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Journal of Bacteriology, February 2006, p. 1295-1300, Vol. 188, No. 4
0021-9193/06/$08.00+0     doi:10.1128/JB.188.4.1295-1300.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

The Brucella abortus xthA-1 Gene Product Participates in Base Excision Repair and Resistance to Oxidative Killing but Is Not Required for Wild-Type Virulence in the Mouse Model

Michael L. Hornback and R. Martin Roop II^*

Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, North Carolina 27834

Received 30 September 2005/ Accepted 3 December 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Exonuclease III, encoded by the xthA gene, plays a central role in the base excision pathway of DNA repair in bacteria. Studies with Escherichia coli xthA mutants have also shown that exonuclease III participates in the repair of oxidative damage to DNA. An isogenic xthA-1 mutant (designated CAM220) derived from virulent Brucella abortus 2308 exhibited increased sensitivity to the alkylating agent methyl methanesulfonate (MMS) compared to the parent strain. In contrast, 2308 and the isogenic xthA-1 mutant displayed similar levels of resistance to the DNA cross-linker mitomycin C. These phenotypic properties are those that would be predicted for a strain defective in base excision repair. The B. abortus xthA-1 mutant also displayed reduced resistance to killing by H₂O₂ and the ONOO^–-generating compound 3-morpholinosydnonimine (SIN-1) compared to strain 2308, indicating that the xthA-1 gene product participates in protecting B. abortus 2308 from oxidative damage. Introducing a plasmid-borne copy of the parental xthA-1 gene into CAM220 restored wild-type resistance of this mutant to MMS, H₂O₂, and SIN-1. Although the B. abortus xthA-1 mutant exhibited increased sensitivity to oxidative killing compared to the parental strain in laboratory assays, CAM220 and 2308 displayed equivalent spleen colonization profiles in BALB/c mice through 8 weeks postinfection and equivalent intracellular survival and replication profiles in cultured murine macrophages. Thus, although the xthA-1 gene product participates in base excision repair and resistance to oxidative killing in B. abortus 2308, XthA-1 is not required for wild-type virulence of this strain in the mouse model.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Brucella abortus is a gram-negative bacterium that causes spontaneous abortions and infertility in cattle and bison (23). In humans, B. abortus infection results in a chronic, febrile illness known as undulant fever. The ability of this organism to cause disease in its natural bovine hosts and in humans is intimately linked with not only its capacity to resist killing by macrophages but also its ability to persist within these host cells (26). Previous studies have shown that oxidative killing plays a primary role in the brucellacidal activity of host macrophages (17). Consequently, gene products that help the brucellae avoid oxidative killing would be expected to assist these bacteria to establish and maintain residence in their intracellular niche.

Reactive oxygen intermediates (ROIs) such as superoxide (O₂^–), hydrogen peroxide (H₂O₂), and hydroxyl radicals (OH^.) are harmful to living cells because they damage DNA, proteins, and lipids (11). Bacterial cells generally have two lines of defense against damage by ROIs. The first line of defense includes enzymes such as catalase and superoxide dismutase that directly detoxify these compounds. The second line of defense comprises enzymes that repair oxidative damage to cellular components and those that degrade the oxidatively damaged components. Such enzymes include DNA repair enzymes and some of the stress response proteases (4, 7).

In Escherichia coli, base excision repair (BER) plays a major role in removing oxidative lesions from DNA (15). DNA glycosylases recognize the damaged nucleotide and remove the nitrogenous base leaving an apurinic/apyrimidinic (AP) site. AP endonucleases then cut the phosphate backbone 5' of the AP site, which produces a free 3' hydroxyl group on the neighboring nucleotide, allowing DNA polymerase I and ligase to repair the lesion. The E. coli xthA gene encodes exonuclease III (XthA), and this enzyme accounts for approximately 90% of the AP endonuclease activity in this bacterium (20). The remainder of the AP endonuclease activity in E. coli is carried out by endonuclease IV, which is encoded by the nfo gene (3). The importance of BER in the repair of oxidative damage to cellular DNA is reflected in the fact that E. coli xthA mutants and xthA nfo double mutants are hypersensitive to H₂O₂ in vitro (3, 6). Recently, it has also been demonstrated that BER plays an important role in protecting Salmonella enterica serovar Typhimurium from oxidative killing by murine macrophages (31).

