Abstract
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.
Highlights
Host innate immunity represents the first line of defense against invading pathogenic microorganisms
Our results reveal that the Salmonella Base Excision Repair system (BER), comprised of DNA glycosylases and AP endonucleases, is able to eliminate excess mutations that accumulate in NO?–exposed cells during their interaction with the host
During Salmonella infections, the BER system protects the bacterium against potentially detrimental DNA damage arising from NO?–exposure
Summary
Host innate immunity represents the first line of defense against invading pathogenic microorganisms. Nitric oxide (NO?) is an essential component of this innate immune system, which is required for the efficient clearance of pathogenic fungi, viruses, parasites and bacteria [1,2]. Exposure can inhibit bacterial growth through the modification of multiple intracellular targets including protein thiols, heme containing proteins, thiol-coordinated metals, lipid bilayers, and DNA [4,5,6,7,8]. Typhimurium encoded on Salmonella Pathogenicity Island 2 (SPI2) impedes trafficking of iNOS to the Salmonella Containing Vacuole (SCV) in host macrophages [9]. Is detoxified by the Hmp flavohemoglobin, which is required for virulence in hosts proficient for inflammatory NO?
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