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Structural and Functional Comparison of Salmonella Flagellar Filaments Composed of Fljb and Flic

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Structural and Functional Comparison of Salmonella Flagellar Filaments Composed of Fljb and Flic biomolecules Article Structural and Functional Comparison of Salmonella Flagellar Filaments Composed of FljB and FliC 1,2, 1, 1,3 1 Tomoko Yamaguchi y , Shoko Toma y, Naoya Terahara , Tomoko Miyata , 1, 1 1,2,4,5, 1, ,§ Masamichi Ashihara z, Tohru Minamino , Keiichi Namba * and Takayuki Kato * 1 Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan; [email protected] (T.Y.); [email protected] (S.T.); [email protected] (N.T.); [email protected] (T.M.); masamichi.ashihara@thermofisher.com (M.A.); [email protected] (T.M.) 2 RIKEN Center for Biosystems Dynamics Research, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan 3 Faculty of Science and Engineering, Chuo University, Tokyo 112-8551, Japan 4 RIKEN SPring-8 Center, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan 5 JEOL YOKOGUSHI Research Alliance Laboratories, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan * Correspondence: [email protected] (K.N.); [email protected] (T.K.); Tel.: +81-6-6879-4625 (K.N. & T.K.) These authors contributed equally to this work. y Current address: Thermo Fisher Scientific K.K., 4-2-8 Shibaura, Minato-ku, Tokyo 108-0023, Japan. z § Current address: Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Received: 27 December 2019; Accepted: 4 February 2020; Published: 6 February 2020 Abstract: The bacterial flagellum is a motility organelle consisting of a long helical filament as a propeller and a rotary motor that drives rapid filament rotation to produce thrust. Salmonella enterica serovar Typhimurium has two genes of flagellin, fljB and fliC, for flagellar filament formation 3 4 and autonomously switches their expression at a frequency of 10− –10− per cell per generation. We report here differences in their structures and motility functions under high-viscosity conditions. A Salmonella strain expressing FljB showed a higher motility than one expressing FliC under high viscosity. To examine the reasons for this motility difference, we carried out structural analyses of the FljB filament by electron cryomicroscopy and found that the structure was nearly identical to that of the FliC filament except for the position and orientation of the outermost domain D3 of flagellin. The density of domain D3 was much lower in FljB than FliC, suggesting that domain D3 of FljB is more flexible and mobile than that of FliC. These differences suggest that domain D3 plays an important role not only in changing antigenicity of the filament but also in optimizing motility function of the filament as a propeller under different conditions. Keywords: bacterial flagellar motility; flagellin; Salmonella; FljB; FliC; electron cryomicroscopy; viscosity; infection 1. Introduction Salmonella infection is one of the four major causes of disease involving diarrheas in the world. Salmonella enterica serovar Typhimurium (hereafter Salmonella) has a wide range of hosts, and it infects not only mouse, which is its original host, but also humans. The infection occurs mainly via oral intake, and flagellar motility plays an important role in infection. The flagella enable bacteria to move through viscous environments such as mucosa, search for the host cell surface, and adhere to the cell membrane for infection [1,2]. Biomolecules 2020, 10, 246; doi:10.3390/biom10020246 www.mdpi.com/journal/biomolecules Biomolecules 2020, 10, 246 2 of 11 The bacterial flagellum consists of three main parts: the basal body, which works as a rotary motor; the filament, which functions as a screw propeller; and the hook as a universal joint connecting the filament to the motor [3]. Salmonella has several peritrichous flagella, and the length of the filament is 10 to 15 µm long. When the cell swims straight, the motors rotate counterclockwise (CCW), and the normally left-handed supercoiled filaments form a bundle behind the cell to produce thrust. When the motors switch their rotation to the clockwise (CW) direction, the filaments switch to a right-handed supercoil in order for the bundle to fall apart so that the cell can change its orientation by tumbling to change the direction of swimming. Salmonella has two flagellin genes, fljB and fliC, and their expression is autonomously regulated to produce either FljB or FliC for filament formation at a frequency of 3 4 10− –10− per cell per generation. This is called phase variation [4], and it is thought to be a mechanism to enable escape from the host immune system by changing flagellar antigenicity. It has been reported that FliC-expressing bacteria display a significant advantage for invasion of most epithelial cell lines of murine and human origin compared to FljB-expressing bacteria. Differences in the swimming behaviors near surfaces have also been observed, and