Surveys of the genome sequences of Brucella melitensis 16M (5), Brucella suis 1330 (25), and B. abortus 9-941 (14) indicate that the Brucella spp. possess two xthA homologs but have no homolog of nfo. These genes are annotated as xthA-1 (BruAb1_0885) and xthA-2 (BruAb1_1979) in the B. abortus 9-941 genome sequence. The studies described in this report were performed to determine if the xthA-1 gene product participates in BER and resistance to oxidative killing in B. abortus 2308 and to evaluate the contribution of XthA-1 to virulence in the mouse model.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Construction of the B. abortus xthA-1 mutant. Oligonucleotide primers designed based upon the gene designated BMEI1093 in the B. melitensis 16M genome were used to amplify a DNA fragment encompassing 500 base pairs upstream and 100 bp downstream of the xthA-1 open reading frame from genomic DNA from B. abortus 2308 using PCR. The PCR product was cloned into pGEM-T Easy (Promega), and the kanamycin resistance gene (aph3) from TnphoA was inserted into a unique StyI site that resides approximately in the center of the xthA-1 gene (Fig. 1). The resulting plasmid was electroporated into B. abortus 2308 using previously described procedures (9), and transformants were selected on Schaedler agar supplemented with 5% defibrinated bovine blood (SBA) and 45 µg/µl kanamycin. A transformant designated CAM220 was selected for further characterization based on its resistance to kanamycin and sensitivity to ampicillin. The genotype of B. abortus CAM220 was confirmed by PCR analysis of genomic DNA from this mutant with xthA-1-, aph3-, and pGEM-specific primer sets.

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FIG. 1. Genetic organization of xthA-1 and the surrounding genes in B. abortus 2308. The designations applied to the corresponding genes in the B. abortus 9-941 genome sequence (e.g., BruAb1_0885) are provided for reference. The small arrows denote the approximate positions and directionality of the primers used to amplify the xthA-1 locus from the B. abortus 2308 genome. The StyI site in xthA-1 into which the aphA gene was inserted during the construction of the B. abortus xthA-1 mutant CAM220 is also shown. The scale bar provides a reference for the approximate sizes of xthA-1 and surrounding genes.

To facilitate genetic complementation studies, a PCR product encompassing 500 bp upstream and 100 bp downstream of the xthA-1 gene from B. abortus 2308 was cloned into the EcoRI/XbaI site of pDARA. pDARA is a derivative of pMR10 (NCBI accession number AJ606312) and was constructed by inserting the bla gene from pBluescript II KS(+) into the ClaI site within the aph3 gene of pMR10 (T. Brown, unpublished data). The resulting plasmid, pMHX2, was introduced into CAM220 by electrotransformation, and the derivative of the B. abortus xthA-1 mutant carrying pMHX2 was given the designation MLH220.

Resistance to the DNA-damaging agents MMS, mitomycin C, and bleomycin. B. abortus strains were grown on SBA for 48 h at 37°C with 5% CO₂ and resuspended in Gerhardt's minimal media (GMM) (13) to a cell density of 10⁹ CFU/ml (optical density of 0.15 at 600 nm). One hundred microliters of these cell suspensions was spread on individual tryptic soy agar (TSA) plates. A 7-mm Whatman no. 3 filter paper disk was placed in the center of each plate and impregnated with 10 µl of a 520-mg/ml solution of methyl methanesulfonate (MMS) in dimethylformamide, a 100-µg/ml solution of mitomycin C, or a 20-mg/ml solution of bleomycin. After 48 h of incubation of the plates at 37°C with 5% CO₂, the zones of growth inhibition around each of the paper disks (in mm) were measured.

