Bacterial Flagella Research Articles

Structure of FlgK reveals the divergence of the bacterial Hook-Filament Junction of Campylobacter

Evolution of a nano-machine consisting of multiple parts, each with a specific function, is a complex process. A change in one part should eventually result in changes in other parts, if the overall function is to be conserved. In bacterial flagella, the filament and the hook have distinct functions and their respective proteins, FliC and FlgE, have different three-dimensional structures. The filament functions as a helical propeller and the hook as a flexible universal joint. Two proteins, FlgK and FlgL, assure a smooth connectivity between the hook and the filament. Here we show that, in Campylobacter, the 3D structure of FlgK differs from that of its orthologs in Salmonella and Burkholderia, whose structures have previously been solved. Docking the model of the FlgK junction onto the structure of the Campylobacter hook provides some clues about its divergence. These data suggest how evolutionary pressure to adapt to structural constraints, due to the structure of Campylobacter hook, causes divergence of one element of a supra-molecular complex in order to maintain the function of the entire flagellar assembly.

Biotemplated flagellar nanoswimmers

In this article, a porous hollow biotemplated nanoscale helix that can serve as a low Reynolds number robotic swimmer is reported. The nanorobot utilizes repolymerized bacterial flagella from Salmonella typhimurium as a nanotemplate for biomineralization. We demonstrate the ability to generate templated nanotubes with distinct helical geometries by using specific alkaline pH values to fix the polymorphic form of flagellar templates. Using uniform rotating magnetic fields to mimic the motion of the flagellar motor, we explore the swimming characteristics of these silica templated flagella and demonstrate the ability to wirelessly control their trajectories. The results suggest that the biotemplated nanoswimmer can be a cost-effective alternative to the current top-down methods used to produce helical nanorobots.

An intrinsically disordered linker controlling the formation and the stability of the bacterial flagellar hook

BackgroundIn a macro-molecular complex, any minor change may prove detrimental. For a supra-molecular nano-machine like the bacterial flagellum, which consists of several distinct parts with specific characteristics, stability is important. During the rotation of the bacterial flagellar motor, which is located in the membrane, the flagella rotate at speeds between 200 and 2000 rpm, depending on the bacterial species. The hook substructure of the bacterial flagellum acts as a universal joint connecting the motor to the flagellar filament. We investigated the formation of the bacterial flagellar hook and its overall stability between the FlgE subunits that make up the hook and attempted to understand how this stability differs between bacteria.ResultsAn intrinsically disordered segment plays an important role for overall hook stability and for its structural cohesion during motor rotation. The length of this linker segment depends on the species of bacteria; for Salmonella enterica and Campylobacter jejuni it is approximately 37 and 54 residues, respectively. Few residues of the linker are conserved and mutating the conserved residues of the linker yields non-flagellated cells. In the case of Campylobacter, which rotates its flagella at a speed much higher than that of Salmonella, shortening the linker leads to a rupture of the hook at its base, decreasing cell motility. Our experiments show that this segment is required for polymerization and stability of the hook, demonstrating a surprising role for a disordered region in one of the most finely tuned and closely studied macromolecular machines.ConclusionsThis study reveals a detailed functional characteristic of an intrinsically disordered segment in the hook protein. This segment evolved to fulfill a specific role in the formation of the hook, and it is at the core of the stability and flexibility of the hook. Its length is important in the case of bacteria with high-speed rotating flagella. Finding a way of disrupting this linker in Campylobacter might help in preventing infections.

Bacteria-inspired nanorobots with flagellar polymorphic transformations and bundling

Wirelessly controlled nanoscale robots have the potential to be used for both in vitro and in vivo biomedical applications. So far, the vast majority of reported micro- and nanoscale swimmers have taken the approach of mimicking the rotary motion of helical bacterial flagella for propulsion, and are often composed of monolithic inorganic materials or photoactive polymers. However, currently no man-made soft nanohelix has the ability to rapidly reconfigure its geometry in response to multiple forms of environmental stimuli, which has the potential to enhance motility in tortuous heterogeneous biological environments. Here, we report magnetic actuation of self-assembled bacterial flagellar nanorobotic swimmers. Bacterial flagella change their helical form in response to environmental stimuli, leading to a difference in propulsion before and after the change in flagellar form. We experimentally and numerically characterize this response by studying the swimming of three flagellar forms. Also, we demonstrate the ability to steer these devices and induce flagellar bundling in multi-flagellated nanoswimmers.

