Architecture and assembly of periplasmic flagellum

Abstract

Periplasmic flagella are complex nanomachines responsible for distinctive morphology and motility of spirochetes. Although bacterial flagella have been extensively studied for several decades in model systems Escherichia coli and Salmonella enterica, our understanding of periplasmic flagella in many disease-causing spirochetes remains incomplete. Recent advances, including molecular genetics, biochemistry, structural biology, and cryo-electron tomography, have greatly increased our understanding of structure and function of periplasmic flagella. In this chapter, we summarize some of the recent findings that provide new insights into the structure, assembly and function of periplasmic flagella.

The flagellum is a major organelle for motility in many bacterial species. It confers locomotion and is often associated with virulence of bacterial pathogens. Flagella from different species share a conserved core, but also exhibit profound variations in flagellar structure, flagellar number and placement (Chen et al., 2011; Zhao et al., 2014), resulting in distinct flagella that appear to be adapted to the specific environments that the bacteria encounter. While many bacteria possess multiple peritrichous flagella such as those found in Escherichia coli and Salmonella enterica, other bacteria such as Vibrio spp. and Pseudomonas aeruginosa normally have a single flagellum at one cell pole (Fig. 1). Spirochetes uniquely assemble flagella that are embedded in periplasmic space between their inner and outer membranes, thus called periplasmic flagella (Charon et al., 2012). Although the flagella of E. coli and Salmonella have been extensively studied for several decades, periplasmic flagella are less understood, despite their profound impact on the distinctive morphology and motility of spirochetes. In this chapter, many aspects of periplasmic flagella will be discussed, with particular focus on their structure and assembly.

Figure 1. Distinctive placement of bacterial flagellum.

(A) Bacteria with flagella distributed all over the cell (e.g. Escherichia coli) are peritrichous. (B) Monotrichous bacteria, such as Vibrio cholera, Pseudomonas aeruginosa, and Caulobacter crescentus have a single flagellum present at one end of the cell. (C). Spirochetes, including species of Borrelia, Treponema, and Leptospira, possess specialized flagella located within the periplasmic space. The rotation of the periplasmic flagella allows this bacterium to swim forward in a corkscrew-like motion.

Spirochetes are a distinctive group of bacteria of significant importance in human health.

Spirochetes cause several major diseases in humans such as Lyme disease (Borrelia burgdorferi), syphilis (Treponema pallidum), leptospirosis (Leptospira interrogans), and periodontitis (Treponema spp.). Lyme disease is the most commonly reported tick-borne illness in the United States, and the incidence is growing rapidly. The number of patients diagnosed with Lyme disease each year in the United States is approximately 300,000. The disease is caused by Borrelia burgdorferi and related organisms, and is transmitted to humans through the bite of infected Ixodes ticks (Radolf et al., 2012). Syphilis is a common sexually transmitted disease in many areas of the world. Leptospirosis is the most common waterborne zoonosis worldwide.

Motility is essential for spirochetes to infect and disseminate in mammalian hosts.

Spirochetal motility is unique, as the entire bacterium is involved in translocation without the involvement of external appendages. The motility is driven by periplasmic flagella, and rotation of the flagella causes a serpentine movement, allowing the organism to very efficiently bore its way through viscous media or tissue (Charon et al., 2012). To complete the host-vector life cycle, B. burgdorferi is able to adapt to divergent host environments and also evade the defense of its mammalian reservoir (Radolf et al., 2012). Several studies provide direct evidence that the unique motility and chemotaxis of B. burgdorferi are essential for the establishment of infection in mammals and the completion of its enzootic cycle (Li et al., 2010; Motaleb et al., 2015; Sultan et al., 2013; Sultan et al., 2015).

Periplasmic flagella are necessary for the flat-wave morphology and distinctive motility of B. burgdorferi.