Resistance to hydrogen peroxide and peroxynitrite. B. abortus strains were grown on SBA for 48 h at 37°C with 5% CO₂, the cells harvested in Gerhardt's minimal media, and the cell suspensions adjusted to a concentration of 5 x 10⁵ CFU/ml (low cell density) or 1 x 10⁹ CFU/ml (high cell density). One-milliliter portions of the bacterial cell suspensions were dispensed into 17- by 100-mm culture tubes and exposed to H₂O₂ at final concentrations of 1, 2.5, and 4 mM. After exposure for 1 h in a 37°C shaking incubator, the cell suspensions were serially diluted in phosphate-buffered saline and plated onto TSA supplemented with 1,400 U/ml of bovine catalase. These plates were incubated for 48 to 72 h at 37°C with 5% CO₂, and the CFU were enumerated.

Low-cell-density B. abortus cell suspensions (e.g., 5 x 10⁵ CFU/ml) prepared as described above were exposed to 25 mM of the peroxynitrite (ONOO^–) generator 3-morpholinosydnonimine (SIN-1) (30). Bovine catalase at a final concentration of 1,900 U/ml was also added to these cell suspensions at the time of addition of the SIN-1 to detoxify any H₂O₂ produced as a side-product of the reaction. Following a 1-h incubation with the SIN-1 at 37°C, the cell suspensions were serially diluted in phosphate-buffered saline and plated onto TSA plates supplemented with 1,400 U/ml bovine catalase, the plates were incubated for 48 to 72 h at 37°C with 5% CO₂, and the number of CFU was determined.

Survival and replication of the Brucella strains in cultured murine macrophages. Previously described procedures (28) were used to obtain proteose peptone-elicited macrophages from the peritoneal cavities of C57BL/6 mice. Cultured macrophages were infected with B. abortus 2308 or CAM220 opsonized with a subagglutinating concentration of Brucella-specific hyperimmune serum at a multiplicity of infection of 100:1, and the number of surviving intracellular brucellae was determined at 2, 24, and 48 h postinfection as described by Gee et al. (12).

Virulence of the Brucella strains in mice. Four-week-old, female C57BL/6 mice were infected with 5 x 10⁴ CFU of B. abortus 2308 or CAM220 via the intraperitoneal route, and the numbers of brucellae present in the spleens of mice at 1, 4, and 8 weeks postinfection were determined using previously described procedures (12).

Statistical analysis. All statistical analyses were performed using the Student two-tailed t test (27). P values 0.05 were considered significant.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The product of the gene (BruAb1_0885) annotated as xthA-1 in the B. abortus 9-941 genome sequence is predicted to be a protein of 260 amino acids that shares 36% amino acid identity with exonuclease III (XthA) of E. coli. More importantly, the putative Brucella XthA-1 protein possesses conserved amino acid domains that are believed to be important for the AP endonuclease activity of the E. coli XthA (Fig. 2). Considering the critical role of XthA in BER in E. coli, the resistance of the B. abortus xthA-1 mutant CAM220 to killing by MMS was compared to the parental 2308 strain. MMS alkylates single nucleotides, and BER plays a key role in the elimination of these types of lesions from cellular DNA in E. coli (18, 20). As shown in Fig. 3A, the xthA-1 mutant was significantly more sensitive to killing by MMS in this assay than 2308. MLH220, a derivative of the B. abortus xthA-1 mutant which carries a cloned copy of the wild-type xthA-1 gene on a pMR10-based plasmid, on the other hand, displayed the same level of resistance to MMS in this assay as the parental 2308 strain.

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FIG. 2. Conservation between the amino acid domains of the E. coli XthA that are believed to be required for AP endonuclease activity and the corresponding regions of the Brucella XthA-1. The amino acid residues in boldface are those that are believed to be associated with the AP endonuclease function of the E. coli XthA based on biophysical studies (24). Biochemical studies have determined that the tyrosine residue at position 215 (*) is necessary for AP endonuclease activity of E. coli XthA (29). The underlined residues are those conserved between the E. coli XthA and Brucella XthA-1 proteins.

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FIG. 3. Sensitivity of B. abortus 2308, CAM220 (2308 xthA-1) (xthA-1-), and MLH220 (CAM220 pMHX2) (xthA-1+) to MMS (A), and sensitivity of B. abortus 2308, CAM220 (2308 xthA-1), MEK5 (2308 uvrA) (uvrA-), and MEK12 (2308 recA) (recA-) to mitomycin C (B) in disk sensitivity assays. Significance values for comparisons of CAM220, MLH220, MEK5, and MEK12 versus 2308: *, P < 0.05; **, P < 0.01.

