Multicopper oxidase from Acinetobacter baumannii heterologous expression characterization and role in virulence

Abstract

The present study was designed with an aim to explore the prevalence and role of multicopper oxidase in survival, metal homeostasis and virulence of A. baumannii. In silico analysis revealed high conservation (more than 98 % similarity) of MCOs among the strains of A. baumannii. In this regard, MCO with accession number EEX03816.1 consisting of 1935 base pairs encoding a protein of 644 amino acids (AbMCO) was identified from the proteome of A. baumannii ATCC 19606. Further, AbMCO was overexpressed in E. coli, purified using Ni-NTA chromatography and characterized. The optimum temperature and pH for activity of purified AbMCO was 50 °C and 6.0 respectively. In vitro assays showed that AbMCO catalyzed the oxidation of phenolic substrates viz. 2,6-dimethoxyphenol, guaiacol, ABTS, catechol and 2,3- dihydroxybenzoic in the presence of CuSO4 (50 and#956;and#924;). Expression of abMCO gene was found to be induced by stress as a significant upregulation (8-fold) in the expression was observed in the presence of 1.5 mM copper sulphate and 700 mM sodium chloride newlinerespectively. However other stress conditions viz. oxidative (hydrogen peroxide,menadione); osmotic (polyethyleneglycol, sorbitol); metals (iron, manganese, cadmium); antibiotics and desiccation does not affect the production of AbMCO, suggesting the protective role of AbMCO against copper and high salinity stress. Survival and virulence assays showed that AbMCO exhibited copper resistance and siderophore oxidase activity, thus depicting its role in copper and iron homeostasis. However expression of abMCO does not affect the survival of A. baumannii inside the macrophages, suggesting no role in the virulence.