金黄色葡萄球菌在生物被膜态与浮游态的转录组差异表达分析

doi:10.11843/j.issn.0366-6964.2022.08.027

摘要/Abstract

摘要： 金黄色葡萄球菌是引起奶牛细菌性乳腺炎的主要原因，生物被膜的形成是金黄色葡萄球菌在不利环境条件下持久性存在的关键因素。探索同一株菌在生物被膜态与浮游态生长状态下的耐药性与其生长状态的相关性，可为进一步探究金黄色葡萄球菌的耐药性机制奠定基础。本研究培养了金黄色葡萄球菌的生物被膜，使用光学显微镜和扫描电镜观察其形成过程。测定并比较了9种抗菌药物对32株金黄色葡萄球菌在生物被膜态和浮游态的最小抑菌浓度，并对两种状态下的金黄色葡萄球菌进行转录组学测序，筛选出具有显著性差异的细胞信号通路和表达基因，同时对主要差异表达的基因进行RT-qPCR验证。结果发现，在生物被膜形成前期，随着培养时间的延长，显微镜下观察到的生物被膜态菌聚集面积越来越大，结构也越来越紧密，培养至72 h后，生物被膜逐渐开始分散。MIC测定结果显示浮游态菌的抑菌浓度低于生物被膜态菌。转录组结果显示两种状态菌的差异表达基因共1 512个，其中，生物被膜态菌中上调基因760个，下调752个。GO与KEGG富集分析显示，相比于浮游态菌，生物被膜态菌中与代谢相关的通路显著富集，其次为氨基酸的生物合成和ABC转运蛋白通路。与生物被膜形成相关的基因，如编码ABC转运蛋白的基因表达上调，而与代谢途径相关的基因下调。RT-qPCR验证了10个主要差异基因，其表达差异趋势与转录组测序结果一致。这些差异可能对金黄色葡萄球菌生物被膜态的高耐药性和细菌毒力的研究有所帮助。

关键词: 转录组测序, 差异表达基因, 生物被膜, 金黄色葡萄球菌

Abstract: Staphylococcus aureus is the main cause of bacterial mastitis in dairy cows, and the formation of biofilm is the key factor of S. aureus persistence under adverse environmental conditions. Exploring the correlation between drug resistance and growth state of the same strain in biofilm and planktonic growth state can lay a foundation for further exploring the drug resistance mechanism of S. aureus. In this study, the biofilm of S. aureus was cultured and its formation was observed by optical microscope and scanning electron microscope. The minimum inhibitory concentrations of 9 kinds of antibiotics against 32 strains of S. aureus in biofilm and planktonic states were measured and compared. Transcriptome sequencing was performed on S. aureus under the two states to screen out cell signaling pathways and expressed genes with significant differences. At the same time, the main differentially expressed genes were verified by RT-qPCR. The results showed that in the early stage of biofilm formation, with the extension of culture time, the aggregation area of biofilm state bacteria observed under the microscope became larger and larger, and the structure was more and more compact. After 72 hours of culture, the biofilm gradually began to disperse. MIC results showed that the inhibitory concentration of planktonic bacteria was lower than that of biofilm bacteria. Transcriptome results showed that there were 1512 differentially expressed genes in the two states, among which 760 genes were up-regulated and 752 genes were down-regulated in the biofilm state. GO and KEGG enrichment analysis showed that, compared with plankplankton, metabolization-related pathways were significantly enriched in biofilm bacteria, followed by amino acid biosynthesis and ABC transporter pathways.Genes associated with biofilm formation, such as those encoding ABC transportersare up-regulated, while genes associated with metabolic pathways are down-regulated.Ten major differentially expressed genes were verified by RT-qPCR, and the differentially expressed trends were consistent with transcriptome sequencing results. These differences may be helpful to study the high drug resistance and virulence of S.aureus in biofilm state.

Key words: transcriptome sequencing, differentially expressed genes, biofilm, Staphylococcus aureus

中图分类号:

S852.611

毛彦妮, 常佳伟, 李娜, 王鑫, 康馨匀, 马强, 马靓, 王桂琴. 金黄色葡萄球菌在生物被膜态与浮游态的转录组差异表达分析[J]. 畜牧兽医学报, 2022, 53(8): 2697-2707.

MAO Yanni, CHANG Jiawei, LI Na, WANG Xin, KANG Xinyun, MA Qiang, MA Liang, WANG Guiqin. Transcriptome Differential Expression Analysis of Staphylococcus aureus in Biofilm State and Planktonic State[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(8): 2697-2707.

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