Article Figures & Data
Figures
- FIG. 1.
The numbers of H. pylori recovered from mice infected with mutants lacking tlpA or tlpC do not differ from those from mice infected with the wild type. Each point represents the bacterial numbers per gram of stomach in mice infected for 2 weeks with SS1 ΔtlpC::cat-1 mutant (left), wild-type SS1 (middle), or SS1 ΔtlpA::cat-1 mutant (right). Mice infected by wild-type SS1 were given 1 × 108 CFU (n = 4), mice receiving SS1 ΔtlpA::cat (n = 9) were inoculated with 4 × 107 to 13 × 107 CFU, and mice receiving SS1 ΔtlpC::cat (n = 6) were given 1 × 107 to 6 × 107 CFU. Two different isolates of ΔtlpA::cat-1 were used but their colonization levels did not differ, so their data are shown together in one column. The short horizontal lines represent the geometric mean CFUs per gram. The levels of colonization do not differ significantly from each other (P > 0.05).
- FIG. 2.
Number of bacteria in the stomachs of mice infected with mixtures of wild-type and mutant H. pylori after 2 weeks. Each point represents one mouse. The competitive index (CI) is the mutant/wild-type ratio for output divided by the same ratio for input. The short horizontal lines represent the geometric means. The number of mice (n) used follows: for ΔtlpA::cat mutant (left), n = 10; for ΔtlpC::cat mutant (middle), n = 8; and for cheY::aphA3 mutant (right), n = 4. We were unable to detect any cheY mutants in the output and used 250 CFU/g (the estimated detection limit) for these calculations. Statistical analysis using the Wilcoxon matched-pair signed-rank test (available at http://www.fon.hum.uva.nl/Service/Statistics/Signed_Rank_Test.html ) showed that all strains are significantly different from a strain with no competition defect (CI = 1) (P < 0.01). On the basis of observations with other mutants and wild-type strains, two strains with equal abilities yield a CI of 1 (data not shown).
Tables
- TABLE 1.
Strains and plasmids used in this study
Strain or plasmid Genotype or description Antibiotic resistance Source or reference E. coli DH10B Cloning strain Lab stock H. pylori G27 Wild type, type I N. Salama (6) SS1 Wild type, type I J. O'Rourke (19) SPM342F Wild type, type I N. Salama (21) TAG101 G27 ΔtlpA::cat-1 Cmr This study TAG102 G27 ΔtlpA::cat-2 Cmr This study TAS101 SS1 ΔtlpA::cat-1 Cmr This study TCG568 G27 ΔtlpC::cat-1 Cmr This study TCS565 SS1 ΔtlpC::cat-1 Cmr This study CYG487 G27 ΔcheY::aphA3-1 Kmr This study CYS481 SS1 ΔcheY::aphA3-1 Kmr This study Plasmids pBluescript Cloning vector Apr Stratagene pBS-cat pBluescript::cat (from Campylobacter coli) Apr Cmr N. Salama pBS-kan pBluescript::aphA3 (from C. coli) Apr Kmr N. Salama pKO112 pBluescript with tlpA (SPM342F) Apr This study pTA101 pKO112 ΔtlpA::cat-1 Apr Cmr This study pTA102 pKO112 ΔtlpA::cat-2 Apr Cmr This study pTC100 pBluescript with tlpC (SS1) Apr This study pTC200 pTC100 ΔtlpC::cat-1 Apr Cmr This study pKO126 pBluescript with cheY (SS1) Apr This study pKO126K pKO126 cheY::aphA3-1 Apr Kmr This study - TABLE 2.
PCR primers used in this study
Primer Sequence Colicat1 5′-GTATAGTCTGCTGTAAACTCAGTCC-3′ tlpA1 5′-TGAAAGATCTGCCTTTTGGGGCGTTG-3′ tlpA3 5′-CCGCAAGCTTGAAACTGCTTTTTATTCACATC-3′ tlpA4 5′-GCTGTTTAAGGACACCCC-3′ tlpA5 5′-TTAGCGAACGATAGCGCG-3′ tlpC-4 5′-ACCCCCAACTAACTCCCCTTAAG-3′ tlpC-5 5′-CAGAGCTTGAATCAATGGTTGGG-3′ pTC2-1 5′-CAAGAAAGGAGTCTGAAAAC-3′ pTC2-2 5′-TTGATTCTTACCCATTAGGGG-3′ cheY6 5′-ATCGCACAAGATAGAAACGG-3′ cheY7 5′-GCTGTTCTAAAACCTCAAATCCATT-3′ - TABLE 3.
In vitro motility of H. pylori tlpA, tlpC, and cheY mutantsa
Strain Swim rate (mm/day) SS1 4.5 ± 0.3 SS1 ΔtlpA::cat-2 4.1 ± 0.8 SS1 ΔtlpC::cat-1 4.1 ± 0.1 SS1 cheY::aphA3-1 0.4 ± 0.1 G27 5.2 ± 0.3 G27 ΔtlpA::cat-1 5.1 ± 0.9 G27 ΔtlpA::cat-2 5.0 ± 0.7 G27 ΔtlpC::cat-1 4.7 ± 0.4 G27 cheY::aphA3-1 0.75 ± 0.15 ↵ a Colonial expansion (swim) rates were determined by measuring the colony diameter each day for 7 days of cells grown in brucella broth, 2.5 to 5% fetal bovine serum, and 0.35% agar.
