Xiaowei FANG, Wei HU, Yu ZHANG, Chen WANG, Qingping LUO, Hui WU, Xiongyan LIANG, Yufang GU, Chun FANG, Yuying YANG

Roles of Histidine Kinase Gene yycG in the Pathogenicity of Listeria monocytogenes

As an opportunistic foodborne pathogen, Listeria monocytogenes could successfully switch self-characteristics in response to environmental conditions. In L. monocytogenes, sixteen two-component systems (TCSs) have been found to contribute the bacteria to sensing and adapting to various conditions. Our previous genomic study showed that mutation was observed in the histidine kinase gene yycG between Lm850658 and M7 among the sixteen TCSs. The YycFG TCS was found playing important roles in many other Gram-positive bacteria. While the roles of YycFG TCS in L. monocytogenes remain poorly known. In this study, we aimed to determine whether yycG play roles in pathogenicity of L. monocytogenes. We created a histidine kinase gene yycG deletion strain (ΔyycG) based on the wild type strain Lm850658 and a complemental strain (CΔyycG). We found the yycG deletion significantly impaired the growth ability and mobility, but enhanced the hemolysis ability in vitro. In addition, infection assays on cell and mice model showed that ΔyycG exhibited significantly defected in infection ability and virulence. All these phenotypes of the ΔyycG could be reversed largely to the levels of the wild type strain Lm850658 by gene complementation. Cell wall-associated and secreted protein analysis showed that the secreted content of ΔyycG was significantly increased. And western blotting revealed that Internalin protein B (InlB) and Listerolysin O (LLO) was markedly increased in the secreted fractions of ΔyycG, which might be responsible for decreased adhesion and invasion ability and increased hemolytic activity, respectively. Overall, we found the histidine kinase yycG played important roles in pathogenicity of L. monocytogenes for the first time. Further investigation is needed to explore how the YycFG TCS modulates the growth, mobility, cell surface proteins translocation and virulence.

Listeria monocytogenes’in Patojenitesinde Histidin Kinaz Geni yycG’nin Rolü

Gıda kaynaklı fırsatçı bir patojen olan Listeria monocytogenes, çevresel koşullara tepki olarak kendi özelliklerini başarılı bir şekilde değiştirebilir. L. monocytogenes’te, onaltı adet iki bileşenli sistemin (TCS) bakterilerin çeşitli durumları algılamasına ve adaptasyonuna katkıda bulunduğu belirlenmiştir. Önceki genomik çalışmamız, on altı TCS’den Lm850658 ve M7 arasındaki histidin kinaz gen yycG’de mutasyon gözlendiğini göstermiştir. YycFG TCS’nin diğer birçok Gram pozitif bakteride önemli roller oynadığı belirlenmiştir. Oysa L. monocytogenes’teki YycFG TCS’nin rolü hala tam olarak bilinmemektedir. Bu çalışmada, yycG’nin L. monositogenes’in patojenisitesinde rol oynayıp oynamadığını belirlemeyi amaçladık. Yaban tipi Lm850658 suşunu ve tamamlayıcı bir suşu (CΔyycG) temel alan bir histidin kinaz geni yycG silme suşu (ΔyycG) oluşturduk. YycG’nin silinmesinin büyüme kabiliyetini ve hareketliliğini önemli ölçüde bozduğunu, ancak in vitro hemoliz yeteneğini artırdığını bulduk. Ek olarak, hücre ve fare modelindeki enfeksiyon deneyleri, ΔyycG’nin enfeksiyon yeteneği ve virülansta önemli ölçüde kusurlu olduğunu gösterdi. ΔyycG’nin tüm bu fenotipleri, gen tamamlaması ile büyük ölçüde yaban tipi Lm850658 suşunun düzeyine tersine çevrilebilir. Hücre duvarı ilişkili ve salgılanan proteinler analizi, ΔyycG’nin salgılanan miktarının önemli ölçüde arttığını gösterdi. Western blot analizi, ΔyycG’nin salgılanan fraksiyonlarında; sırasıyla azalmış yapışma ve invazyon yeteneğinden ve artmış hemolitik aktiviteden sorumlu olabilecek Internalin protein B (InlB) ve Listerolysin O’nun (LLO) belirgin şekilde arttığını ortaya koydu. Histidin kinaz yycG’nin L. monocytogenes patojenitesinde önemli roller oynadığı ilk kez belirlendi. YycFG TCS’nin büyümeyi, hareketliliği, hücre yüzeyi proteinlerinin translokasyonunu ve virülansı nasıl değiştirdiğini belirlemek için ileri araştırmalara ihtiyaç vardır.

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