Substrate targeting and recognition in the flagellar type III secretion pathway

The bacterial flagellum is a complex molecular machine that enables bacteria to move as well as having additional roles in biofilm formation, adhesion and host cell invasion. The sequential assembly of the flagellar rod, hook and filament requires the export of thousands of structural subunits across the bacterial cell membrane by a specialised flagellar type III secretion system (fT3SS) located at the base of each flagellum. These subunits are unfolded and exported into a narrow 20Å wide channel in the centre of each flagellum energised by the proton motive force (PMF) and facilitated by a cytoplasmic ATPase complex comprising FliH, FliI and FliJ, which are evolutionarily related to components of the F1 ATPase. Unfolded subunits transit the length of the channel to the flagellum tip where they fold into the nascent structure. The order of subunit export is determined by the stage of flagellum assembly. Early subunits are exported to assemble the rod and hook substructures after which a substrate specificity switch in the fT3SS allows late subunit export. The first aim of this study was to determine how the export signals that reside within early flagellar subunits contribute to export. In this thesis it was found that early flagellar subunits dock at the cytoplasmic domain of FlhB to correctly position an export signal at the subunit extreme N-terminus. The distance between the extreme N-terminal signal and the FlhB gate recognition motif (GRM) was found to be critical for subunit export. Evidence is presented to show that the extreme N-terminal signal of early flagellar subunits converts the FliPQR components of the export gate from a closed to open conformation, allowing subunits to enter the central channel within the flagellum. Further evidence was presented to show that the FlhA component of the flagellar export machinery is involved in export gate opening. Increasing the proton-motive force (PMF) improved subunit export by a strain encoding a FlhA variant defective in export gate opening, indicating that the PMF energises opening of the export gate. The data are compatible with the view that early subunits dock at the cytoplasmic domain of FlhB to correctly position the extreme N-terminal export signal to trigger PMF-driven opening of the export gate. The late filament structural subunits are delivered from their site of synthesis in the cytoplasm to the flagellar export machinery at the membrane by their cognate chaperones. Using motility and export assays, and in vitro and in vivo affinity chromatography pull down assays, data are provided that build on what is currently known about the sequence of binding events between chaperoned subunits and the flagellar export machinery. Specifically, evidence is presented to show that the FlgN chaperone binds the FliJ component of the ATPase complex after FlgN has docked and been released from the cytoplasmic domain of FlhA. The final aim of this study was to determine whether the flagellar export chaperones regulate activation of the PMF-driven fT3SS export machinery. The FliJ stalk component of the ATPase binds the export gate protein FlhA, allowing it to utilise ΔΨ to drive highly efficient subunit export. This thesis showed that the flagellar export chaperones regulate FliJ activation of FlhA. The data showed that chaperones and FlhA compete for a common binding site on FliJ, and that unladen chaperones, which would be present in the cell when subunit levels are low, disrupt the FliJ-FlhA interaction, preventing activation of the export gate. This provides a mechanism whereby the export gate is only activated when subunits are available.Department of Patholog

Regulation of bacterial Type III Secretion System export gate opening by substrates and the FliJ stalk of the flagellar ATPase.

Type III Secretion Systems (T3SS) transport proteins from the bacterial cytosol for assembly into cell surface nanomachines or direct delivery into target eukaryotic cells. At the core of the flagellar T3SS, the FlhAB-FliPQR export gate regulates protein entry into the export channel whilst maintaining the integrity of the cell membrane. Here, we identify critical residues in the export gate FliR plug that stabilise the closed conformation, preserving the membrane permeability barrier, and we show that the gate opens and closes in response to export substrate availability. Our data indicate that FlhAB-FliPQR gate opening, which is triggered by substrate export signals, is energised by FlhA in a proton motive force-dependent manner. We present evidence that the export substrate and the FliJ stalk of the flagellar ATPase provide mechanistically distinct, non-redundant gate-activating signals that are critical for efficient export

Modifying bacterial flagellin to evade Nod-like Receptor CARD 4 recognition enhances protective immunity against Salmonella.

Pattern recognition receptors (PRRs) expressed in antigen-presenting cells are thought to shape pathogen-specific immunity by inducing secretion of costimulatory cytokines during T-cell activation, yet data to support this notion in vivo are scarce. Here, we show that the cytosolic PRR Nod-like Receptor CARD 4 (NLRC4) suppresses, rather than facilitates, effector and memory CD4+ T-cell responses against Salmonella in mice. NLRC4 negatively regulates immunological memory by preventing delayed activation of the cytosolic PRR NLR pyrin domain 3 (NLRP3) that would otherwise amplify the production of cytokines important for the generation of Th1 immunity such as intereukin-18. Consistent with a role for NLRC4 in memory immunity, primary challenge with Salmonella expressing flagellin modified to largely evade NLRC4 recognition notably increases protection against lethal rechallenge. This finding suggests flagellin modification to reduce NLRC4 activation enhances protective immunity, which could have important implications for vaccine development against flagellated microbial pathogens.Wellcome Trus

Inhibiting Mycobacterium tuberculosis CoaBC by targeting an allosteric site.

