DNA Alkylation Damage as a Sensor of Nitrosative Stress in Mycobacterium tuberculosis

Abstract

One of the cellular consequences of nitrosative stress is alkylation damage to DNA. To assess whether nitrosative stress is registered on the genome of Mycobacterium tuberculosis, mutants lacking an alkylation damage repair and reversal operon were constructed. Although hypersensitive to the genotoxic effects of N-methyl-N′-nitro-N-nitrosoguanidine in vitro, the mutants displayed no phenotype in vivo, suggesting that permeation of nitrosative stress to the level of cytotoxic DNA damage is restricted.

During the course of an infection, Mycobacterium tuberculosis is expected to sustain significant levels of nitrosative stress. Nitrate reductase activity provides endogenous nitrosative stress through the production of  · NO (33). Although expressed under aerobic and anaerobic conditions, this activity is required at elevated levels during metabolic adaptation of the organism to microaerophilia (31) and may serve a respiratory function within the burgeoning granuloma (32). Exogenous nitrosative stress increases significantly after the onset of acquired immunity via the production of  · NO by activated macrophages (4). The reaction of  · NO with O₂ produces nitrous anhydride (3), which nitrosates amines and amides to produce compounds that are metabolically activated to form potent DNA alkylating agents (10, 13, 15, 30). As such, alkylation damage of DNA can be considered a downstream sensor of the level of nitrosative stress to which a cell is exposed (Fig. 1).

FIG. 1.

Major types of DNA damage caused by exposure to  · NO as a function of the growth stage of M. tuberculosis. The major DNA damage pathways are indicated by arrows. The relative levels of the precursors of nitrous anhydride (N₂O₃) and peroxynitrite (ONOO-) are shown for the acute and stationary phases of growth of M. tuberculosis in the mouse model of infection, where + denotes low and +++ denotes high.

In order to assess the permeation of nitrosative stress to this level in M. tuberculosis, we set out to develop mutant strains that would be defective in dealing with the consequences of alkylation damage. Alkylation of DNA can form cytotoxic (N-alkylation) or promutagenic (O-alkylation) lesions (30, 34). Since M. tuberculosis contains an operon of genes predicted to be involved in the repair (Rv1317c) and reversal (Rv1316c) of such lesions (5, 9, 16), we constructed deletion mutants in this operon by two independent strategies (Fig. 2) and investigated the growth characteristics of the mutant strains in vitro and in vivo. The SID-H and BS-SK strains both lacked the region of Adl that contains the acceptor site (in AdaA) for methyl groups from methyl phosphotriesters and regulates the adaptive response (24), as well as the entire AlkA domain. They also lacked the region of Ogt containing the acceptor site for alkyl groups from O-alkylated bases (29).

FIG. 2.

Construction of Δadl-ogt mutants of M. tuberculosis. (A) Genetic organization at the Rv1317c-Rv1316c locus of M. tuberculosis H37Rv, where rrf and rrl are the 5S and 23S rRNA genes, respectively (5). Intergenic distances (base pairs) are shown beneath the map. Rv1317c is annotated in the EMBL and Tuberculist databases as “alkA” (5). However, Rv1317c is chimeric (9, 16), comprising 5′-terminal adaA (18, 24) and 3′-terminal alkA components, and we have thus designated this gene “adl” to denote this fact. (B) Restriction maps and genotypes of the wild-type and mutant alleles (not drawn to scale). The Δadl-ogt::hyg allele was delivered on the vector pSDVM, which is a subclone of pGEM3Zf(+) that also contained a lacZ gene, whereas the Δadl-ogt::aph allele was delivered on pBSEB, which is a subclone of ptrpA-1-rpsL (25). Allelic exchange in M. tuberculosis H37Rv to form the SID-H mutant was performed by using UV-pretreated pSDVM (20), whereas that in streptomycin-resistant H37Rv to form the BS-SK mutant was carried out with pBSEB by using previously described methods (27). The flanking homologous sequences carried on the delivery vectors are shown as thick horizontal lines of the indicated sizes (base pairs). The mutant alleles differed in that an additional 246 bp of the ogt gene were deleted in Δadl-ogt::aph compared to Δadl-ogt::hyg. The selectable markers are shown as stippled boxes, and the thick dashed line in the H37Rv map represents the 1,592-bp KpnI-NotI fragment used as the probe for the Southern blot shown in panel C. Shown above the wild-type allele is the 4,207-bp KpnI fragment carrying the adl-ogt operon and flanking sequences that was used to genetically complement the SID-H mutant by single-copy insertion at the attB locus via the integrative delivery vector pAINT (1). Relevant restriction sites are shown and abbreviated as follows: E, EcoRI; K, KpnI; N, NotI, Ns, NsiI; S, SacI; Sm, SmaI; Sp, SpeI. (C) Genotypic confirmation of the SID-H mutant by Southern blot analysis. Genomic DNA was digested with SmaI and probed by using the 1,592-bp KpnI-NotI fragment. WT, wild type.

