagellar building blocks from the cytoplasm to the distal end of the growing flagellar structure. The C-terminal cytoplasmic domain of FlhA (FlhAC) serves as a docking platform for flagellar chaperones in complex with their cognate substrates and ensures the strict order of protein export for efficient flagellar assembly. FlhAC adopts open and closed conformations, and the chaperones bind to the open form, allowing the fT3SS to transport the substrates to the cell exterior. To clarify the role of the closed form in flagellar protein export, we isolated pseudorevertants from the flhA(G368C/K549C) mutant, in which the closed conformation is stabilized to inhibit the protein transport activity of the fT3SS. Each of M365I, R370S, A446E and P550S substitutions in FlhAC identified in the pseudorevertants affected hydrophobic side-chain interaction networks in the closed FlhAC structure, thereby restoring the protein transport activity to a considerable degree. We propose that a cyclic open-close domain motion of FlhAC is required for rapid and efficient flagellar protein export where a structural transition from the open to the closed form induces the dissociation of empty chaperones from FlhAC.

The FlhA linker mediates flagellar protein export switching during flagellar assembly

The flagellar protein export apparatus switches substrate specificity from hook-type to filament-type upon hook assembly completion, thereby initiating filament assembly at the hook tip. The C-terminal cytoplasmic domain of FlhA (FlhAC) serves as a docking platform for flagellar chaperones in complex with their cognate filament-type substrates. Interactions of the flexible linker of FlhA (FlhAL) with its nearest FlhAC subunit in the FlhAC ring is required for the substrate specificity switching. To address how FlhAL brings the order to flagellar assembly, we analyzed the flhA(E351A/W354A/D356A) ΔflgM mutant and found that this triple mutation in FlhAL increased the secretion level of hook protein by 5-fold, thereby increasing hook length. The crystal structure of FlhAC(E351A/D356A) showed that FlhAL bound to the chaperone-binding site of its neighboring subunit. We propose that the interaction of FlhAL with the chaperon-binding site of FlhAC suppresses filament-type protein export and facilitates hook-type protein export during hook assembly.

Membrane voltage-dependent activation mechanism of the bacterial flagellar protein export apparatus

The proton motive force (PMF) consists of the electric potential difference (Δψ), which is measured as membrane voltage, and the proton concentration difference (ΔpH) across the cytoplasmic membrane. The flagellar protein export machinery is composed of a PMF-driven transmembrane export gate complex and a cytoplasmic ATPase ring complex consisting of FliH, FliI, and FliJ. ATP hydrolysis by the FliI ATPase activates the export gate complex to become an active protein transporter utilizing Δψ to drive proton-coupled protein export. An interaction between FliJ and a transmembrane ion channel protein, FlhA, is a critical step for Δψ-driven protein export. To clarify how Δψ is utilized for flagellar protein export, we analyzed the export properties of the export gate complex in the absence of FliH and FliI. The protein transport activity of the export gate complex was very low at external pH 7.0 but increased significantly with an increase in Δψ by an upward shift of external pH from 7.0 to 8.5. This observation suggests that the export gate complex is equipped with a voltage-gated mechanism. An increase in the cytoplasmic level of FliJ and a gain-of-function mutation in FlhA significantly reduced the Δψ dependency of flagellar protein export by the export gate complex. However, deletion of FliJ decreased Δψ-dependent protein export significantly. We propose that Δψ is required for efficient interaction between FliJ and FlhA to open the FlhA ion channel to conduct protons to drive flagellar protein export in a Δψ-dependent manner.

A positive charge region of Salmonella FliI is required for ATPase formation and efficient flagellar protein export

The FliH2FliI complex is thought to pilot flagellar subunit proteins from the cytoplasm to the transmembrane export gate complex for flagellar assembly in Salmonella enterica. FliI also forms a homo-hexamer to hydrolyze ATP, thereby activating the export gate complex to become an active protein transporter. However, it remains unknown how this activation occurs. Here we report the role of a positively charged cluster formed by Arg-26, Arg-27, Arg-33, Arg-76 and Arg-93 of FliI in flagellar protein export. We show that Arg-33 and Arg-76 are involved in FliI ring formation and that the fliI(R26A/R27A/R33A/R76A/R93A) mutant requires the presence of FliH to fully exert its export function. We observed that gain-of-function mutations in FlhB increased the probability of substrate entry into the export gate complex, thereby restoring the export function of the ∆fliH fliI(R26A/R27A/R33A/R76A/R93A) mutant. We suggest that the positive charge cluster of FliI is responsible not only for well-regulated hexamer assembly but also for substrate entry into the gate complex.

The FlgN chaperone activates the Na+-driven engine of the Salmonella flagellar protein export apparatus

The bacterial flagellar protein export machinery consists of a transmembrane export gate complex and a cytoplasmic ATPase complex. The gate complex has two intrinsic and distinct H+-driven and Na+-driven engines to drive the export of flagellar structural proteins. Salmonella wild-type cells preferentially use the H+-driven engine under a variety of environmental conditions. To address how the Na+-driven engine is activated, we analyzed the fliJ(Δ13–24) fliH(Δ96–97) mutant and found that the interaction of the FlgN chaperone with FlhA activates the Na+-driven engine when the ATPase complex becomes non-functional. A similar activation can be observed with either of two single-residue substitutions in FlhA. Thus, it is likely that the FlgN-FlhA interaction generates a conformational change in FlhA that allows it to function as a Na+ channel. We propose that this type of activation would be useful for flagellar construction under conditions in which the proton motive force is severely restricted.

