A structure-function study of C-terminal residues predicted to line the export channel in Salmonella Flagellin
Introduction
Over 80% of bacterial species propel themselves in liquid environments by rotating thin helical filaments called flagella [1]. The majority of a flagellar filament is composed of a single protein called flagellin that polymerizes to form a helical tubular filament approximately 24 nm in diameter with a 2 nm diameter central channel. A flagellar filament can grow to a length of up to 15 μm, extending from the cell membrane where it is attached to the basal body motor [2,3]. Structural and sequence analysis studies predicted the central channel is lined with polar and charged amino acids, and that the hydrophilic lining of the central channel is conserved [2,4].
The process of bacterial flagellin export and assembly into flagella is of fundamental interest in microbiology. Furthermore, engineered flagella displaying peptides and proteins have potential applications in biotechnology and nanotechnology [[5], [6], [7]]; flagellar bionanotubes with various molecular recognition, catalytic, and nanomaterial functions have been demonstrated [[8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19]]. Furthermore, bacterial flagellins have medical relevance, including as an immunomodulatory agent recognized by the mammalian host immune system via Toll-like receptor-5 [20]. Flagellin monomers are synthesized inside the cell as soluble monomers, prevented from self-assembly by binding the FliS chaperone protein in a 1:1 ratio [21], and exported into the central channel by a type III secretion system (T3SS) [22], similar to the T3SS used for secretion of virulence factors by a wide variety of pathogenic bacteria [23]. Flagellin monomers then diffuse to the distal end and are incorporated into the growing flagellar filament in their final, native structure with the help of the FliD chaperone protein [24,25]. The N- and C-terminal regions that comprise the α-helical, coiled-coil D0 domain in the filament are intrinsically disordered in the soluble flagellin monomer [26,27].
The first complete structure of the flagellin protein in its polymeric flagellar form at a 4 Å resolution was described by Yonekura et al. [2]. The 2 nm (20 Å) diameter of the central channel is too narrow to accommodate a fully folded flagellin monomer, so it is thought that monomers must enter the central channel in an unfolded state. The disordered flagellin monomers may have exposed non-polar side chains that could strongly interact with the surface of the central channel, potentially disrupting the export process. Analysis of the flagellin structure (PDB ID: 1UCU) indicated that the inner surface of the internal channel is lined with the solvent-exposed side chains of one charged residue and three polar residues from the C-terminal region, Gln 484, Asn 488, Ser 491, and Arg 494 [2,4]. The hydrophilic surface of the central channel should minimize retention of unfolded monomers via hydrophobic interactions, facilitating their rapid diffusion to the distal end of the filament for assembly.
Experimental evidence for the mechanism of flagellin monomer transport has been conflicting. In the originally proposed passive diffusion mechanism, it would be expected that the rate of filament growth would decrease as the filament grows longer, which was observed via electron microscopy [28] and shown in a mathematical simulation [29]. In contrast to the original diffusion model, several recent studies indicated that bacterial flagella grow at a constant rate independent of filament length [30,31]. Furthermore, a chain mechanism involving linked monomers being pulled through the channel was also proposed to explain the length-independent growth [32]. However, a more recent study by Renault et al. [33] indicated that flagella in Salmonella grow in a length-dependent manner, consistent with the originally observed diffusion-limited transport mechanism through the channel. Regardless of the flagellin monomer transport mechanism, i.e., length-dependent or length-independent, the polar nature of the central channel may be important for efficient transport of monomer proteins as previous studies have postulated [2,4].
Furthermore, channel-lining residues may also play a role in filament stability and morphology. Previous studies have shown that deleting the disordered N- and C-terminal regions of flagellin caused flagellar filaments to become less stable and adopt a straight morphology rather than the typical helical morphology [[34], [35], [36]]. Additionally, studies have shown that point mutations in flagellin monomers can change the filament morphology as well as the handedness of the helix [37,38], providing evidence that key residues may exist in the flagellin monomer that influence the physical properties of the assembled filament.
Although previous studies have predicted the importance of the hydrophilic nature of the flagellar channel, there is currently no experimental evidence regarding this fundamental question. This study describes an experimental structure-function investigation of this hypothesis by site-directed mutagenesis of the putative channel-lining residues in Salmonella enterica serovar Typhimurium (henceforth referred to as S. Typhimurium).
Section snippets
Key resources table
The bacterial strains and plasmids used in this study are summarized in Table 1. The Salmonella strains and plasmids were described previously [39]. The plasmid pTH890 is derived from pTrc99A and carries ampicillin resistance and the S. Typhimurium phase 1 flagellin fliC gene under control of the hybrid Trc-lac promoter. Flagellin is expressed at a constant rate due to a leaky promoter and does not require an inducing agent. pTH890 was used to compliment the flagellin-deficient S. Typhimurium
Swimming agar motility assay
It was previously suggested by Yonekura et al. that the hydrophilic solvent-exposed surface of several putative channel-lining residues is essential in facilitating the transport of unfolded flagellin monomers to the distal end of the flagellar filament [2]. It follows that disruption of the hydrophilic nature of the channel surface should have a significant effect on flagellin monomer export, flagellar assembly, and consequently, cellular motility. We attempted to investigate this hypothesis
Discussion
Analysis of the first complete flagellin structure resulted in the hypothesis that hydrophilic channel-lining residues are required to facilitate the transport of unfolded flagellin monomers to the distal end of the filament. It should be noted that the N- and C-terminal regions that form the coiled-coil D0 domain of the FliC structure were determined at a lower resolution of ~4 Å resolution via cryoelectron microscopy [2,42]. Thus, there is some uncertainty in the side chain conformations and
Conclusions
In conclusion, this study provides the first experimental evidence that the hydrophilic character of the four putative channel-lining residues of flagellin is not strictly required for flagellin export and assembly. The four C-terminal flagellin residues investigated in this study, Gln 484, Asn 488, Ser 491, and Arg 494, may function as channel-lining residues; however, substitutions of these residues can affect the stability and morphology of the flagellar filament. A general observation is
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The authors would like to thank Dr. Marc Erhardt for technical advice on the reagents used for immunofluorescence imaging, and Dr. Yusuke Morimoto for information regarding the flagella staining protocol. Financial support for this work was provided by a grant from the Faculty Research and Creative Activities Award program at Western Michigan University.
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