FliC-expressing bacteria more frequently “stop” [5]. In order to understand the differences in their antigenicity and motility function, structural and functional characterization is necessary. However, structural information is available only for the FliC filament [6–8]. The three-dimensional (3D) structures of FliC and the FliC filament have been studied by X-ray crystallography and electron cryomicroscopy (cryoEM) helical image analysis, respectively [6–8]. Salmonella FliC from the strain SJW1103 consists of 494 amino acid residues, and the molecular mass is about 50 kDa. The molecule consists of four domains, D0, D1, D2, and D3, arranged from the inner core to the outer surface of the filament. Domains D0 and D1 form the inner core of the filament and are made of α-helical coiled coils. These domains play a critical role in forming the supercoiled structure of the filament as a helical propeller. In addition, a β-hairpin structure in domain D1 is considered to be important for switching the conformation of flagellin subunits between the two states to produce various types of supercoiled filaments in left- and right-handed forms for swimming and tumbling [6,8]. Domains D2 and D3 are found in the outer part of the filament structure. These two domains increase the stability of the filament structure as well as the drag force of the filament as a propeller by increasing the diameter [6,9]. The outermost domain, D3, is thought to contain epitopes for antibodies, determining the antigenicity of the flagella. To understand the differences in the antigenicity between FljB and FliC, structural information for the FljB and FljB filaments is necessary. Functional characterization of cell motility is also necessary to investigate the potentially different physiological roles played by these two types of filament, if any. In the present study, we therefore investigated differences in the FljB and FliC filament structures and their motility functions. A Salmonella strain expressing FljB showed a higher motility than the one expressing FliC under high-viscosity conditions. The structure of the FljB filament analyzed by cryoEM image analysis was nearly identical to that of the FliC filament, except for the position and orientation of the outermost domain D3. Domain D3 of FljB also showed a higher flexibility and mobility than that of FliC. These differences suggest that domain D3 plays an important role not only in changing antigenicity, but also in optimizing motility function of the filament as a propeller under different conditions. We have discussed the relationship between the structure and motility function by comparing FljB and FliC. 2. Materials and Methods 2.1. Salmonella Strains Bacterial strains of Salmonella enterica serovar Typhimurium used in this study are listed in Table1. SJW1103 lacks the fljB operon and so expresses only the FliC flagellin. To express FljB from the fliC promoter on the chromosome, the DfliC::tetRA allele was replaced by the fljB allele using the λ Red homologous recombination system [10], as described previously [11], to generate the SJW1103B strain. Biomolecules 2020, 10, 246 3 of 11 For cryoEM structural analyses, the strain expressing the flagellar filament of the R-type straight form (the right-handed helical symmetry) was used. Table 1. Strains and plasmids used in this study. Salmonella Strains Relevant Characteristics Source or Reference FliC wild-type SJW1103 Yamaguchi et al. 1984 [12] Dhin-fljB-fljA SJW1103DC SJW1103 DfliC::tetRA This study FljB wild-type SJW1103B This study DfliC::fljB FljB_R-type straight filament SJW590 This study fljB(A461V), DfliC 2.2. Swimming Motility Assay Salmonella strains, SJW1103B (only expressing FljB) and SJW1103 (only expressing FliC), were pre-cultured in 5 mL of Luria–Bertani broth (LB, 1% (w/v) tryptone, 0.5% (w/v) yeast extract, 0.5% (w/v) NaCl) with overnight shaking at 37 ◦C. Bacterial growth was measured via optical density at 600 nm (OD600). A 5 µL measure of the culture medium was inoculated into 5 mL of fresh LB, and it was incubated for 6 h at 37 ◦C with shaking. The cells were diluted in the motility buffer (10 mM potassium phosphate pH 7.0, 0.1 mM EDTA, 10 mM sodium D-lactate). The viscosity of the motility buffer was adjusted by adding Ficoll PM400 (GE healthcare, Chicago, IL, USA) to a final concentration of 5%, 10%, or 15%. Swimming motility was observed using a bright-field microscope, CX-41 (Olympus, Tokyo, Japan) with a 40 objective (PlanC N 40 NA 0.65 Olympus) and a 1.25 variable magnification lens (U-CA 1.5 × × × × Olympus) and recorded using a high-speed camera (GEV-B0620M-TC000 IMPREX, Boca Raton, FL, USA). The swimming speed was calculated using the software Move-tr2/2D (Library Co., Tokyo, Japan). 2.3. Fluorescence Labeling of Flagellin Antibodies IgG antibodies against FliC and FljB were purified from rabbit serum using a MelonTM Gel IgG Spin Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA).
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