A live attenuated vaccine model confers cross-protective immunity against different species of the Leptospira genus

Leptospirosis is the leading zoonotic disease in terms of morbidity and mortality worldwide. Effective prevention is urgently needed as the drivers of disease transmission continue to intensify. The key challenge has been developing a widely-applicable vaccine that protects against the >300 serovars that can cause leptospirosis. Live attenuated mutants are enticing vaccine candidates and poorly explored in the field. We evaluated a recently characterized motility-deficient mutant lacking the expression of a flagellar protein, FcpA. Although the fcpA- mutant has lost its ability to cause disease, transient bacteremia was observed. In two animal models, immunization with a single dose of the fcpA- mutant was sufficient to induce a robust anti-protein antibodies response that promoted protection against infection with different pathogenic Leptospira species. Furthermore, characterization of the immune response identified a small repertoire of biologically relevant proteins that are highly conserved among pathogenic Leptospira species and potential correlates of cross-protective immunity.

FLAgellum Member 8 modulates extravascular trypanosome distribution in the mammalian host

The African trypanosome flagellum is essential in multiple aspects of the parasite development. In the mammalian infective form of this protist, FLAgellar Member 8 (FLAM8) is a large protein distributed along the entire flagellum that is suspected to be involved in host-parasite interactions. Analyses of knockdown and knockout trypanosomes demonstrated that FLAM8 is not essential in vitro for survival, growth, motility and slender to stumpy differentiation. Functional investigations in experimental infections showed that FLAM8 -deprived trypanosomes are able to establish and maintain the infection in the blood circulation, and to differentiate into transmissible stumpy forms. However, bioluminescence imaging revealed that FLAM8 -null parasites exhibit an impaired dissemination in the extravascular compartment, especially in the skin, that is partially restored by the addition of a single rescue copy of FLAM8 . To our knowledge, FLAM8 is the first example of a flagellar protein that modulates T. brucei parasite distribution in the host tissues, contributing to the maintenance of extravascular parasite populations in mammalian anatomical niches.

The FlhA linker mediates flagellar protein export switching during flagellar assembly

The flagellar protein export apparatus switches export specificity from hook-type to filament-type upon completion of hook assembly, thereby initiating filament assembly at the hook tip. The C-terminal cytoplasmic domain of FlhA (FlhAC) forms a homo-nonameric ring structure that serves as a docking platform for flagellar export chaperones in complex with their cognate filament-type substrates. Interactions of the flexible linker of FlhA (FlhAL) with its nearest FlhAC subunit in the ring allow the chaperones to bind to FlhAC to facilitate filament-type protein export, but it remains unclear how it occurs. Here, we report that FlhAL acts as a switch that brings the order to flagellar assembly. The crystal structure of FlhAC(E351A/D356A) showed that Trp-354 in FlhAL bound to the chaperone-binding site of its neighboring subunit. We propose that FlhAL binds to the chaperon-binding site of FlhAC to suppress the interaction between FlhAC and the chaperones until hook assembly is completed.

Membrane voltage-dependent activation of the flagellar protein export engine

Ion motive force (IMF) consists of the electric potential difference (ΔΨ) and the ion concentration difference (ΔpI) across the cytoplasmic membrane. The flagellar protein export machinery is an ion/protein antiporter utilizing IMF to drive ion-coupled protein export, but it remains unknown how. Here, we report a ΔΨ-dependent activation mechanism of the transmembrane export gate complex. Depletions of both H+ and Na+ gradients nearly diminished flagellar protein export in the absence of the cytoplasmic ATPase complex, but an increase in ΔΨ by an upward shift of external pH from 7.5 to 8.5 dramatically recovered it. An increase in the cytoplasmic level of export substrates and gain-of-function mutations in FlhA enhanced protein export at external pH 7.5 in the absence of Na+ in a similar manner to ΔΨ increase. We propose that the export gate complex has a voltage-gated mechanism to activate the ion/protein antiporter of the flagellar protein export engine.

The FlgN chaperone activates the Na+-driven engine of the flagellar protein export apparatus

The bacterial flagellar protein export machinery promotes H+-coupled translocation of flagellar proteins to the cell exterior. When the cytoplasmic ATPase complex does not function, the transmembrane export gate complex opens its Na+ channel and continues protein transport. However, it remains unknown how. Here we report that the FlgN chaperone acts as a switch to activate a backup export mechanism for the ATPase complex by activating the Na+-driven engine. Impaired interaction of FlhA with the FliJ subunit of the ATPase complex increased Na+-dependence of flagellar protein export. Deletion of FlgN inhibited protein export in the absence of the ATPase complex but not in its presence. Gain-of-function mutations in FlhA restored not only the FlgN defect but also the FliJ defect. We propose that the interaction of FlgN with FlhA opens the Na+ channel in the export engine, thereby maintaining the protein export activity in the absence of the active ATPase complex.