To determine if the increased sensitivity to DNA damage displayed by the B. abortus xthA-1 mutant is specific for the type of alkylation damage induced by MMS, the mutant and parental strains were also examined for their resistance to killing by mitomycin C. Mitomycin C produces intra- and interstrand cross-linking between residues in DNA, resulting in the production of "bulky lesions" (32). Because of their size, bulky lesions are repaired by nucleotide excision repair and recombinational repair pathways rather than BER in bacteria (16). As expected, the B. abortus uvrA mutant MEK5 (which is defective in nucleotide excision repair) and the isogenic recA mutant MEK12 (defective in recombinational repair) (M. E. Kovach and R. M. Roop II, Proc. 77th Annu. Conf. Res. Work. Anim. Dis., abstr. 2, 1996) displayed increased sensitivity to mitomycin C in this assay compared to the parental 2308 strain (Fig. 3B). The B. abortus xthA-1 mutant CAM220, on the other hand, did not. These experimental findings suggest that the B. abortus XthA-1 acts in a manner similar to its E. coli counterpart as an authentic AP endonuclease that participates in base excision repair.

As noted previously, base excision repair plays an important role in protecting bacterial cells from oxidative damage to their DNA and E. coli xthA mutants are hypersensitive to H₂O₂ in laboratory assays (6). To determine if the xthA-1 gene product plays a role in oxidative defense in B. abortus, 2308 and the isogenic xthA-1 mutant were exposed to H₂O₂ at final concentrations of 1, 2.5, and 4 mM. The B. abortus xthA-1 mutant was much more susceptible to killing by H₂O₂ under these conditions than the parental strain, and introduction of a cloned copy of the Brucella xthA-1 into the xthA-1 mutant restored the resistance of this strain to H₂O₂ to the same level as that displayed by the parental 2308 strain (Fig. 4A). Because H₂O₂ is an uncharged molecule and biological membranes do not present a barrier to its diffusion, bacterial cell density has a dramatic effect on the capacity of various cellular defense mechanisms to protect these organisms from damage mediated by H₂O₂. Catalase, for example, is very effective at protecting bacterial cells against exposure to H₂O₂ when the cell density is high, but not when it is low (21). In contrast, DNA repair mechanisms such as base excision repair play critical roles in protecting bacteria from H₂O₂-mediated damage when cell densities are low (1). To determine if bacterial cell density influences the resistance of the B. abortus xthA-1 mutant to H₂O₂, the same experiment was repeated with bacterial cell suspensions at a density of 10⁹ CFU/ml. As shown in Fig. 4B, at the higher cell density B. abortus 2308 and the isogenic xthA-1 mutant exhibited equivalent levels of resistance to H₂O₂ when challenged with 1, 2.5, and 5 mM of hydrogen peroxide. Thus, the B. abortus xthA-1 mutant displays the same sensitivity to oxidative killing at low cell density that has been described as being characteristic of DNA repair mutants of S. enterica serovar Typhimurium (1).

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FIG. 4. Susceptibility of B. abortus 2308 (black bars), CAM220 (2308 xthA-1) (white bars), and MLH220 (CAM220 pMHX2) (hatched bars) cell suspensions at (A) low cell density (5 x 105 CFU/ml) and (B) high cell density (109 CFU/ml) to killing by H2O2. Significance value for comparisons of CAM220 and MLH220 versus 2308: **, P < 0.01.

Exposure of DNA to peroxynitrite (ONOO^–) in vitro produces lesions that serve as substrates for formamidopyrimidine glycosylase (Fpg), which initiates BER (10). Additionally, peroxynitrite has been proposed to be an important component of the oxidative killing pathway of host phagocytes (33). To determine if the Brucella XthA-1 participates in resistance to peroxynitrite, B. abortus 2308, the xthA-1 mutant, and a derivative of the xthA-1 mutant carrying a plasmid-borne copy of the wild-type xthA-1 were exposed to the ONOO^– generator SIN-1 (30). Exogenous catalase was included in this assay to prevent toxicity from H₂O₂ generated as a by-product of the auto-oxidation of SIN-1. Figure 5 shows that the B. abortus xthA-1 mutant was more sensitive to exposure to SIN-1 than the parental 2308 strain and that wild-type resistance was restored to this mutant when xthA-1 was supplied in trans on a low-copy-number plasmid. These results suggest that the B. abortus xthA-1 gene product is involved with resistance to peroxynitrite-mediated killing.