The mutant strains grew normally in vitro (data not shown) but were hypersensitive to the genotoxic effects of the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) (Fig. 3). The viability of both the H37Rv and SID-H strains was reduced in a dose-dependent manner by lower concentrations of MNNG, but the mutant strain was ca. 100-fold more sensitive than the wild type to the cytotoxic effects of this compound (Fig. 3A). The BS-SK strain was found to be similarly hypersensitive to the cytotoxic effects of MNNG (data not shown). The damage-hypersensitive phenotype of strain SID-H was consistent with loss of the DNA glycosylase (alkA) component of adl (14) in the mutant strains. This effect was possibly compounded by inactivation of the adaA component of adl, which would abrogate the ability of M. tuberculosis to mount an adaptive response to alkylation damage (24). However, the functionality of this regulatory pathway has yet to be investigated in M. tuberculosis.

FIG. 3.

Response of M. tuberculosis strains to treatment with MNNG. Logarithmic-phase cultures (10 ml; ca. 10⁷ CFU/ml) were mixed for 1 h at 37°C with an equal volume of MNNG dissolved in Middlebrook 7H9 broth containing 0.05% Tween. Bacteria were washed four times in phosphate-buffered saline containing 0.1% Tween 80 and resuspended at ca. 10⁶ CFU/ml in 100 ml of 7H9 medium. (A) Survival. Treated and washed cells were serially diluted, and aliquots from three or more serial dilutions were plated in duplicate on 7H10 agar. The viability of treated cultures is shown as a percentage of untreated controls, and the data represent the mean and standard deviation from three independent experiments. (B) Mutation induction. Treated and washed cells were grown standing at 37°C for 8 to 10 days with daily manual agitation before being serially diluted and plated on 7H10 agar with or without rifampin at 1 μg/ml. Mutation frequencies are shown as the ratio of mutants resistant to rifampin to the total number of CFU. The data are presented as means and standard errors and are representative of two independent experiments.

The mutagenic effects of MNNG were assessed from levels of induced mutation to rifampin resistance in the various strains. Although the average spontaneous mutation frequency of the SID-H mutant strain in untreated controls was ca. twofold higher than that of H37Rv, this difference was not statistically significant. Both H37Rv and SID-H showed an increase in the frequency of rifampin resistance following MNNG treatment, but the magnitude of the increase was ca. 100-fold higher in SID-H over the concentration range tested (Fig. 3B). This finding is consistent with the functional inactivation of ogt in the mutant strains, since Rv1316c is the only recognizable gene for reversing promutagenic, O-alkylation lesions (22) in M. tuberculosis (16). Rifampin-resistant mutants randomly selected from MNNG-treated and untreated control cultures of the wild-type and mutant strains were genotyped by PCR amplification and sequencing of the rifampin resistance-determining region of the rpoB gene (21). The spectrum of rpoB mutations induced in SID-H closely matched those observed in rifampin-resistant clinical isolates of M. tuberculosis (21) and in mutants selected in vitro (17) and was consistent with the exacerbation of transition mutagenesis as the predicted consequence of persisting promutagenic lesions (data not shown).