Structural analysis of variant of Helicobacter pylori MotB in its activated form, engineered as chimera of MotB and leucine zipper

Rotation of the bacterial flagellum is powered by a proton influx through the peptidoglycan (PG)-tethered stator ring MotA/B. MotA and MotB form an inner-membrane complex that does not conduct protons and does not bind to PG until it is inserted into the flagellar motor. The opening of the proton channel involves association of the plug helices in the periplasmic region of the MotB dimer into a parallel coiled coil. Here, we have characterised the structure of a soluble variant of full-length Helicobacter pylori MotB in which the plug helix was engineered to be locked in a parallel coiled coil state, mimicking the open state of the stator. Fluorescence resonance energy transfer measurements, combined with PG-binding assays and fitting of the crystal structures of MotB fragments to the small angle X-ray scattering (SAXS) data revealed that the protein’s C-terminal domain has a PG-binding-competent conformation. Molecular modelling against the SAXS data suggested that the linker in H. pylori MotB forms a subdomain between the plug and the C-terminal domain, that ‘clamps’ the coiled coil of the plug, thus stabilising the activated form of the protein. Based on these results, we present a pseudo-atomic model structure of full-length MotB in its activated form.

Synchronization and Collective Dynamics of Flagella and Cilia as Hydrodynamically Coupled Oscillators

Cooperative motion of flagella and cilia faciliates swimming of microorganisms and material transport in the body of multicellular organisms. Using minimal models, we address the roles of hydrodynamic interaction in synchronization and collective dynamics of flagella and cilia. Collective synchronization of bacterial flagella is studied with a model of bacterial carpets. Cilia and eukaryotic flagella are characterized by periodic modulation of their driving forces, which produces various patterns of two-body synchronization and metachronal waves. Long-range nature of the interaction introduces novel features in the dynamics of these model systems. The flagella of a swimmer synchronize also by a viscous drag force mediated through the swimmer’s body. Recent advance in experimental studies of the collective dynamics of flagella, cilia and related artificial systems are summarized.

Treponema pallidum flagellins stimulate MMP-9 and MMP-13 expression via TLR5 and MAPK/NF-κB signaling pathways in human epidermal keratinocytes

Syphilis is a chronic disease caused by Treponema pallidum and the pathogenesis is still unclear. T. pallidum infection induced inflammatory responses are involved in the immunopathological damage in skin and other tissues. Flagellin, the monomeric subunit of bacterial flagella, is a classic pathogen associated molecular patterns (PAMPs) that interacts to TLR5 and induces inflammatory responses. Keratinocytes, as immune sentinels recognize the PAMPs via TLRs, play an important role in skin innate immune response. Matrix metalloproteinases (MMPs) expressed by keratinocytes are involved in skin inflammatory responses and promoting pathogens invasion. In this study, we demonstrate that FlaB1, FlaB2 and FlaB3, the flagellins of T. pallidum, induced MMP-9 and MMP-13 production in human immortalized keratinocytes cell line HaCaT. Silencing of TLR5, but not TLR2 and TLR4 attenuated MMP-9 and MMP-13 expressions induced by T. pallidum flagellins. MMP-9 and MMP-13 expressions were also be abrogated by transfection with a dominant negative (DN) plasmid of MyD88. We also found that treatment of HaCaT cells with FlaB1, FlaB2 and FlaB3 activate the MAPK and NF-κB signaling pathways. Inhibited of ERK, JNK, p38 and NF-κB suppressed MMP-9 expression induced by the FlaB1. MMP-13 expression was found to be suppressed by pretreatment with inhibitors of ERK, JNK and NF-κB, but not p38. These findings demonstrate that T. pallidum flagellins (FlaB1, FlaB2 or FlaB3) can stimulate MMP-9 and MMP-13 expression through TLR5 and MAPK/NF-κB signaling pathways in human epidermal keratinocytes, which could contribute to the pathogenesis of T. pallidum infection.

Autonomously responsive pumping by a bacterial flagellar forest: A mean-field approach.

