Dual nuclease and helicase activities of Helicobacter pylori AddAB are required for DNA repair, recombination, and mouse infectivity

Abstract

Helicobacter pylori infection of the human stomach is associated with disease-causing inflammation that elicits DNA damage in both bacterial and host cells. Bacteria must repair their DNA to persist. The H. pylori AddAB helicase-exonuclease is required for DNA repair and efficient stomach colonization. To dissect the role of each activity in DNA repair and infectivity, we altered the AddA and AddB nuclease (NUC) domains and the AddA helicase (HEL) domain by site-directed mutagenesis. Extracts of Escherichia coli expressing H. pylori addA(NUC)B or addAB(NUC) mutants unwound DNA but had approximately half of the exonuclease activity of wild-type AddAB; the addA(NUC)B(NUC) double mutant lacked detectable nuclease activity but retained helicase activity. Extracts with AddA(HEL)B lacked detectable helicase and nuclease activity. H. pylori with the single nuclease domain mutations were somewhat less sensitive to the DNA-damaging agent ciprofloxacin than the corresponding deletion mutant, suggesting that residual nuclease activity promotes limited DNA repair. The addA(NUC) and addA(HEL) mutants colonized the stomach less efficiently than the wild type; addB(NUC) showed partial attenuation. E. coli DeltarecBCD expressing H. pylori addAB was recombination-deficient unless H. pylori recA was also expressed, suggesting a species-specific interaction between AddAB and RecA and also that H. pylori AddAB participates in both DNA repair and recombination. These results support a role for both the AddAB nuclease and helicase in DNA repair and promoting infectivity.

FIGURE 1.

The AddA^HEL alteration eliminates DNA unwinding activity, but the AddA^NUC and AddB^NUC alterations do not. Extracts were prepared from E. coli strain V3060 (ΔrecBCD2731 DE3) carrying derivatives of pSA405 expressing H. pylori AddAB proteins as indicated. The indicated amounts of protein were reacted at 37° for 2 min with ³²P 5′-end labeled linearized pBR322 in reaction buffer containing 5 mm ATP and 2 mm Mg²⁺. The products of the reaction were separated by agarose gel electrophoresis and autoradiographed. The positions of the dsDNA substrate (ds) and unwound ssDNA (ss) are indicated.

FIGURE 2.

Both AddAB nuclease and helicase activities are required for H. pylori wild-type resistance to ciprofloxacin. The minimal inhibitory concentration of ciprofloxacin for each strain was determined using E-test strips; the mean ± S.E. is indicated (n = 3–5). Strains are derivatives of H. pylori strain NSH57. The wild-type or mutant NUC or HEL addAB allele was integrated at the rdx locus, and the corresponding endogenous locus was deleted.

FIGURE 3.

H. pylori AddAB and RecA restore partial UV resistance to an E. coli ΔrecBCD strain. Strains are transformants of E. coli strain V3060 with the indicated proteins expressed from plasmids. Cultures were grown to mid-log phase at 37 °C in LB with appropriate antibiotics, harvested by centrifugation, resuspended in 10 mm MgSO₄, exposed to UV light, and plated on LB agar plates containing appropriate antibiotics. Survival is the fraction of initial colony-forming units surviving after exposure to the indicated fluence of UV light. The data shown are representative of three independent experiments.

FIGURE 4.

H. pylori AddAB and RecA are required to restore partial ciprofloxacin resistance to an E. coli ΔrecBCD strain. Strains are transformants of E. coli strain V3060 with the indicated alleles on plasmids. Cultures were grown to mid-log phase at 37 °C in LB with appropriate antibiotics and spread on LB plates. The minimal inhibitory concentration (mean ± S.E.; n = 3) of ciprofloxacin as determined with E-test strips is indicated.

FIGURE 5.

AddAB nuclease and helicase activities are required for wild-type H. pylori stomach colonization. Mice were orally infected with a 1:1 mixture of the indicated H. pylori strains. After 1 week the bacteria colonizing the stomach were harvested and the competitive index determined (ratio of nuclease or helicase mutant to deletion or wild type (WT) in the output corrected for the input ratio). Each datum is the CI from one mouse. Open symbols indicate mice from which only wild-type or deletion mutant bacteria were recovered and represent an upper limit on the competitive index with 95% confidence (Poisson distribution). Similarly, black symbols indicate animals in which only the nuclease or helicase mutant was recovered and represent a lower limit on the competitive index with 95% confidence. Gray symbols indicate animals where both strains in the inoculum were recovered at 1 week. The results of infections with two different addA^HEL clones are indicated by circles and squares. CI < 1 indicates a colonization defect, and CI > 1 indicates out-competition.