B. burgdorferi possesses 7–11 periplasmic flagella that are inserted at each cell pole and wrap around the cell cylinder to produce the spirochete’s distinctive flat-wave morphology. Periplasmic flagella are crucial not only for motility but also for the overall shape of B. burgdorferi, as mutant cells lacking flagella are non-motile and exhibit a rod-shaped morphology (Motaleb et al., 2000). Similar to the external flagella found in the model organisms E. coli and S. enterica, periplasmic flagella are composed of the flagellar motor, the hook and the filament. The flagellar motor is a rotary motor that anchors the flagellum to the inner membrane. The motors of the periplasmic flagella are noticeably larger than those of other external flagella (Fig. 2). Importantly, the motor possesses a spirochete-specific “collar” (Fig. 2) (Kudryashev et al., 2009; Liu et al., 2009; Moon et al., 2016; Murphy et al., 2006). The motor can be further divided into two parts: the rotor and the stator. The rotation of the motor is driven by the torque generated by the stator-rotor interaction, utilizing energy generated by the flow of protons through the stator channel. The rotor is composed of the MS-ring, the C-ring, and the rod. The MS-ring is the base of the rotor and it is formed by multiple copies of FliF. The C-ring is located in the cytoplasm and is also known as the switch complex. It consists of the proteins FliG, FliM and FliN and controls the direction of flagellar rotation. The rod serves as a drive shaft and consists of multiple different proteins (FlgB, FlgC, FlgF and FlgG). The hook of the periplasmic flagellum is located in periplasmic space, in contrast to the externally localized hook in E. coli and S. enterica. A recent study indicates that the hook proteins are cross-linked by a covalent bond, an unusual property necessary for transmission of high rotational torque from the motor to the filament (Miller et al., 2016). The filament is the longest component of the periplasmic flagella. Multiple filaments arising from both poles form flat ribbons that wrap around the spirochete cell body in a right-handed fashion (Charon et al., 2009). Flagellar-specific type III secretion system (fT3SS), which is embedded in the flagellar motor, is responsible for the transport and assembly of the protein compoments of the rod, the hook and the filament (Zhao et al., 2013).

Figure 2. Comparison of motor structures from E. coli, Vibrio, H. pylori, and Borrelia.

(A) A central section of an E. coli flagellar motor. (B) A central section from a non-sheathed Vibrio flagellar motor. (C) A central section from a sheathed Vibrio flagellar motor. (D) A central section from a sheathed flagellar motor of H. pylori. (E) A central section from Borrelia flagellar motor. (F-J) Schematic models derived from the central sections (A-E), respectively. Adapted from prior publications (Qin et al., 2016; Zhao et al., 2013; Zhu et al., 2017), with permission.

Characterization of the unique periplasmic structure of spirochetal flagella.

The collar is a unique spirochete-specific component that has not been found in other bacterial flagella reported to date (Chen et al., 2011; Zhao et al., 2014). The collar in the B. burgdorferi periplasmic flagellar motor is a large complex with ~71 nm in diameter and ~24 nm in height, presumably composed of many different proteins. However, there is limited information regarding its structure, function and protein components. Recently, a hypothetical membrane protein FlbB was identified as a candidate involved in collar assembly (Moon et al., 2016). In addition, the novel tetratricopeptide repeat protein BB0236 was also proposed to contribute to the collar assembly (Moon et al., 2018). Mutants deficient in either FlbB or BB0236 are non-motile and their periplasmic flagella lack the collar, its associated proteins (including FliL), and the stator (Fig. 3). This finding provides direct evidence that the collar is indeed an important (as well as unique) component of periplasmic flagella (Fig. 2). Although additional unknown proteins are likely involved in the collar assembly, it is evident that the periplasmic collar provides a static framework promoting the recruitment and stable association of stator units, which could in turn facilitate the higher torques to rotate the periplasmic flagellum. The rotation of the flagella within the confinements of the periplasm enables the spirochete to bore its way through complex, viscous environment in vertebrate and tick tissues.

Figure 3. Characterization of the unique features in periplasmic flagella, as examined through mutational analysis.

(A) Central section from a mutant lacking FlbB. (B) Central section from a mutant lacking BB0236. (C) Central section from a class average of a mutant lacking FliL. (D) Central section from another class average of a mutant lacking fliL. (E) A central section from WT flagellar motor. (F-J) Schematic models derived from panels (A-E), respectively. Adapted from prior publication (Moon et al., 2018), with permission.

Stator-rotor interaction.

