| In the field of infectious diseases, drug-resistant bacteria infection is one of the important issues throughout the world. At present, in addition to the reasonable application of antibiotics to reduce the drug resistance mutation rate of pathogenic bacteria, corresponding strategies involve exploring new antibacterial biochemical pathways and developing novel antimicrobial agents for the resistance against pan-drug resistant bacteria.Through methylation modification, changing the structure of antibacterial drug targets is a significant drug-resistance mechanism for E.coli(as a representative of pathogenic bacteria). As a important enzyme, S-adenosylhomocysteine nucleosidase(hereafter referred to as SAHN) participates in activated methyl cycle. Furthermore, in the methylation reaction regulated by S-adenosine methionine(hereafter called SAM), methionine and adenine are regenerated and the signal molecule Autoinducer 2(AI-2) of Quorum sensing(QS) is produced.As an important protection mode of bacteria growth ad division, biofilm affected by quorum sensing can guarantee the survival of bacteria in the harsh environment and enhance the resistance of bacteria against antibiotics. Biofilm formation becomes one of the significant mechanisms of bacterial resistance, which is the main reason for repeated episodes and uncontrollability of numerous chronic infectious diseases. Besides, the declining activity of SAHN exerts serious influence on bacteria growth and division in the cells. Firstly, SAHN activity decrease will cause the accumulation of intracellular S-adenosylhomocysteine(hereafter called SAH). However, as an effective feedback inhibitor of SAM-dependent methyltransferase, intracellular SAH accumulation of low concentration will thoroughly suppress methyl transfer reaction. As a result, gene expression regulation and protein modification are affected. On the other hand, pathogenic bacteria adaptability is reduced to stop bacterial cells growth and division. Secondly, SAHN inhibition will lead to the fact that downstream AI-2 precursor is deleted and pathogens cannot synthesize AI-2 and therefore bacterial quorum sensing effects can be effectively controlled, such as pathogen virulence factors expression and bacterial biofilm formation. In addition, SAHN inhibition will cause methyl cycle interruption and important compounds(including methionine and SAM) are unable to regenerate in the methyl cycle. Provided that no methionine is supplied in the environment, SAM cannot penetrate bacterial cell membranes and pathogens simply cannot grow. Consequently, bacteria growth and division may be influenced by different mechanisms. As this type of nucleosidase does not exist in mammalian cells, the analysis and research concerning the function of SAHN has provided a number of ideas for broad spectrum bactericide development.In this paper, the gene sahn was amplified with the genome of E.coli as template by PCR then was linked to vector to pET-28a-SAHN and transformed into Escherichia coli BL21. SAHN was expressed as IPTG-inducible His-tagged recombinant enzymes purified using Ni-NTA chromatography. After purification, it was concentrated to 348 μg/m L, which was measured by coomassie brilliant blue method. The determination of SAHN enzyme activity was carried out based on coupling of xanthine oxidase enzyme. By inducing E.coli that has been built, the effect of SAHN activity at different expression levels on E.coli growth, division and quorum sensing is investigated, which provides theoretical and experimental basis and reference for the study of developing novel SAHN-targeted anti-infection drugs. |
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