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| Influence of Antisense LNA Targeted LuxS Transcription on Biofilm Formation in Staphylococcus epidermidis |
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Abstract Abstract Bacterial biofilm is the leading cause of bacterial drug resistance increases. Staphylococcus epidermidis is an opportunistic pathogen, once it formed the biofilm, it will adhere to the surfaces of medical device and make the diseases recurrent and difficult to cure. Quorum sensing system has been recently regarded as the research target to suppress the bacterial biofilm formation. S-ribosyl homocysteine lyase (LuxS) is the key enzyme for the synthesis of autoinducer-2 (AI-2), they constitute the AI-2/LuxS quorum sensing system in bacteria. In this study, the biofilm formed in the early stage was chosen as the experiment subject, then antisense locked nucleic acid (LNA) technology was used to block the expression of LuxS gene in S. epidermidis AI-2/LuxS quorum sensing system, the inhibitory effect of 2 antisense locked oligonucleotides named LNA81 and LNA352 was compared by the transcription level of LuxS gene, Using real-time PCR measured the relative expression quantity of biofilm formation related genes, including sigma factor B (sigB), intercellular adhesion AB (icaAB), staphylococal accessory regulator A (sarA), accumulation-associated protein (aap), fibrinogen-binding protein (fbe) and autolysin E (atlE). AI-2 signaling molecule activity was measured by Vibrio harveyi bioluminescence assay, and the formation and characteristic parameters of biofilm were analyzed by confocal laser scanning microscope (CLSM) and image structure analyzer (ISA) software. Results showed that between the 2 antisense locked oligonucleotides, LNA81 and LNA352, 100 nmol/L LNA81 mixed with 2.5 μL RNAiMAX transfection reagent exhibited better inhibition in expression of LuxS through targeting LuxS mRNA, it could get a 43% inhibition ratio in LuxS transcription at 6 h. When the transcription of LuxS was inhibited partially, the transcript level of gene sigB and icaAB increased, sarA, aap, fbe and atlE decreased. The activity of AI-2 signaling molecule was decreased in 9 h compared to control group, at the time of 6 h its activity got a minimum, and after 9 h its activity was closed to the control group, this tendency was consistent with the transcript level of gene LuxS which was inhibited to a minimum at 6 h and its inhibition ratio became unsteady after 6 h. Finally, the ability of biofilm formation was decreased, the structure of biofilm was more simple than control group. This study is focus on the regulating effect of AI-2/LuxS quorum sensing system on the early biofilm formation, antisense strategy was used to target LuxS mRNA, which is the critical gene for the formation of AI-2, it not only has the ablity of quorum sensing system but also plays an important role in sulfur metabolism and glycometabolism in bacteria. It can be seen that AI-2 signaling molecule activity and expression of genes involved in biofilm formation were affected when the LuxS gene expression was partial blocked. The biofilm formation is a dynamic process, it is affected by various factors, the changes of above factors were finally resulting in inhibition of the biofilm formation. These results will provide a theoretical basis for the further study on AI-2/LuxS quorum sensing system and its regulatory effects on the biofilm formation in S. epidermidis, they also can provide more potential targets and therapeutic schedule to prevent S. epidermidis infection.
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Received: 31 August 2016
Published: 13 January 2017
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