Powered by the electrochemical gradient across the cytoplasmic membrane, the flagellar motor can rotate the filament at high speed. It is believed that the flagellar rotation is mediated by the interaction between the cytoplasmic loop region of MotA, and the C-terminal domain of FliG in the C-ring. However, there is limited structural information on stator-rotor interaction in model systems E. coli and S. enterica, largely because the stator is poorly resolved due to its dynamic nature and low occupancy (Fukuoka et al., 2009; Leake et al., 2006; Paulick et al., 2009). In contrast, the in situ flagellar motor structures of B. burgdorferi and other spirochetes determined by cryo-electron tomography (cryo-ET) reveal more detailed information regarding the stators and their interactions with the C-ring (Chen et al., 2011; Kudryashev et al., 2010; Liu et al., 2010; Liu et al., 2009; Murphy et al., 2006; Raddi et al., 2012). The presence of the collar in spirochetes is likely essential for the better visualization of the stator and its interaction with the rotor, because the collar provides a stable framework to recruit and stabilize the stators.

CheY-P binding and flagellar switching of rotational direction.

The flagellar motor in many bacteria species can rotate in both counter-clockwise (CCW) and clockwise (CW) directions to achieve swimming towards attractants or away from repellents. The rotation direction is controlled by a sophisticated chemotactic system. In the signaling pathway, CheY is phosphorylated by CheA kinase, then the phosphorylated CheY binds to the FliM protein in the C-ring and induces conformational changes that alter the stator-rotation interaction and cause switching (Welch et al., 1993). Studies in E. coli of the correlation between the CW rotation and the intracellular level of the phosphorylated CheY in individual cells indicated that binding and switching are highly cooperative (Cluzel et al., 2000). The switching spreads from one or more nucleation points on the C-ring, a phenomenon referred to as ‘conformational spread’ (Bai et al., 2010). Recent experiments revealed that the flagellar motor can adapt to varied levels of phosphorylated CheY by increasing the content of FliM (Yuan et al., 2012). Additional experiments suggested that it is not CheY-P binding, but rather the direction of motor rotation, that has the largest effect on remodeling of the FliM (Lele et al., 2012). It was suggested that there are ~34 molecules of FliM in a motor with exclusively CW rotation and ~44 molecules in a motor with CCW rotation. These E. coli studies also indicate that motors with even more FliM molecules may exist. It is unclear how the C-ring can accommodate such a large change, and if similar C-ring modifications also occur in spirochetes.

Because periplasmic flagellar motors are located at the two cell poles, it was hypothesized that spirochetal motors rotate asymmetrically at one end relative to the other during a run (Charon and Goldstein, 2002; Li et al., 2002). CheX is the only CheY-P phosphatase identified in the B. burgdorferi genome. A cheX mutant constantly flexes and is not able to run or reverse (Motaleb et al., 2005), while both cheA2 and cheY3 mutant constantly run in one direction (Li et al., 2002; Motaleb et al., 2011). A comparison of the motor structures from two different motions (flex and run) will likely shed new light upon the mechanisms underlying CheY-P binding and the switching of rotational direction.

Flagellar assembly.

The bacterial flagellum is built from the inside out, from proximal to distal structures, in a temporally and spatially regulated fashion. Detailed insights into the flagellar assembly have been well established in E. coli and S. enterica (Chevance and Hughes, 2008; Macnab, 2003). In these organisms, multiple copies of FliF form the MS-ring (Suzuki et al., 2004), which serves as the initial base for flagellar assembly, structural maturation, and function. The MS-ring also serves as a scaffold for the assembly of the C-ring. FliG proteins directly associate with the cytoplasmic face of the MS-ring and form the FliG ring (Lee et al., 2010; Minamino et al., 2011). FliM and FliN proteins form a stable complex with a stoichiometry of 1:4 (Brown et al., 2005; Delalez et al., 2014). The FliM-FliN₄ complex will bind to the FliG ring to form the completed C-ring. The export apparatus, which is assembled inside the MS-ring and the C-ring, is responsible to export flagellar axial protein components from the cytoplasm to the distal end of the nascent flagellar apparatus. FliE are likely assembled first and form a junction between the MS-ring and the rod to overcome their symmetry mismatch. Then multiple copies of FlgB,C,F,G form the rod, FlgI form the P-ring and FliH form the L-ring. FlgD cap assembles at the rod tip to support the assembly of the hook. Then the filament-cap (FliD) is formed after the hook assembly to support the assembly of the filament (Zhang et al., 2019). By arresting assembly with a series of genetic mutations, cryo-ET imaging of the motor in B. burgdorferi provided snapshots of the sequential assembly of periplasmic flagella (Zhao et al., 2013).

