The Base Excision Repair System of Salmonella enterica serovar Typhimurium Counteracts DNA Damage by Host Nitric Oxide

Intracellular pathogens must withstand nitric oxide (NO·) generated by host phagocytes. Salmonella enterica serovar Typhimurium interferes with intracellular trafficking of inducible nitric oxide synthase (iNOS) and possesses multiple systems to detoxify NO·. Consequently, the level of NO· stress encountered by S. Typhimurium during infection in vivo has been unknown. The Base Excision Repair (BER) system recognizes and repairs damaged DNA bases including cytosine and guanine residues modified by reactive nitrogen species. Apurinic/apyrimidinic (AP) sites generated by BER glycosylases require subsequent processing by AP endonucleases. S. Typhimurium xth nfo mutants lacking AP endonuclease activity exhibit increased NO· sensitivity resulting from chromosomal fragmentation at unprocessed AP sites. BER mutant strains were thus used to probe the nature and extent of nitrosative damage sustained by intracellular bacteria during infection. Here we show that an xth nfo S. Typhimurium mutant is attenuated for virulence in C3H/HeN mice, and virulence can be completely restored by the iNOS inhibitor L-NIL. Inactivation of the ung or fpg glycosylase genes partially restores virulence to xth nfo mutant S. Typhimurium, demonstrating that NO· fluxes in vivo are sufficient to modify cytosine and guanine bases, respectively. Mutants lacking ung or fpg exhibit NO·–dependent hypermutability during infection, underscoring the importance of BER in protecting Salmonella from the genotoxic effects of host NO·. These observations demonstrate that host-derived NO· damages Salmonella DNA in vivo, and the BER system is required to maintain bacterial genomic integrity

NO·–sensitivity of <i>S.</i> Typhimurium Base Excision Repair mutants.

<p>(A) Growth of <i>S.</i> Typhimurium 14028s and mutant derivatives after the addition of NO· from 2 mM SperNO (at arrow). (B) Survival of Wild Type and <i>xth nfo</i> after exposure to 1 mM SperNO. (C) Rescue of <i>xth nfo</i> cells by additional inactivation of indicated glycosylases after 2 hr exposure to 1 mM SperNO. Asterisks represent statistical significance. (p<0.05.)</p

The attenuation of <i>xth nfo</i> mutant <i>S.</i> Typhimurium is due to NO·–mediated base damage.

<p>(A) Full virulence can be restored to <i>xth nfo S.</i> Typhimurium (14028s) in C3H/HeN mice by administration of the NOS2 inhibitor L-NIL. (B) Livers were harvested from female C3H/HeN infected with a mixed inoculum of Wild Type and mutant <i>S.</i> Typhimurium at indicated days. Ratios of mutant to Wild Type viable cfu·g⁻¹ were determined and C.I. = Mutant_OUT:WT_OUT/Mutant_IN:WT_IN. (C) The attenuation of <i>xth nfo S.</i> Typhimurium is suppressed by further inactivation of DNA glycosylases <i>ung</i> and <i>fpg</i>.</p

Conceptual representation of mutation avoidance by BER during nitrosative stress.

<p>NO· can be oxidized to nitrous anhydride (N₂O₃) and/or peroxynitrite (ONOO⁻) depending in the presence or absence of superoxide. Nitrous anhydride and peroxynitrite mediate DNA base deamination and oxidation, respectively. The Ung repair pathway removes dU from DNA via an AP site, thereby avoiding the MUTATION pathway. Similarly, Fpg removes oxidized dG, limiting peroxynitrite-mediated hypermutation, but increased mutability in cells lacking Fpg can be ameliorated by MutY. NO·-induced mutations can also be avoided by pathways that do not produce AP site intermediates (e.g., Nfi).</p

Mutability of <i>S.</i> Typhimurium strain 14028s derivatives upon exposure to nitric oxide (NO·) or peroxynitrite (ONOO⁻).

<p>Frequency of Rif^R×10⁻⁸.</p>a<p>Mutability significantly higher than unexposed cells (Wilcoxon Rank Sum, p≤.05).</p>b<p>Mutability significantly higher than exposed WT cells (Wilcoxon Rank Sum, p≤.05).</p

Bacterial strains and DNA primers used in this study.

<p>Bacterial strains and DNA primers used in this study.</p

The base excision repair system of <i>S.</i> Typhimurium protects against the mutagenic effects of host NO· during infection.

<p>Bacteria isolated from livers 7d post inoculation were diluted and plated to determine total viable cfu·g⁻¹, and 5FC resistance rates were determined as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000451#s4" target="_blank">Materials and Methods</a>. For comparison, C3H/HeN mice were administered L-NIL orally to inhibit NO· production. Asterisks indicate rates significantly different compare to Wild Type cells from untreated mice as determined by Wilcoxon Rank Sum test.( * p = 0.05 **p = 0.01.).</p