title: Nitrosative damage to free and zinc-bound cysteine thiols underlies nitric oxide toxicity in wild-type Borrelia burgdorferi.
abstract: Borrelia burgdorferi encounters potentially harmful reactive nitrogen species (RNS) throughout its infective cycle. In this study, diethylamine NONOate (DEA/NO) was used to characterize the lethal effects of RNS on B. burgdorferi. RNS produce a variety of DNA lesions in a broad spectrum of microbial pathogens; however, levels of the DNA deamination product, deoxyinosine, and the numbers of apurinic/apyrimidinic (AP) sites were identical in DNA isolated from untreated and DEA/NO-treated B. burgdorferi cells. Strains with mutations in the nucleotide excision repair (NER) pathway genes uvrC or uvrB treated with DEA/NO had significantly higher spontaneous mutation frequencies, increased numbers of AP sites in DNA and reduced survival compared with wild-type controls. Polyunsaturated fatty acids in B. burgdorferi cell membranes, which are susceptible to peroxidation by reactive oxygen species (ROS), were not sensitive to RNS-mediated lipid peroxidation. However, treatment of B. burgdorferi cells with DEA/NO resulted in nitrosative damage to several proteins, including the zinc-dependent glycolytic enzyme fructose-1,6-bisphosphate aldolase (BB0445), the Borrelia oxidative stress regulator (BosR) and neutrophil-activating protein (NapA). Collectively, these data suggested that nitrosative damage to proteins harbouring free or zinc-bound cysteine thiols, rather than DNA or membrane lipids underlies RNS toxicity in wild-type B. burgdorferi.
authors.affiliation: Laboratory of Zoonotic Pathogens, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 903 South 4th Street, Hamilton, MT 59840, USA.
authors.forename: Travis J
authors.initials: TJ
authors.lastname: Bourret
authors.forename: Julie A
authors.initials: JA
authors.lastname: Boylan
authors.forename: Kevin A
authors.initials: KA
authors.lastname: Lawrence
authors.forename: Frank C
authors.initials: FC
authors.lastname: Gherardini
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D025065:Borrelia burgdorferi / Q000187:drug effects*; D019743:Chemokines, CXC / Q000378:metabolism; D003545:Cysteine / Q000031:analogs & derivatives* / Q000378:metabolism; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism; D009569:Nitric Oxide / Q000633:toxicity*; D015538:Nitrosation; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D019743:Chemokines, CXC; C534091:NapA protein, Borrelia burgdorferi; D013438:Sulfhydryl Compounds; D015534:Trans-Activators; C545158:cysteine thiolate; D009569:Nitric Oxide; D005634:Fructose-Bisphosphate Aldolase; D015032:Zinc; D003545:Cysteine