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FIG. 5. Susceptibility of B. abortus 2308, CAM220 (2308 xthA-1) (xthA-1-), and MLH220 (CAM220 pMHX2) (xthA-1+) to killing by SIN-1. Black bars represent no SIN-1 treatment, and white bars represent treatment with 25 mM SIN-1. Significance value for comparison of CAM220 and MLH220 versus 2308: *, P < 0.05.

Bleomycin is an antineoplastic agent that binds DNA and ferrous iron, and its toxicity is based on its capacity to generate OH^., which causes localized oxidative damage to the DNA molecule (2). Biochemical studies have shown that the E. coli XthA can repair DNA lesions induced by bleomycin in vitro (19). To determine if the B. abortus xthA-1 mutant exhibits an increased sensitivity to bleomycin compared to the parent strain, a disk inhibition assay was performed. As shown in Fig. 6, the B. abortus xthA-1 mutant displayed significantly increased sensitivity to bleomycin compared to the parental 2308 strain in this assay, and this increased sensitivity to bleomycin was relieved by the introduction of a plasmid-borne copy of xthA-1 into the xthA-1 mutant. These experimental findings suggest that the B. abortus xthA-1 gene product plays a role similar to that of its E. coli counterpart in repairing the oxidative lesions in DNA generated by exposure to bleomycin. Moreover, they provide further evidence for the participation of XthA-1 in BER in B. abortus 2308.

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FIG. 6. Sensitivity of B. abortus 2308, CAM220 (2308 xthA-1) (xthA-1-), and MLH220 (CAM220 pMHX2) (xthA-1+) to bleomycin in a disk sensitivity assay. Significance value for comparisons of CAM220 and MLH220 versus 2308; *, P < 0.05.

Experimental evidence suggests that DNA repair mechanisms play an important role in allowing bacterial pathogens to resist oxidative killing in host macrophages (1). More importantly, Salmonella enterica serovar Typhimurium mutants defective in BER display attenuation in infected primary murine macrophages and in experimentally infected mice (31). Consequently, to determine if the increased susceptibility to oxidative killing displayed by the B. abortus xthA-1 mutant in laboratory assays translated into attenuation in an experimental host, the virulence of this mutant in cultured murine macrophages and experimentally infected C57BL/6 mice was examined. As shown in Fig. 7, the B. abortus xthA-1 mutant and the virulent parental strain 2308 displayed equivalent survival and replication profiles in cultured murine macrophages and spleen colonization profiles in experimentally infected C57BL/6 mice. These experimental findings indicate that the xthA-1 gene product is not required for the wild-type virulence of B. abortus 2308 in the mouse model.

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FIG. 7. (A) Intracellular survival and replication of B. abortus 2308 and CAM220 (2308 xthA-1) (xthA-1-) in cultured murine macrophages and (B) spleen colonization profiles of 2308 and CAM220 in experimentally infected C57BL/6 mice.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The genetic evidence obtained in this study suggests that the product of the xthA-1 gene is an authentic AP endonuclease that participates in BER in B. abortus 2308. Moreover, like its E. coli counterpart, XthA-1 appears to play an important role in protecting this bacterium from oxidative damage to its DNA. The capacity of the brucellae to resist the oxidative killing pathways of host macrophages has been proposed to be of critical importance in their ability to establish and maintain chronic infections in their mammalian hosts (17). Although in laboratory assays the B. abortus xthA-1 mutant exhibits increased susceptibility to ROIs and ROI/reactive nitrogen intermediate hybrids of the type that the brucellae would be expected to encounter during their interactions with host phagocytes, the phenotype displayed by the xthA-1 mutant in the mouse model indicates that the corresponding gene product is not required for wild-type virulence.