This study is motivated by a microfluidic device that imparts a magnetic torque on an array of bacterial flagella. Bacterial flagella can transform their helical geometry autonomously in response to properties of the background fluid, which provides an intriguing mechanism allowing their use as an engineered element for the regulation or transport of chemicals in microscale applications. The synchronization of flagellar phase has been widely studied in biological contexts, but here we examine the synchronization of flagellar tilt, which is necessary for effective pumping. We first examine the effects of helical geometry and tilt on the pumping flows generated by a single rotating flagellum. Next, we explore a mean-field model for an array of helical flagella to understand how collective tilt arises and influences pumping. The mean-field methodology allows us to take into account possible phase differences through a time-averaging procedure and to model an infinite array of flagella. We find array separation distances, magnetic field strengths, and rotation frequencies that produce nontrivial self-consistent pumping solutions. For individual flagella, pumping is reversed when helicity or rotation is reversed; in contrast, when collective effects are included, self-consistent tilted pumping solutions become untilted nonpumping solutions when helicity or rotation is reversed.

Discovery of a proteolytic flagellin family in diverse bacterial phyla that assembles enzymatically active flagella

Bacterial flagella are cell locomotion and occasional adhesion organelles composed primarily of the polymeric protein flagellin, but to date have not been associated with any enzymatic function. Here, we report the bioinformatics-driven discovery of a class of enzymatic flagellins that assemble to form proteolytically active flagella. Originating by a metallopeptidase insertion into the central flagellin hypervariable region, this flagellin family has expanded to at least 74 bacterial species. In the pathogen, Clostridium haemolyticum, metallopeptidase-containing flagellin (which we termed flagellinolysin) is the second most abundant protein in the flagella and is localized to the extracellular flagellar surface. Purified flagellar filaments and recombinant flagellin exhibit proteolytic activity, cleaving nearly 1000 different peptides. With ~ 20,000 flagellin copies per ~ 10-μm flagella this assembles the largest proteolytic complex known. Flagellum-mediated extracellular proteolysis expands our understanding of the functional plasticity of bacterial flagella, revealing this family as enzymatic biopolymers that mediate interactions with diverse peptide substrates.

A trans-acting leader RNA from a Salmonella virulence gene

Bacteria use flagella to move toward nutrients, find its host, or retract from toxic substances. Because bacterial flagellum is one of the ligands that activate the host innate immune system, its synthesis should be tightly regulated during host infection, which is largely unknown. Here, we report that a bacterial leader mRNA from the mgtCBR virulence operon in the intracellular pathogen Salmonella enterica serovar Typhimurium binds to the fljB coding region of mRNAs in the fljBA operon encoding the FljB phase 2 flagellin, a main component of bacterial flagella and the FljA repressor for the FliC phase 1 flagellin, and degrades fljBA mRNAs in an RNase E-dependent fashion during infection. A nucleotide substitution of the fljB flagellin gene that prevents the mgtC leader RNA-mediated down-regulation increases the fljB-encoded flagellin synthesis, leading to a hypermotile phenotype inside macrophages. Moreover, the fljB nucleotide substitution renders Salmonella hypervirulent, indicating that FljB-based motility must be compromised in the phagosomal compartment where Salmonella resides. This suggests that this pathogen promotes pathogenicity by producing a virulence protein and limits locomotion by a trans-acting leader RNA from the same virulence gene during infection.

Self-propelled nanofluids a path to a highly effective coolant

We propose a new self-propelled nanofluid having advantageous thermal and rheological properties at the same time. The nanofluid consists of a low volume fraction of self-propelled particles known as Artificial Bacterial Flagella (ABF), which will swim as pushers in a manner similar to the swimming of E. coil microorganisms with flagella. A theoretical model is introduced, describing the mechanisms responsible for the reduction of viscosity. The model shows that the swimming velocity of the particle and its geometry play an essential role in the reduction of the suspension viscosity. The results obtained from the theoretical model compare qualitatively with experiments in the literature. The model shows a significant decrease in viscosity at very low volume fractions, and that the viscosity of the suspension is reduced as the volume fraction of the particles increases. Using an in-house finite volume code, we numerically simulate natural convection effects in our ABF self-propelled nanofuid inside a square cavity heated from its vertical sides. Simulations are conducted at volume fractions of 0.7%, 0.8% and 0.83%, comparing the performance of a self-propelled nanofluid with conventional non-active nanofluids (i.e. carbon nanotubes in water). The results show that the heat transfer rate measured by the Nusselt number is three times higher than for the case of classical nanofluids and pure water at the same operating conditions and 0.83% volume fraction of particles. Also, due to the very dilute volume fractions of particles in the proposed nanofluid, their stability can endure for long operating times. There is also a significant decrease in the viscosity (around 25 times lower than water) which will result in a significant reduction in the pumping power.

