title: Ohr plays a central role in bacterial responses against fatty acid hydroperoxides and peroxynitrite.
abstract: Organic hydroperoxide resistance (Ohr) enzymes are unique Cys-based, lipoyl-dependent peroxidases. Here, we investigated the involvement of Ohr in bacterial responses toward distinct hydroperoxides. In silico results indicated that fatty acid (but not cholesterol) hydroperoxides docked well into the active site of Ohr from Xylella fastidiosa and were efficiently reduced by the recombinant enzyme as assessed by a lipoamide-lipoamide dehydrogenase-coupled assay. Indeed, the rate constants between Ohr and several fatty acid hydroperoxides were in the 107-108M-1⋅s-1range as determined by a competition assay developed here. Reduction of peroxynitrite by Ohr was also determined to be in the order of 107M-1⋅s-1at pH 7.4 through two independent competition assays. A similar trend was observed when studying the sensitivities of a ∆ohr mutant of Pseudomonas aeruginosa toward different hydroperoxides. Fatty acid hydroperoxides, which are readily solubilized by bacterial surfactants, killed the ∆ohr strain most efficiently. In contrast, both wild-type and mutant strains deficient for peroxiredoxins and glutathione peroxidases were equally sensitive to fatty acid hydroperoxides. Ohr also appeared to play a central role in the peroxynitrite response, because the ∆ohr mutant was more sensitive than wild type to 3-morpholinosydnonimine hydrochloride (SIN-1 , a peroxynitrite generator). In the case of H2O2insult, cells treated with 3-amino-1,2,4-triazole (a catalase inhibitor) were the most sensitive. Furthermore, fatty acid hydroperoxide and SIN-1 both induced Ohr expression in the wild-type strain. In conclusion, Ohr plays a central role in modulating the levels of fatty acid hydroperoxides and peroxynitrite, both of which are involved in host-pathogen interactions.
authors.affiliation: Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, 05508-090, Sao Paulo, Brazil.
authors.forename: Thiago G P
authors.initials: TG
authors.lastname: Alegria
authors.affiliation: Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, 05508-090, Sao Paulo, Brazil.
authors.forename: Diogo A
authors.identifier: 0000-0002-8451-2083
authors.initials: DA
authors.lastname: Meireles
authors.affiliation: Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, 05508-090, Sao Paulo, Brazil.
authors.forename: José R R
authors.identifier: 0000-0001-5909-8215
authors.initials: JR
authors.lastname: Cussiol
authors.affiliation: Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, 11800 Montevideo, Uruguay.
authors.forename: Martín
authors.initials: M
authors.lastname: Hugo
authors.affiliation: Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, 11800 Montevideo, Uruguay.
authors.forename: Madia
authors.initials: M
authors.lastname: Trujillo
authors.affiliation: Departamento de Biologia, Universidade Estadual Paulista Júlio de Mesquita Filho, Campus do Litoral Paulista, 11330-900, Sao Vicente, Brazil.
authors.forename: Marcos Antonio
authors.initials: MA
authors.lastname: de Oliveira
authors.affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, 05508-000, Sao Paulo, Brazil.
authors.forename: Sayuri
authors.initials: S
authors.lastname: Miyamoto
authors.affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, 05508-000, Sao Paulo, Brazil.
authors.forename: Raphael F
authors.initials: RF
authors.lastname: Queiroz
authors.affiliation: Instituto de Física de São Carlos, Universidade de São Paulo, 13566-570, Sao Carlos, Brazil.
authors.forename: Napoleão Fonseca
authors.initials: NF
authors.lastname: Valadares
authors.affiliation: Instituto de Física de São Carlos, Universidade de São Paulo, 13566-570, Sao Carlos, Brazil.
authors.forename: Richard C
authors.initials: RC
authors.lastname: Garratt
authors.affiliation: Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, 11800 Montevideo, Uruguay; rradi@fmed.edu.uy nettoles@ib.usp.br.
authors.forename: Rafael
authors.initials: R
authors.lastname: Radi
authors.affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, 05508-000, Sao Paulo, Brazil.
authors.forename: Paolo
authors.initials: P
authors.lastname: Di Mascio
authors.affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, 05508-000, Sao Paulo, Brazil.
authors.forename: Ohara
authors.initials: O
authors.lastname: Augusto
authors.affiliation: Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, 05508-090, Sao Paulo, Brazil; rradi@fmed.edu.uy nettoles@ib.usp.br.
authors.forename: Luis E S
authors.identifier: 0000-0002-4250-9177
authors.initials: LE
authors.lastname: Netto
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000502:physiology*; D005227:Fatty Acids / Q000737:chemistry* / Q000378:metabolism; D054884:Host-Pathogen Interactions; D006861:Hydrogen Peroxide / Q000737:chemistry* / Q000378:metabolism; D062105:Molecular Docking Simulation; D009566:Nitrates / Q000737:chemistry* / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D005227:Fatty Acids; D009566:Nitrates; C112257:organic hydroperoxide resistance protein, Xanthomonas campestris; C039642:peroxynitric acid; D006861:Hydrogen Peroxide
keywords: Cys-based peroxidase; Pseudomonas aeruginosa; hydroperoxides; pathogenic bacteria; thiols