Sisson Gary, Goodwin Avery, Raudonikiene Ausra, Hughes Nicky J, Mukhopadhyay Asish K, Berg Douglas E, Hoffman Paul S
Department of Microbiology and ImmunologyFaculty of Medicine, Dalhousie University, HaliFax, Nova Scotia B3H 4H7, Canada.
Antimicrob Agents Chemother. 2002 Jul;46(7):2116-23. doi: 10.1128/AAC.46.7.2116-2123.2002.
Nitazoxanide (NTZ) is a redox-active nitrothiazolyl-salicylamide prodrug that kills Helicobacter pylori and also many anaerobic bacterial, protozoan, and helminthic species. Here we describe development and use of a spectrophotometric assay, based on nitroreduction of NTZ at 412 nm, to identify H. pylori enzymes responsible for its activation and mode of action. Three enzymes that reduce NTZ were identified: two related NADPH nitroreductases, which also mediate susceptibility to metronidazole (MTZ) (RdxA and FrxA), and pyruvate oxidoreductase (POR). Recombinant His-tagged RdxA, FrxA, and POR, overexpressed in nitroreductase-deficient Escherichia coli, each rapidly reduced NTZ, whereas only FrxA and to a lesser extent POR reduced nitrofuran substrates (furazolidone, nitrofurantoin, and nitrofurazone). POR exhibited no MTZ reductase activity either in extracts of H. pylori or following overexpression in E. coli; RdxA exhibited no nitrofuran reductase activity, and FrxA exhibited no MTZ reductase activity. Analysis of mutation to rifampin resistance (Rif(r)) indicated that NTZ was not mutagenic and that nitrofurans were only weakly mutagenic. Alkaline gel DNA electrophoresis indicated that none of these prodrugs caused DNA breakage. In contrast, MTZ caused DNA damage and was strongly mutagenic. We conclude that POR, an essential enzyme, is responsible for most or all of the bactericidal effects of NTZ against H. pylori. While loss-of-function mutations in rdxA and frxA produce a Mtz(r) phenotype, they do not contribute much to the innate susceptibility of H. pylori to NTZ or nitrofurans.
硝唑尼特（NTZ）是一种具有氧化还原活性的硝基噻唑基水杨酰胺前体药物，可杀死幽门螺杆菌以及许多厌氧细菌、原生动物和蠕虫物种。在此，我们描述了一种基于NTZ在412 nm处的硝基还原反应的分光光度法的开发和应用，以鉴定负责其激活和作用模式的幽门螺杆菌酶。鉴定出三种还原NTZ的酶：两种相关的NADPH硝基还原酶，它们也介导对甲硝唑（MTZ）的敏感性（RdxA和FrxA），以及丙酮酸氧化还原酶（POR）。在缺乏硝基还原酶的大肠杆菌中过表达的重组His标签RdxA、FrxA和POR，每种都能迅速还原NTZ，而只有FrxA以及程度较轻的POR能还原硝基呋喃底物（呋喃唑酮、呋喃妥因和呋喃西林）。POR在幽门螺杆菌提取物中或在大肠杆菌中过表达后均未表现出MTZ还原酶活性；RdxA未表现出硝基呋喃还原酶活性，FrxA未表现出MTZ还原酶活性。对利福平耐药突变（Rif(r)）的分析表明，NTZ没有致突变性，而硝基呋喃只有微弱的致突变性。碱性凝胶DNA电泳表明，这些前体药物均未导致DNA断裂。相比之下，MTZ会导致DNA损伤且具有很强的致突变性。我们得出结论，POR作为一种必需酶，负责NTZ对幽门螺杆菌的大部分或全部杀菌作用。虽然rdxA和frxA中的功能丧失突变会产生Mtz(r)表型，但它们对幽门螺杆菌对NTZ或硝基呋喃的固有敏感性贡献不大。