A flagellum-specific chaperone facilitates assembly of the core type III export apparatus of the bacterial flagellum.

Many bacteria move using a complex, self-assembling nanomachine, the bacterial flagellum. Biosynthesis of the flagellum depends on a flagellar-specific type III secretion system (T3SS), a protein export machine homologous to the export machinery of the virulence-associated injectisome. Six cytoplasmic (FliH/I/J/G/M/N) and seven integral-membrane proteins (FlhA/B FliF/O/P/Q/R) form the flagellar basal body and are involved in the transport of flagellar building blocks across the inner membrane in a proton motive force-dependent manner. However, how the large, multi-component transmembrane export gate complex assembles in a coordinated manner remains enigmatic. Specific for most flagellar T3SSs is the presence of FliO, a small bitopic membrane protein with a large cytoplasmic domain. The function of FliO is unknown, but homologs of FliO are found in >80% of all flagellated bacteria. Here, we demonstrate that FliO protects FliP from proteolytic degradation and promotes the formation of a stable FliP–FliR complex required for the assembly of a functional core export apparatus. We further reveal the subcellular localization of FliO by super-resolution microscopy and show that FliO is not part of the assembled flagellar basal body. In summary, our results suggest that FliO functions as a novel, flagellar T3SS-specific chaperone, which facilitates quality control and productive assembly of the core T3SS export machinery.

Structure and in situ organisation of the Pyrococcus furiosus archaellum machinery.

The archaellum is the macromolecular machinery that Archaea use for propulsion or surface adhesion, enabling them to proliferate and invade new territories. The molecular composition of the archaellum and of the motor that drives it appears to be entirely distinct from that of the functionally equivalent bacterial flagellum and flagellar motor. Yet, the structure of the archaellum machinery is scarcely known. Using combined modes of electron cryo-microscopy (cryoEM), we have solved the structure of the Pyrococcus furiosus archaellum filament at 4.2 Å resolution and visualise the architecture and organisation of its motor complex in situ. This allows us to build a structural model combining the archaellum and its motor complex, paving the way to a molecular understanding of archaeal swimming motion.

Flagellum Length Control: How Long Is Long Enough?

The bacterial flagellum is an organelle that self-assembles outside the cell body. Recent work has uncovered the mechanism for length control of this self-assembly process.

Flagella bending affects macroscopic properties of bacterial suspensions.

To survive in harsh conditions, motile bacteria swim in complex environments and respond to the surrounding flow. Here, we develop a mathematical model describing how flagella bending affects macroscopic properties of bacterial suspensions. First, we show how the flagella bending contributes to the decrease in the effective viscosity observed in dilute suspension. Our results do not impose tumbling (random reorientation) as was previously done to explain the viscosity reduction. Second, we demonstrate how a bacterium escapes from wall entrapment due to the self-induced buckling of flagella. Our results shed light on the role of flexible bacterial flagella in interactions of bacteria with shear flow and walls or obstacles.

Variability in bacterial flagella re-growth patterns after breakage

Many bacteria swim through liquids or crawl on surfaces by rotating long appendages called flagella. Flagellar filaments are assembled from thousands of subunits that are exported through a narrow secretion channel and polymerize beneath a capping scaffold at the tip of the growing filament. The assembly of a flagellum uses a significant proportion of the biosynthetic capacities of the cell with each filament constituting ~1% of the total cell protein. Here, we addressed a significant question whether a flagellar filament can form a new cap and resume growth after breakage. Re-growth of broken filaments was visualized using sequential 3-color fluorescent labeling of filaments after mechanical shearing. Differential electron microscopy revealed the formation of new cap structures on broken filaments that re-grew. Flagellar filaments are therefore able to re-grow if broken by mechanical shearing forces, which are expected to occur frequently in nature. In contrast, no re-growth was observed on filaments that had been broken using ultrashort laser pulses, a technique allowing for very local damage to individual filaments. We thus conclude that assembly of a new cap at the tip of a broken filament depends on how the filament was broken.

