Crystal Structure of Flagellar Export Chaperone FliS in Complex With Flagellin and HP1076 of Helicobacter pylori

Abstract

Functional flagella formation is a widespread virulence factor that plays a critical role in survival and host colonization. Flagellar synthesis is a complex and highly coordinated process. The assembly of the axial structure beyond the cell membrane is mediated by export chaperone proteins that transport their cognate substrates to the export gate complex. The export chaperone FliS interacts with flagellin, the basic component used to construct the filament. Unlike enterobacteria, the gastric pathogen Helicobacter pylori produces two different flagellins, FlaA and FlaB, which exhibit distinct spatial localization patterns in the filament. Previously, we demonstrated a molecular interaction between FliS and an uncharacterized protein, HP1076, in H. pylori. Here, we present the crystal structure of FliS in complex with both the C-terminal D0 domain of FlaB and HP1076. Although this ternary complex reveals that FliS interacts with flagellin using a conserved binding mode demonstrated previously in Aquifex aeolicus, Bacillus subtilis, and Salmonella enterica serovar Typhimurium, the helical conformation of FlaB in this complex was different. Moreover, HP1076 and the D1 domain of flagellin share structural similarity and interact with the same binding interface on FliS. This observation was further validated through competitive pull-down assays and kinetic binding analyses. Interestingly, we did not observe any detrimental flagellation or motility phenotypes in an hp1076-null strain. Our localization studies suggest that HP1076 is a membraneassociated protein with a cellular localization independent of FliS. As HP1076 is uniquely expressed in H. pylori and related species, we propose that this protein may contribute to the divergence of the flagellar system, although its relationship with FliS remains incompletely elucidated.

Cryo-electron Tomography Reveals the Roles of FliY in Helicobacter pylori Flagellar Motor Assembly

“…2A ). In line with previous phenotypic studies ( 7 , 30 ), the cryo-EM images of the Δ fliY and FliY N strains revealed that none of the cells has flagellar filament. In addition, deformation of the cell membranes was observed in the ΔfliY strain (∼82% images) and the FliY N strain (∼80% images), as shown in Fig.…”

“…The H. pylori wild-type strain G27, the Δ fliY strain, and the FliY N and FliY C complementation strains expressed with the N- or C-terminal domain of FliY, respectively ( 7 , 30 ) were inoculated into 10 mL of brucella broth supplemented with 10% heat-inactivated fetal bovine serum. The bacterial culture was grown at 37°C under microaerophilic conditions for about 24 h until the optical density at 600 nm reached ∼2.…”

Glycosyltransferase Jhp0106 (PseE) contributes to flagellin maturation in Helicobacterpylori

“…However, the export process of glycosylated and non‐glycosylated FlaA was not tightly coupled with FliS chaperone binding, which was reported in a recent study in H . pylori G27 strain 35 . Their observation on the formation of short flagellar filaments in the Δ fliS mutant suggested a less efficient secretion of FlaA and impaired flagellar assembly.…”

“…pylori and other pathogens, 16,17,32‐37 it is likely that in the wild‐type strain (Figure 6, the left panel), PseE would first bind to FlaA, followed by sugar transfer to glycosylate FlaA. The glycosylated FlaA within this FlaA‐PseE complex would possibly be recognized by a flagellin‐specific chaperone FliS 33‐35 . With the chaperone‐mediated protection, the unfolded glycosylated FlaA would be transported efficiently via a flagellar type three secretion system (fT3SS) to extracellular surface.…”

Slimy partners: the mucus barrier and gut microbiome in ulcerative colitis

“…However, for many bacterial pathogens, binding to mucus is a crucial step in their colonization. Flagella, composed of flagellin arranged in helical chains, are an important evolved strategy for mucus adhesion during infection by some pathogens, and they play a critical role in biofilm formation 17 . Enterotoxigenic E. coli (ETEC) strains are major causes of morbidity and mortality due to diarrheal illness in developing countries.…”