Öztaş Gülmüş Ebru, Akçelik Nefise, Özdemir Caner, Akçelik Mustafa
Ankara University, Department of Biology, Ankara, Türkiye.
Ankara University, Institue of Biotechnology, Ankara, Türkiye.
Mikrobiyol Bul. 2024 Jul;58(3):225-238. doi: 10.5578/mb.20240038.
In recent years, as the paradigm of communication between cells has been clarified, the ability of bacteria to change their gene expression patterns in response to various extracellular signals has attracted great interest. In particular, intracellular and intercellular communication between bacterial populations, called quorum sensing (QS), is essential for coordinating physiological and genetic activities. QS studies are critical, particularly in elucidating the regulatory mechanisms of infectious processes in food-borne pathogens. Elucidating the QS mechanisms in Salmonella is effective in silencing the virulence factors in the fight against this bacterium. The aims of this study were; to create luxS gene mutants that play a vital role in the QS activity of Salmonella and to determine the effect of this mutation on the expression of virulence genes in the bacteria and to determine the impact of synthetic N-hexanoyl-homoserine lactone (C6HSL) on biofilm formation and AI-2 signaling pathway of Salmonella wild strain and luxS gene mutants. luxS gene mutants were constructed by recombining the gene region with the chloramphenicol gene cassette based on homologous region recombination. In the luxS mutants obtained in this way, the expression of eight different virulence genes (hilA, invA, inv, glgC, fimF, fliF, lpfA, gyrA), which have essential roles in Salmonella pathogenicity, was determined by quantitative real-time reverse transcriptase polymerase chain reaction (rRT-qPCR) method and compared with natural strains. As a result of these studies, it was determined that the expression of each gene examined was significantly reduced in luxS mutant strains. The relative AI-2 activities of Salmonella strains were analyzed depending on time. It was determined that the highest activity occurred at the fourth hour and the AI-2 activities of luxS mutants were reduced compared to the wild strain. Finally, it was determined that C6HSL increased the biofilm activity of Salmonella Typhimurium DMC4, SL1344 wild strains, and mutants, mainly at the 72nd hour. In conclusion, our results proved that C6HSL stimulated QS communication in all strains and increased biofilm of Salmonella formation and autoinducer activity. This situation determines that Salmonella responds to external signals by using QS systems. In addition, this research contributed to provide additional information on interspecies communication mechanisms to develop strategies to prevent biofilm formation of this pathogen.
近年来，随着细胞间通讯模式的阐明，细菌响应各种细胞外信号改变其基因表达模式的能力引起了极大关注。特别是，细菌群体之间的细胞内和细胞间通讯，即群体感应（QS），对于协调生理和遗传活动至关重要。QS研究至关重要，尤其是在阐明食源性病原体感染过程的调控机制方面。阐明沙门氏菌中的QS机制对于在对抗这种细菌的过程中使毒力因子失活是有效的。本研究的目的是：创建在沙门氏菌QS活性中起关键作用的luxS基因突变体，确定这种突变对细菌中毒力基因表达的影响，并确定合成的N-己酰基高丝氨酸内酯（C6HSL）对沙门氏菌野生菌株和luxS基因突变体生物膜形成和AI-2信号通路的影响。基于同源区域重组，通过将基因区域与氯霉素基因盒重组构建luxS基因突变体。通过定量实时逆转录聚合酶链反应（rRT-qPCR）方法测定以这种方式获得的luxS突变体中在沙门氏菌致病性中起重要作用的八个不同毒力基因（hilA、invA、inv、glgC、fimF、fliF、lpfA、gyrA）的表达，并与天然菌株进行比较。这些研究的结果表明，在luxS突变菌株中，所检测的每个基因的表达均显著降低。根据时间分析沙门氏菌菌株的相对AI-2活性。确定最高活性出现在第四小时，并且luxS突变体的AI-2活性与野生菌株相比有所降低。最后，确定C6HSL主要在第72小时增加了鼠伤寒沙门氏菌DMC4和SL1344野生菌株及突变体的生物膜活性。总之，我们的结果证明C6HSL刺激了所有菌株中的QS通讯，并增加了沙门氏菌生物膜的形成和自诱导活性。这种情况表明沙门氏菌通过使用QS系统对外部信号作出反应。此外，这项研究有助于提供关于种间通讯机制的更多信息，以制定预防这种病原体生物膜形成的策略。