Flagellar export apparatus and its evolutionarily related injectisome.

The fT3SS consists of five integral membrane proteins (FlhA, FlhB, FliP, FliQ and FliR) and three soluble proteins (FliH, FliI and FliJ), and is located at the center of the cytoplasmic face of the MS-ring. The ATP complex promotes the export process by binding and delivering substrates to the export apparatus (Fraser et al., 2003; Minamino and Imada, 2015). FliI is an ATPase and shows structural similarity with the α and β subunits of the F₀F₁-ATP synthase (Ibuki et al., 2011); it exhibits its full ATPase activity when it self-assembles into a homo-hexamer (Imada et al., 2007). FliH, FliI, and FliJ coordinately deliver a chaperone-substrate complex to the export gate by binding to the docking platform of the fT3SS for substrate export. FliP, FliQ and FliR form an export gate complex with helical symmetry (Kuhlen et al., 2018).

fT3SSs in different bacterial species are highly conserved. In addition, they are evolutionally related to virulence T3SSs. The evolutionary relationship between the flagellum and the injectisome has garnered significant debate. The latest phylogenomic and comparative analyses of fT3SSs and vT3SSs suggest that the vT3SS derived from a flagellar ancestor. The loss of flagellum-specific genes led to an eventual loss in the motility function, but this system presumably kept the ability to secrete proteins (Abby and Rocha, 2012)

The overall organization of the fT3SS machine in periplasmic flagella shares many similar features as those observed in the vT3SS machine (Hu et al., 2017; Hu et al., 2015; Kawamoto et al., 2013) (Fig 4). However, the ATPase complex of the periplasmic flagella is noticeably different from those observed in the injectisome (Fig 4). There are 23 spokes and one hub in the ATPase complex of the B. burgdorferi periplasmic flagella. Only 6 spokes and one hob were observed in Salmonella injectisome, presumably optimizing for substrate recruitment and export. In contrast, the ATPase complex in the periplasmic flagella not only facilitates substrate recruitment and secretion, but also supports the integrity of the C-ring, which undergoes rotation and switches rotational direction between CW and CCW.

Figure 4. Comparison of the fT3SS from B. burgdorferi and the vT3SS from Salmonella.

(A) A central section from the B. burgdorferi motor. (B) The fT3SS in the spirochete motor includes the ATPase complex (orange) and the export apparatus (purple) underneath the MS-ring. (C, D) The vT3SS from Salmonella injectisome is modeled in a similar color scheme. The difference between the two T3SSs is striking in a comparison of the cross-sections of their ATPase complexes. Note that the C-ring from the B. burgdorferi motor is a continuous ring with ~46 copies of FliN tetramer. There are 23 visible FliH spokes (E, F). There are six pods in Salmonella injectisome. Only six spokes of the FliH homolog OrgB connect the ATPase complex to the SpaO molecules that compose the pod of the injectisome. Adapted from prior publication (Qin et al., 2018), with permission.

Outlook and perspective.

Although the structure and functions of bacterial flagellum have been studied for several decades, many important questions remain to be addressed. For example, how does the stator couple proton gradient to generate the torque? How does the C-ring change its conformation to generate rotation or switch rotational direction? How does the proton gradient power the expert apparatus and facilitate protein transport across inner membrane? Periplasmic flagella also raise additional questions: how do the periplasmic flagella coordinate their rotation from two cell poles? how does the spirochete specific collar assemble? Given that periplasmic flagella play critical roles in many bacterial pathogens, it will be important to understand not only the conserved structure and function among bacterial flagella, but also the specific features that distinct periplasmic flagella from others. Emerging techniques such as cryo-ET will be increasingly valuable to address many of these fundamental questions in periplasmic flagellum.