The HP0256 gene product is involved in motility and cell envelope architecture of Helicobacter pylori

Abstract

Background: Helicobacter pylori is the causative agent for gastritis, and peptic and duodenal ulcers. The bacterium displays 5-6 polar sheathed flagella that are essential for colonisation and persistence in the gastric mucosa. The biochemistry and genetics of flagellar biogenesis in H. pylori has not been fully elucidated. Bioinformatics analysis suggested that the gene HP0256, annotated as hypothetical, was a FliJ homologue. In Salmonella, FliJ is a chaperone escort protein for FlgN and FliT, two proteins that themselves display chaperone activity for components of the hook, the rod and the filament.

Results: Ablation of the HP0256 gene in H. pylori significantly reduced motility. However, flagellin and hook protein synthesis was not affected in the HP0256 mutant. Transmission electron transmission microscopy revealed that the HP0256 mutant cells displayed a normal flagellum configuration, suggesting that HP0256 was not essential for assembly and polar localisation of the flagella in the cell. Interestingly, whole genome microarrays of an HP0256 mutant revealed transcriptional changes in a number of genes associated with the flagellar regulon and the cell envelope, such as outer membrane proteins and adhesins. Consistent with the array data, lack of the HP0256 gene significantly reduced adhesion and the inflammatory response in host cells.

Conclusions: We conclude that HP0256 is not a functional counterpart of FliJ in H. pylori. However, it is required for full motility and it is involved, possibly indirectly, in expression of outer membrane proteins and adhesins involved in pathogenesis and adhesion.

Figure 1

Gene synteny of HP0256 is conserved in Helicobacter genus (Panel A). Most HP0256 homologs were found in epsilonproteobacteria (Panel B). Schematics were generated using STRING from EMBL (string.embl.de/).

Figure 2

Multiple sequence alignments of the H. pylori HP0256 sequences and orthologues. The alignment was created using the GENEDOC programme. Residues in colour are conserved in sequences. Sequence regions labelled abcdefg have a high likelihood of forming coiled-coil domains. ALME, gene encoding the flagellar export protein FliJ of Alkaliphilus metalliredigens; PECA, gene encoding a putative flagellar biosynthesis chaperone FliJ of Pelobacter carbinolicus; BASU, gene encoding a flagellar biosynthesis chaperone of Bacillus subtilis; CLDI, gene encoding a flagellar protein of Clostridium difficile; LAIN, gene encoding a flagellar biosynthesis chaperone of Lawsonia intracellularis; SATY, gene encoding a flagellar biosynthesis chaperone of Salmonella enterica subsp. enterica serovar; PSAT, gene encoding the flagellar export protein FliJ of Pseudoalteromonas atlantica; LEPN, gene encoding the flagellar protein FliJ of Legionella pneumophila; XACA, gene encoding the protein FliJ of Xanthomonas axonopodis; Cacu_0256, gene encoding a PP-loop family protein of Campylobacter curvus; Cafe_0256, gene encoding an hypothetical protein of Campylobacter fetus; Caje_0256, gene encoding an hypothetical protein of Campylobacter jejuni; Cala_0256, gene encoding an hypothetical protein of Campylobacter lari; Hepy_0256, gene encoding an hypothetical protein of Campylobacter jejuni; Hehe_0256, gene encoding an hypothetical protein of Helicobacter hepaticus; Wosu_0256, gene encoding an hypothetical protein of Wolinella succinogenes; Thde_0256, gene encoding a conserved hypothetical protein of Thiomicrospira denitrificans.

Figure 3

The ablation of the HP0256 gene impairs motility in H. pylori that may be restored by complementation, when hp0256 is put under the control of the promoter of flaA. Motility plate assay were performed four times. A. CCUG17874 wild-type strain; B. CCUG17874-hp0256KO; C. P79 wild-type strain; D. P79-hp0256KO; E. P79-hp0256KO complemented with pIR0601; F. P79-hp0256KO with empty vector (control).

Figure 4

Mutation of HP0256 does not affect flagellin and hook protein production. Flagellin and hook protein levels in the HP0256-KO mutant and the wild-type were analyzed by SDS-PAGE and immunoblotting. Two independent immunoblottings were performed. Panel A, Coomassie blue staining protein gel, Panel B, immunobloting, Lane 1, Protein marker; lane 2, CCUG17874 cytoplasmic fraction; lane 3, CCUG17874 cell envelope fraction; lane 4, cytoplasmic fraction of CCUG17874 derivative HP0256-KO mutant and lane 5, cell envelope fraction of CCUG17874 derivative HP0256-KO mutant.

Figure 5

An HP0256 mutant has a normally assembled flagellum filament. The arrows indicate the localisation of the flagella in the cell. The transmission electron microscopy was performed on 50 cells for each strain. Panel A, CCUG17874 wild-type; panel B, P79 wild-type; panel C, CCUG17874-hp0256KO and panel D, P79-hp0256KO.

Figure 6

Confirmation of transcriptional changes in selected flagellar genes in the HP0256 mutant using qRT-PCR. Fold changes and standard deviations were calculated using the era transcript abundance as reference. qRT-PCRs were performed on at least two biological replicates.

Figure 7

The HP0256 mutant has lower adhesion ability compared to the wild-type and significantly induces a weaker IL-8 secretion in AGS cells. Panel A shows that the HP0256 mutant adheres significantly less to the AGS host cells compared to the wild-type. Panel B shows that the HP0256 mutant induces a lower IL-8 secretion of AGS cells compared to the wild-type cells. (*) indicates results with a p-value of less than 0.05.