Coenzyme A (CoA) is a fundamental co-factor for all life, involved in numerous metabolic pathways and cellular processes, and its biosynthetic pathway has raised substantial interest as a drug target against multiple pathogens including Mycobacterium tuberculosis. The biosynthesis of CoA is performed in five steps, with the second and third steps being catalysed in the vast majority of prokaryotes, including M. tuberculosis, by a single bifunctional protein, CoaBC. Depletion of CoaBC was found to be bactericidal in M. tuberculosis. Here we report the first structure of a full-length CoaBC, from the model organism Mycobacterium smegmatis, describe how it is organised as a dodecamer and regulated by CoA thioesters. A high-throughput biochemical screen focusing on CoaB identified two inhibitors with different chemical scaffolds. Hit expansion led to the discovery of potent and selective inhibitors of M. tuberculosis CoaB, which we show to bind to a cryptic allosteric site within CoaB

Ten-year mortality, disease progression, and treatment-related side effects in men with localised prostate cancer from the ProtecT randomised controlled trial according to treatment received

Background The ProtecT trial reported intention-to-treat analysis of men with localised prostate cancer randomly allocated to active monitoring (AM), radical prostatectomy, and external beam radiotherapy. Objective To report outcomes according to treatment received in men in randomised and treatment choice cohorts. Design, setting, and participants This study focuses on secondary care. Men with clinically localised prostate cancer at one of nine UK centres were invited to participate in the treatment trial comparing AM, radical prostatectomy, and radiotherapy. Intervention Two cohorts included 1643 men who agreed to be randomised and 997 who declined randomisation and chose treatment. Outcome measurements and statistical analysis Analysis was carried out to assess mortality, metastasis and progression and health-related quality of life impacts on urinary, bowel, and sexual function using patient-reported outcome measures. Analysis was based on comparisons between groups defined by treatment received for both randomised and treatment choice cohorts in turn, with pooled estimates of intervention effect obtained using meta-analysis. Differences were estimated with adjustment for known prognostic factors using propensity scores. Results and limitations According to treatment received, more men receiving AM died of PCa (AM 1.85%, surgery 0.67%, radiotherapy 0.73%), whilst this difference remained consistent with chance in the randomised cohort (p = 0.08); stronger evidence was found in the exploratory analyses (randomised plus choice cohort) when AM was compared with the combined radical treatment group (p = 0.003). There was also strong evidence that metastasis (AM 5.6%, surgery 2.4%, radiotherapy 2.7%) and disease progression (AM 20.35%, surgery 5.87%, radiotherapy 6.62%) were more common in the AM group. Compared with AM, there were higher risks of sexual dysfunction (95% at 6 mo) and urinary incontinence (55% at 6 mo) after surgery, and of sexual dysfunction (88% at 6 mo) and bowel dysfunction (5% at 6 mo) after radiotherapy. The key limitations are the potential for bias when comparing groups defined by treatment received and changes in the protocol for AM during the lengthy follow-up required in trials of screen-detected PCa. Conclusions Analyses according to treatment received showed increased rates of disease-related events and lower rates of patient-reported harms in men managed by AM compared with men managed by radical treatment, and stronger evidence of greater PCa mortality in the AM group. Patient summary More than 95 out of every 100 men with low or intermediate risk localised prostate cancer do not die of prostate cancer within 10 yr, irrespective of whether treatment is by means of monitoring, surgery, or radiotherapy. Side effects on sexual and bladder function are better after active monitoring, but the risks of spreading of prostate cancer are more common

Identification of a new export signal that targets early subunits to the flagellar Type III secretion export machinery.

Type III secretion systems (T3SS) are essential for motility and virulence in many bacterial pathogens. Proteins destined for the flagellar T3SS (fT3SS) contain at least two export signals in their N-terminal D0 domain. Here, we describe a third carboxy (C)-terminal signal in early flagellar subunits that facilitates subunit targeting to the export machinery. Mutational analysis identified critical residues within the flagellar hook subunit C-terminal export signal. The flagellar ATPase and C-ring components were not required for this targeting, indicating that core export machinery components facilitate substrate targeting via the C-terminal export signal. More broadly, these results demonstrate that multiple distinct export signals within type III secretion substrates facilitate distinct export events at the T3SS export machinery. Our data establish key events in the export mechanism of Type III secretion systems; targeting of subunits to and their sequential interactions with key components of the export machinery.University of Cambridge John Lucas Walker Studentshi

Identification of a new export signal that targets early subunits to the flagellar type III secretion export machinery