To determine whether the mutation in SID-H exerted polar effects on neighboring genes, a fragment carrying a wild-type copy of the adl-ogt operon and flanking sequences (Fig. 2) was integrated at the attB chromosomal locus of SID-H and the damage sensitivity and induced mutation frequency of the resulting complemented strain in response to treatment with 3 μM MNNG were assessed alongside those of SID-H. Comparison with the data of Fig. 3 revealed that genetic complementation of SID-H restored the damage sensitivity and the induced mutation frequency to wild-type levels (95% ± 36% survival of the treated culture versus the untreated control and an induced mutation frequency of 5.7 ×10⁻⁷ ± 0.7 ×10⁻⁷, respectively). This result confirmed that the phenotype of the SID-H mutant was solely attributable to the mutation in the adl-ogt operon.

To examine whether the alkylation damage repair and reversal defects of the Δadl-ogt mutants would be manifested in a growth phenotype in vivo, C57BL/6 mice were infected with SID-H or H37Rv and bacillary loads in the lungs, liver, and spleen were measured at a relatively early stage of stationary infection, i.e., 30 days after the infection entered stationary phase in the lungs. However, no significant differences were observed in the organ counts from mice infected with the mutant strain compared to those of the wild-type control (Fig. 4). The BS-SK mutant displayed a similar phenotype in BALB/c mice: no significant differences in bacillary loads were observed between the mutant and wild-type strains at relatively early (days 30 and 55 postinfection) or later (day 104) stages of stationary infection (data not shown). The phenotype of the Δadl-ogt mutant strains thus parallels that of a recA mutant of M. bovis BCG, which was hypersensitive to DNA damage in vitro but also displayed no growth impairment in mice (26).

FIG. 4.

Virulence of wild-type H37Rv versus that of the SID-H mutant strain in C57BL/6 mice. Infections were carried out as described by Dunn and North (7). Eight-week-old C57BL/6 mice, purchased from Jackson Laboratories (Bar Harbor, Maine), were inoculated via a lateral tail vein with 10⁵ CFU of the H37Rv or SID-H strain in 0.1 ml of phosphate-buffered saline containing 0.01% Tween 80 (PBS-Tween). Both strains were subcultured in Proskauer and Beck medium in the presence of 0.01% Tween 80, harvested as a dispersion culture in log phase (approximately 10⁸ bacteria per ml), subjected to 5 s of ultrasound to break up aggregates, and diluted in PBS-Tween for injection. At 50 days of infection, livers, spleens, and lungs were harvested and homogenized in PBS-Tween and whole-organ homogenates were subjected to 10-fold serial dilution. The dilutions were plated on Middlebrook 7H11 agar. CFU were scored after incubation of the plates for 2 to 3 weeks at 37°C. The results show the average and standard deviation of four mice per time point.

The results of this study suggest that permeation of nitrosative stress to the level of cytotoxic alkylation damage to the genome of M. tuberculosis is restricted in vivo, at least up to 15 weeks postinfection in the murine model. Restriction in the levels of cytotoxic alkylation damage sustained in vivo might be attributable to the action of defense or detoxification systems, which protect against nitrosative stress (2, 8, 11, 12, 19, 23). Alternatively, the environments encountered by M. tuberculosis during the course of an infection may not favor alkylation damage; specifically, at the times of bacillary load determination,  · NO may be preferentially sequestered by O₂ · ⁻, which would increase the potential for oxidative (6, 28) rather than alkylative damage (Fig. 1). An assessment of the growth phenotypes of mutant strains defective in the ability to repair replication-blocking oxidative lesions would be revealing in this respect. Finally, the alkylating agent(s) generated in M. tuberculosis might be of the promutagenic rather than the cytotoxic type, which could lead to a mutator phenotype without affecting the bacillary load. The extent of promutagenic damage that may occur and its role in genomic diversification and the evolution of drug resistance thus remain important areas for future investigation.