Development of a PCR-restriction fragment length polymorphism assay using the nucleotide sequence of the Helicobacter hepaticus urease structural genes ureAB

Abstract

Infection with Helicobacter hepaticus causes chronic active hepatitis in certain strains of mice and is associated with hepatocellular carcinoma in A/JCr mice. Like the gastric helicobacters, H. pylori and H. mustelae, H. hepaticus possesses a high level of urease activity. However, the H. hepaticus urease structural gene sequences have not been previously determined, and the role of the urease enzyme in colonization and in pathogenesis is not known. PCR was used to amplify a portion of the urease structural genes from H. hepaticus genomic DNA. Amplified DNA fragments were cloned, and the nucleotide sequence was determined. The deduced amino acid sequence of the partial H. hepaticus ureA gene product was found to exhibit 60% identity and 75% similarity to the predicted H. pylori UreA. The deduced amino acid sequence of a partial H. hepaticus ureB gene product exhibited 75% identity and 87% similarity to the predicted H. pylori UreB. Diversity among H. hepaticus isolates was evaluated by means of a restriction fragment length polymorphism (RFLP) assay. The 1.6-kb fragments within the ureAB open reading frames, amplified from 11 independent isolates, were digested with the restriction endonuclease HhaI. Three distinct RFLP patterns were observed. Identical RFLP profiles were noted in sequential isolates of one strain of H. hepaticus during an 18 month in vivo colonization study, suggesting that the urease genes of H. hepaticus are stable. The urease genes among H. hepaticus strains were also well conserved, showing 98.8 to 99% nucleotide sequence identity among three isolates analyzed. These findings indicate that H. hepaticus has urease structural genes which are homologous to those of the gastric Helicobacter species and that these gene sequences can be used in a PCR and RFLP assay for diagnosis of this important murine pathogen.

FIG. 1

PCR-amplified products of ureAB genes from H. pylori (H.p) (open bars) and H. hepaticus (shaded bars).

FIG. 2

Predicted amino acid sequences of UreA and UreB from H. hepaticus aligned with the corresponding predicted sequences from H. felis, H. pylori, H. heilmannii, and H. mustelae. ., sequence identity; ∗, sequence difference.

FIG. 3

Restriction digest patterns of the amplified urease gene on a 1.6-kb PCR product from 11 H. hepaticus isolates. Amplified DNA was digested with HhaI and separated by electrophoresis on a 6% Visigel. Shown are a 100-bp DNA ladder (lane 1), pattern A (lanes 2 to 4, 6 to 9, and 11), pattern B (lanes 5 and 12), and pattern C (lane 10).

FIG. 4

(A) Restriction digest patterns of amplified urease genes of H. hepaticus isolates cultured from mice during a long-term colonization study. H. hepaticus was isolated from the mouse cecum 3 (lane 1), 6 (lane 2), 9 (lane 3), 12 (lane 4), 15 (lane 5), and 18 months (lane 6) postinoculation. Lane 7, H. hepaticus strain used for dosing; lane 8, 100-bp DNA ladder. (B) Restriction digest patterns of amplified urease genes of H. hepaticus isolated from different strains of mice housed within the same facility. Lane 1, DBA/2J; lane 2, B6D2F₁/D; lane 3, BALB/J; lane 4, B₆SJLF₁; lane 5, BALB/cByj-nu; lane 6, BALB/cBj; lane 7, C57BL/6c-nu; lane 8, 100-bp DNA ladder.

FIG. 5

Sequence comparison of the H. hepaticus urease genes of different strains. A dash indicates sequence identity; sequence differences are illustrated in boldface type. H.H-T, H. hepaticus type strain (ATCC 51449).

FIG. 6

Results of PCR with urease gene primers specific for H. hepaticus. (A) Lanes 1 to 11, 11 strains of H. hepaticus from 11 sources; lane 12, 1-kb DNA ladder. (B) Lane 1, H. pylori; lane 2, H. bilis; lane 3, H. felis; lane 4, H. muridarum; lane 5, Flexispira rappini; lane 6, H. trogontum; lane 7, H. rodentium; lane 8, H. mustelae; lane 9, H. hepaticus; lane 10, reagent control; lane 11, 1-kb DNA ladder.