Molecular cloning and characterization of the Helicobacter pylori fliD gene, an essential factor in flagellar structure and motility

Abstract

Helicobacter pylori colonizes the human stomach and can cause gastroduodenal disease. Flagellar motility is regarded as a major factor in the colonizing ability of H. pylori. The functional roles of flagellar structural proteins other than FlaA, FlaB, and FlgE are not well understood. The fliD operon of H. pylori consists of flaG, fliD, and fliS genes, in the order stated, under the control of a sigma(28)-dependent promoter. In an effort to elucidate the function of the FliD protein, a hook-associated protein 2 homologue, in flagellar morphogenesis and motility, the fliD gene (2,058 bp) was cloned and isogenic mutants were constructed by disruption of the fliD gene with a kanamycin resistance cassette and electroporation-mediated allelic-exchange mutagenesis. In the fliD mutant, morphologically abnormal flagellar appendages in which very little filament elongation was apparent were observed. The fliD mutant strain was completely nonmotile, indicating that these abnormal flagella were functionally defective. Furthermore, the isogenic fliD mutant of H. pylori SS1, a mouse-adapted strain, was not able to colonize the gastric mucosae of host mice. These results suggest that H. pylori FliD is an essential element in the assembly of the functional flagella that are required for colonization of the gastric mucosa.

FIG. 1

Restriction maps of the recombinant plasmids pFLID and pFLIDKm-1. The locations of three ORFs (flaG, fliD, and fliS genes) are indicated by arrows. pFLIDKm-1 was constructed by inserting a kanamycin resistance cassette into the NsiI restriction site of pFLID. Only the inserts of the plasmids are depicted.

FIG. 2

Characterization of H. pylori KCTC0217BP wild type and isogenic fliD mutant by PCR (A) and Western blotting (B). (A) Lanes 1 and 2, DNA amplified by PCR with primers (fliDseq#2 and fliDseq#5) annealing up- and downstream of the kanamycin cassette insertion site in wild-type and mutant strains, respectively; lanes 3 and 4, DNA amplified by PCR with primers (fliDseq#4 and fliD/CBamHI) annealing downstream of the insertion site in wild-type and mutant strains, respectively; lane M, DNA size markers (in kilobases). (B) Western blotting of whole-cell lysates of H. pylori KCTC0217BP wild type (lane 1) and isogenic fliD mutant (lane 2). The blot was developed with anti-recombinant FliD antibody. Molecular mass markers (in kilodaltons) are shown on the left. Expression of the 76-kDa FliD protein was observed only in the wild-type strain.

FIG. 3

Electron microscopy of H. pylori KCTC0217BP wild type and isogenic fliD mutant, negatively stained with potassium phosphotungstate (pH 6.8). In wild-type cells, full-length sheathed flagella are observed (A). Within the wild-type flagellar sheath, filaments are elongated to the tips of the flagella and terminal bulbs are observed (B). In the fliD mutant, truncated flagella, variable in number and size, are observed (C), and terminal bulbs are not apparent. Filament elongation is not observed in the fliD mutant even under higher magnification (compare panels B and D). Typical sheathed flagella with truncated filaments are occasionally seen in the fliD mutant strains (E). The end points of the truncated filaments are indicated by arrows (F). Bars, 500 nm.

FIG. 4

Flagellin gene expression in wild-type and fliD mutant H. pylori strains. (A) Equal amounts of whole-cell lysates (20 μg of total protein) from wild-type (lane W) and fliD mutant (lane M) H. pylori KCTC0217BP were separated by SDS-PAGE. Two protein bands of around 60 kDa, showing different levels of expression, are indicated by arrowheads. The same gel was also analyzed by Western blotting with polyclonal antibody anti-Fla. Note the significant reduction of FlaA in the fliD mutant. The positions of the hook (FlgE) and major (FlaA) and minor (FlaB) flagellin proteins are indicated on the right. (B) Northern blotting demonstrated that flaA RNA levels were also reduced in the fliD mutant.

FIG. 5

Motility testing of H. pylori KCTC0217BP wild-type and isogenic fliD mutant strains. (A and B) Single-colony motilities of wild-type and fliD mutant strains, respectively. The wild-type strain formed diffuse colonies with large swarming halos, whereas the fliD mutant formed dense colonies. (C) Stab agar test of wild type (left) and fliD mutant (right).