Helicobacter pylori AddAB helicase-nuclease and RecA promote recombination-related DNA repair and survival during stomach colonization

Abstract

Helicobacter pylori colonization of the human stomach is characterized by profound disease-causing inflammation. Bacterial proteins that detoxify reactive oxygen species or recognize damaged DNA adducts promote infection, suggesting that H. pylori requires DNA damage repair for successful in vivo colonization. The molecular mechanisms of repair remain unknown. We identified homologues of the AddAB class of helicase-nuclease enzymes, related to the Escherichia coli RecBCD enzyme, which, with RecA, is required for repair of DNA breaks and homologous recombination. H. pylori mutants lacking addA or addB genes lack detectable ATP-dependent nuclease activity, and the cloned H. pylori addAB genes restore both nuclease and helicase activities to an E. coli recBCD deletion mutant. H. pylori addAB and recA mutants have a reduced capacity for stomach colonization. These mutants are sensitive to DNA damaging agents and have reduced frequencies of apparent gene conversion between homologous genes encoding outer membrane proteins. Our results reveal requirements for double-strand break repair and recombination during both acute and chronic phases of H. pylori stomach infection.

Figure 1

Epsilon Proteobacteria AddA and AddB homologs with conserved nuclease domains. Nuclease domains of AddA (A) and AddB (B) proteins from epsilon Proteobacteria with fully sequenced genomes show highly conserved residues indicated by shading. Sites where mutations in B. subtilis addA and addB abolish nuclease activity (Kooistra et al., 1997) are indicated by an asterisk. Species names, and first and last residue numbers in the displayed alignment are shown on the left. Alignment to the nuclease domains of E. coli RecB and B. subtilis AddA (A) and E. coli RecB and B. subtilis AddB (B) are shown for comparison (bottom of each alignment). The corresponding locus tags and (GI numbers) for the epsilon Proteobacterial AddA proteins are (top to bottom) CCV52592_0910 (154174808), CFF8240_0386 (118474774), CHAB381_1391 (154148488), CJE1654 (57238504), JJD26997_1828 (153951355), CJJ81176_1474 (121612796), Cj1481c (15792796), CLA0743 (57240914), CUP1844 (57242577), Hac_0287 (109946901), HH1643 (32267142), HP1553 (15646160), HPAG1_1502 (108563927), jhp1446 (15612511), NIS_0366 (152990115), SUN_0187 (152991783), Tmden_0090 (78776291), WS1252 (34557616). The corresponding locus tags and GI numbers for the epsilon Proteobacterial AddB proteins are (top to bottom) Ccur5_02000280 (145956769), CFF8240_0385 (118475773), CHAB381_1392 (154149234), CJE1655 (57238505), Cjejd_02001773 (145960086), CJJ81176_1475 (121613506), Cj1482c (15792797), CLA0742 (57240913), CUP1843 (57242576), Hac_0793 (109947353), HH0025 (=(32265524), HP1089 (15645703), HPAG1_0358 (108562783), jhp0336 (15611404), NIS_0365 (152990114), SUN_0185 (152991781), Tmden_0088 (78776289), WS0562 (34556981).

Figure 2

AddAB promotes ATP-dependent DNA unwinding. Extracts were prepared from strain V3060 (ΔrecBCD2731 DE3) carrying vector pETDuet-1 with or without insertion of the H. pylori addA and addB genes or pBR322 with recBCD (pMR3). The indicated amount of extract protein was incubated with pBR322 linearized and 5′ end-labeled with ³²P. Reactions contained ATP (5 mM) as indicated. The positions of ds DNA substrate (ds), unwound ss DNA (ss) produced by boiling or enzymatic reaction, and degraded DNA (nuc) are shown.

Figure 3

AddAB confers modest protection against UV damage. The indicated bacterial strains (wild-type, mutant, and complemented mutant strains) were exposed to UV irradiation, and the fraction of bacteria surviving was determined. The data plotted represent averages of three separate experiments, and bars indicate 1 standard deviation.

Figure 4

AddAB and RecA promote stomach colonization. Mice were orally infected with a 1:1 mixture of the indicated strains. The actual ratio in the inoculum of mutant to wildtype or complemented mutants is indicated. After one week the bacteria colonizing the stomach were harvested and the competitive index determined (ratio of mutant to wild type in the output corrected for the input ratio) as described in the Experimental Procedures. Each data point is from one mouse, and the geometric means are indicated by horizontal bars. Infections with two independent ΔrecA::cat clones are indicated by open and filled circles. Bracketed data points represented an upper limit on the competitive index with 95% confidence because of failure to isolate mutant clones from the stomach as described in the Experimental Procedures. Competitions of mutant clones with wild type were repeated with similar results (data not shown). A colonization defect is indicated by a competitive index of less than one.

Figure 5

AddA and RecA promote babA to babB gene conversion. Quantitative PCR was used to determine the number of copies of babA and babB at the babA locus for genomic DNA prepared from each of three cultures started from individual colonies as described in the Experimental Procedures. The geometric mean of the frequency of babB/babA at the babA locus is reported and the bars indicate 1 standard deviation. The difference in means between groups was considered significant (one-way analysis of variance, p = 0.0002). Significant P values for pair-wise comparisons between groups are indicated **p <0.001, *p <0.01.