Nanoscale-length control of the flagellar driveshaft requires hitting the tethered outer membrane.

The bacterial flagellum exemplifies a system where even small deviations from the highly regulated flagellar assembly process can abolish motility and cause negative physiological outcomes. Consequently, bacteria have evolved elegant and robust regulatory mechanisms to ensure that flagellar morphogenesis follows a defined path, with each component self-assembling to predetermined dimensions. The flagellar rod acts as a driveshaft to transmit torque from the cytoplasmic rotor to the external filament. The rod self-assembles to a defined length of ~25 nanometers. Here, we provide evidence that rod length is limited by the width of the periplasmic space between the inner and outer membranes. The length of Braun's lipoprotein determines periplasmic width by tethering the outer membrane to the peptidoglycan layer.

Optimization of Tail Geometry for the Propulsion of Soft Microrobots

Recent advances in smart materials and microfabrication techniques lead to the development of microrobots for on-demand and targeted therapy. Self-folded hydrogel tubes are particularly promising vehicles as they provide relatively large surface area-to-volume ratio and cargo space for therapeutic agents. In this paper, we decorate these microstructures with an artificially approximated bacterial flagellum to enable efficient swimming in fluidic environments. Flexibility enhances overall motility of the soft microrobot through synergistic propulsion generated by the tubular body and the flagellum, a feature that has not been observed in conventional microrobots manufactured from rigid materials. While the flagellum is applying forward thrust, a precession is induced on the body due to wobbling of the tail that can provide extra speed depending on the tail design. A simple model based on resistive force theory explains the direction-dependent changes in swimming motility and the role of tail geometry.

Bacterial flagella grow through an injection-diffusion mechanism.

The bacterial flagellum is a self-assembling nanomachine. The external flagellar filament, several times longer than a bacterial cell body, is made of a few tens of thousands subunits of a single protein: flagellin. A fundamental problem concerns the molecular mechanism of how the flagellum grows outside the cell, where no discernible energy source is available. Here, we monitored the dynamic assembly of individual flagella using in situ labelling and real-time immunostaining of elongating flagellar filaments. We report that the rate of flagellum growth, initially ∼1,700 amino acids per second, decreases with length and that the previously proposed chain mechanism does not contribute to the filament elongation dynamics. Inhibition of the proton motive force-dependent export apparatus revealed a major contribution of substrate injection in driving filament elongation. The combination of experimental and mathematical evidence demonstrates that a simple, injection-diffusion mechanism controls bacterial flagella growth outside the cell.

Effects of inhibitors of transcription factors, nuclear factor-κB and activator protein 1, on the expression of proinflammatory cytokines and chemokines induced by stimulation with Toll-like receptor ligands in hen vaginal cells

The goal of this study was to determine whether nuclear factor-κB (NFκB) and activator protein 1 (AP-1) were the responsible transcription factors for the induction of proinflammatory cytokines in hen vaginal cells stimulated by different Toll-like receptor (TLR) ligands. Cultured vaginal cells were treated with or without poly I:C (TLR3 ligand; dsRNA virus), lipopolysaccharide (LPS) (TLR4 ligand; gram-negative bacteria), flagellin (TLR5 ligand; bacterial flagellum), R848 (TLR7 ligand; ssRNA virus), and CpG-oligodeoxynucleotide (CpG-ODN) (TLR21 ligand; bacteria and DNA virus) in the presence or absence of different doses of BAY11-7085 (NFκB inhibitor) and tanshinone IIA (AP-1 inhibitor). Then, gene expressions of IL1B, IL6, and CXCLi2 were examined by real-time PCR analysis. The results showed that the induction of the expression of IL1B, IL6 and CXCLi2 by poly I:C, LPS, and CpG-ODN were suppressed by Bay11-7085, but not by tanshinone IIA. IL1B expression was upregulated by flagellin and R848, and the increase in its expression was suppressed by Bay11-7085, but not by tanshinone. These results suggest that NFκB is the responsible transcription factor for the expression of proinflammatory cytokines and chemokines, including I IL1B, IL6, and CXCLi2 in response to the ligands of TLR3, 4, and 21, and IL1B in response to the ligands of TLR5 and 7 in the vaginal cells.