Exonuclease III (XthA) and endonuclease IV (Nfo) are the AP endonucleases that carry out the second step in BER in Escherichia coli and Salmonella (3, 20, 31). Although genetic and biochemical studies indicate that XthA is responsible for 90% of the AP endonuclease activity in these bacteria, experimental evidence suggests that Nfo can compensate for the loss of XthA in BER in both E. coli and Salmonella. More importantly from the perspective of the results presented here, significant attenuation of Salmonella enterica serovar Typhimurium in the mouse model appears to require mutation of both the xthA and nfo genes (31). A survey of the Brucella genome sequences available (5, 14, 25) indicates that these bacteria do not possess an Nfo homolog, but they do have a second XthA. The xthA-2 gene is predicted to encode a 268-amino-acid protein sharing 30% amino acid identity with the E. coli XthA. Moreover, the putative Brucella XthA-2 also possesses all of the conserved amino acid motifs shown in Fig. 2 that are believed to be important for the AP endonuclease activity of the E. coli XthA protein. This raises the question of whether or not XthA-2 works together with XthA-1 to provide the AP endonuclease activity required for efficient BER in the Brucella spp. If this is the case, then residual AP endonuclease activity provided by XthA-2 in the B. abortus xthA-1 mutant CAM220 may be sufficient to protect the DNA of this strain from oxidative damage as the result of exposure to the NADPH oxidase and inducible nitric oxide synthase activity of host phagocytes. This in turn, could explain why the B. abortus xthA-1 mutant exhibits no attenuation in mice or cultured murine macrophages. Another possibility is that other cellular defenses against ROI-mediated damage are sufficient to protect the B. abortus xthA-1 mutant from the oxidative stress encountered during its interaction with host phagocytes.

The most straightforward approach to evaluating the possibility that XthA-1 and XthA-2 perform overlapping function in BER in B. abortus 2308 would be to compare the phenotypic characteristics of B. abortus 2308, isogenic xthA-1 and xthA-2 mutants, and an xthA-1 xthA-2 double mutant in relevant laboratory assays. A comparison of these strains in mice and cultured murine macrophages would also address the possibility that residual AP endonuclease activity provided by XthA-2 underlies the lack of attenuation of the xthA-1 mutant. However, repeated attempts to construct an isogenic xthA-2 mutant from B. abortus 2308 in our laboratory have been unsuccessful. The genetic locus within which the Brucella xthA-2 gene resides has a complex organization. Reverse transcription-PCR analysis of total RNA from B. abortus 2308 indicates that xthA-2 is the second gene in an operon and is cotranscribed with a homolog of the lolA gene of E. coli (M. Hornback, unpublished). The latter gene encodes a chaperone that is involved with transport of lipoproteins from the cytoplasmic membrane to the outer membrane, and lolA is essential in E. coli (22). The 3' end of the Brucella xthA-2 is also only 6 bp removed from the 3' end of another gene on the opposite DNA strand that is predicted to encode the sensor kinase of a two-component regulator. Whether or not the problems associated with the construction of a B. abortus xthA-2 mutant result from disruption of the normal expression patterns of one of the genes adjacent to xthA-2 or from the fact that xthA-2 represents an essential gene in Brucella remains to be experimentally determined. Should xthA-2 turn out to be an essential gene, then alternative strategies will be required to determine if the corresponding gene product is an authentic AP endonuclease that participates in BER in B. abortus 2308 and to what extent XthA-1 and XthA-2 perform overlapping functions. Such approaches might include genetic complementation of well-characterized E. coli or Salmonella xthA nfo mutants (3, 31), biochemical characterization of purified Brucella XthA1 and XthA2 proteins, and/or the use of an antisense-based gene silencing strategy (8) to attenuate xthA2 expression in B. abortus 2308.

 
This work was supported by a grant from the National Institutes of Health (AI-48499).

We thank Michael Kovach for providing the B. abortus recA and uvrA mutants utilized in this study.

 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, East Carolina University School of Medicine, 600 Moye Boulevard, Greenville, NC 27834. Phone: (252) 744-1357. Fax: (252) 744-3535. E-mail: roopr{at}ecu.edu.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Bacteriology, February 2006, p. 1295-1300, Vol. 188, No. 4
0021-9193/06/$08.00+0     doi:10.1128/JB.188.4.1295-1300.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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