ABSTRACTType III secretion systems (T3SSs) are essential for motility and virulence in many bacterial pathogens. Proteins destined for the flagellar T3SS contain at least two export signals in their N-terminal D0 domain. Here, we describe a third carboxy (C)-terminal signal in early flagellar subunits that facilitates subunit targeting to the export machinery. Mutational analysis identified critical residues within the flagellar hook subunit C-terminal export signal. The flagellar ATPase and cytoplasmic ring components were not required for this targeting, indicating that core export machinery components facilitate substrate targeting via the C-terminal export signal. More broadly, these results demonstrate that multiple distinct export signals within type III secretion substrates facilitate distinct export events at the T3SS export machinery. Our data establish key events in the export mechanism of type III secretion systems: targeting of subunits to and their sequential interactions with key components of the export machinery.IMPORTANCEMany bacterial pathogens utilize T3SS to inject virulence proteins (effectors) into host cells or to assemble flagella on the bacterial cell surface. Bacterial flagella present a paradigm for how cells build and operate complex cell-surface “nanomachines.” Efficient subunit targeting from the bacterial cytosol to type III secretion systems is essential for rapid assembly and secretion by T3SSs. Subunits are thought to dock at the export machinery before being unfolded and translocated into the export channel. However, little is known about how subunits dock at the export machinery and the events that occur post docking. Here, we identified a new export signal within the C-termini of subunits that is essential for targeting of subunits to the type III export machinery. We show that this new export signal and previously identified export signals are recognized separately and sequentially, revealing a pathway for subunit transit through the type III export machinery in which sequential recognition events carry out different roles at major steps in the export pathway

Recognition of discrete export signals in early flagellar subunits during bacterial Type III secretion

Funder: The Wellcome Trust; FundRef: http://dx.doi.org/10.13039/100010269Type III Secretion Systems (T3SS) deliver subunits from the bacterial cytosol to nascent cell surface flagella. Early flagellar subunits that form the rod and hook substructures are unchaperoned and contain their own export signals. A gate recognition motif (GRM) docks them at the FlhBc component of the FlhAB-FliPQR export gate, but the gate must then be opened and subunits must be unfolded to pass through the flagellar channel. This induced us to seek further signals on the subunits. Here, we identify a second signal at the extreme N-terminus of flagellar rod and hook subunits and determine that key to the signal is its hydrophobicity. We show that the two export signal elements are recognised separately and sequentially, as the N-terminal signal is recognised by the flagellar export machinery only after subunits have docked at FlhBC via the GRM. The position of the N-terminal hydrophobic signal in the subunit sequence relative to the GRM appeared to be important, as a FlgD deletion variant (FlgDshort), in which the distance between the N terminal signal and the GRM was shortened, ‘stalled’ at the export machinery and was not exported. The attenuation of motility caused by FlgDshort was suppressed by mutations that destabilised the closed conformation of the FlhAB-FliPQR export gate, suggesting that the hydrophobic N-terminal signal might trigger opening of the flagellar export gate.University of Cambridge John Lucas Walker studentshi

Chaperone‐mediated coupling of subunit availability to activation of flagellar Type III secretion

Abstract: Bacterial flagellar subunits are exported across the cell membrane by the flagellar Type III Secretion System (fT3SS), powered by the proton motive force (pmf) and a specialized ATPase that enables the flagellar export gate to utilize the pmf electric potential (ΔΨ). Export gate activation is mediated by the ATPase stalk, FliJ, but how this process is regulated to prevent wasteful dissipation of pmf in the absence of subunit cargo is not known. Here, we show that FliJ activation of the export gate is regulated by flagellar export chaperones. FliJ binds unladen chaperones and, by using novel chaperone variants specifically defective for FliJ binding, we show that disruption of this interaction attenuates motility and cognate subunit export. We demonstrate in vitro that chaperones and the FlhA export gate component compete for binding to FliJ, and show in vivo that unladen chaperones, which would be present in the cell when subunit levels are low, sequester FliJ to prevent activation of the export gate and attenuate subunit export. Our data indicate a mechanism whereby chaperones couple availability of subunit cargo to pmf‐driven export by the fT3SS

Chaperone‐mediated coupling of subunit availability to activation of flagellar Type III secretion

Abstract: Bacterial flagellar subunits are exported across the cell membrane by the flagellar Type III Secretion System (fT3SS), powered by the proton motive force (pmf) and a specialized ATPase that enables the flagellar export gate to utilize the pmf electric potential (ΔΨ). Export gate activation is mediated by the ATPase stalk, FliJ, but how this process is regulated to prevent wasteful dissipation of pmf in the absence of subunit cargo is not known. Here, we show that FliJ activation of the export gate is regulated by flagellar export chaperones. FliJ binds unladen chaperones and, by using novel chaperone variants specifically defective for FliJ binding, we show that disruption of this interaction attenuates motility and cognate subunit export. We demonstrate in vitro that chaperones and the FlhA export gate component compete for binding to FliJ, and show in vivo that unladen chaperones, which would be present in the cell when subunit levels are low, sequester FliJ to prevent activation of the export gate and attenuate subunit export. Our data indicate a mechanism whereby chaperones couple availability of subunit cargo to pmf‐driven export by the fT3SS