Table_3_Transcriptomic Changes of Piscirickettsia salmonis During Intracellular Growth in a Salmon Macrophage-Like Cell Line.xlsx

Piscirickettsia salmonis is the causative agent of Piscirickettsiosis, a systemic infection of salmonid fish species. P. salmonis infects and survives in its host cell, a process that correlates with the expression of virulence factors including components of the type IVB secretion system. To gain further insights into the cellular and molecular mechanism behind the adaptive response of P. salmonis during host infection, we established an in vitro model of infection using the SHK-1 cell line from Atlantic salmon head kidney. The results indicated that in comparison to uninfected SHK-1 cells, infection significantly decreased cell viability after 10 days along with a significant increment of P. salmonis genome equivalents. At that time, the intracellular bacteria were localized within a spacious cytoplasmic vacuole. By using a whole-genome microarray of P. salmonis LF-89, the transcriptome of this bacterium was examined during intracellular growth in the SHK-1 cell line and exponential growth in broth. Transcriptome analysis revealed a global shutdown of translation during P. salmonis intracellular growth and suggested an induction of the stringent response. Accordingly, key genes of the stringent response pathway were up-regulated during intracellular growth as well as at stationary phase bacteria, suggesting a role of the stringent response on bacterial virulence. Our results also reinforce the participation of the Dot/Icm type IVB secretion system during P. salmonis infection and reveals many unexplored genes with potential roles in the adaptation to intracellular growth. Finally, we proposed that intracellular P. salmonis alternates between a replicative phase and a stationary phase in which the stringent response is activated.杀鲑立克次体（Piscirickettsia salmonis）是鲑立克次体病（Piscirickettsiosis）的病原菌，该疾病可引发鲑形目鱼类的全身性感染。该菌可侵染宿主细胞并在其中存活，这一过程与包括IVB型分泌系统（type IVB secretion system）组分在内的毒力因子的表达密切相关。为深入解析杀鲑立克次体在宿主侵染过程中适应性反应的细胞与分子机制，本研究利用源自大西洋鲑头肾的SHK-1细胞系构建了体外侵染模型。结果显示，与未感染的SHK-1细胞相比，侵染10天后细胞活性显著下降，同时杀鲑立克次体的基因组当量显著升高。此时，胞内细菌定位于宽阔的细胞质空泡内。本研究利用杀鲑立克次体LF-89的全基因组微阵列（whole-genome microarray），检测了该菌在SHK-1细胞系内胞内生长以及肉汤培养基中指数生长期的转录组。转录组分析显示，杀鲑立克次体在胞内生长期间出现全局性的翻译通路抑制，并提示严谨反应（stringent response）被激活。相应地，严谨反应通路的关键基因在胞内生长阶段以及稳定期细菌中均呈现上调表达，表明严谨反应在细菌毒力调控中发挥重要作用。本研究结果进一步证实了Dot/Icm IVB型分泌系统（Dot/Icm type IVB secretion system）在杀鲑立克次体侵染过程中的参与作用，并揭示了诸多未被探索的、可能在胞内生长适应中发挥功能的基因。最后，本研究提出，胞内的杀鲑立克次体可在复制阶段与激活严谨反应的稳定阶段之间交替切换。