Data_Sheet_1_Legionella pneumophila Infection Rewires the Acanthamoeba castellanii Transcriptome, Highlighting a Class of Sirtuin Genes.docx

Legionella pneumophila is an environmental bacterium that has evolved to survive predation by soil and water amoebae such as Acanthamoeba castellanii, and this has inadvertently led to the ability of L. pneumophila to survive and replicate in human cells. L. pneumophila causes Legionnaire's Disease, with human exposure occurring via the inhalation of water aerosols containing both amoebae and the bacteria. These aerosols originate from aquatic biofilms found in artifical water sources, such as air-conditioning cooling towers and humidifiers. In these man-made environments, A. castellanii supports L. pneumophila intracellular replication, thereby promoting persistence and dissemination of the bacteria and providing protection from external stress. Despite this close evolutionary relationship, very little is known about how A. castellanii responds to L. pneumophila infection. In this study, we examined the global transcriptional response of A. castellanii to L. pneumophila infection. We compared A. castellanii infected with wild type L. pneumophila to A. castellanii infected with an isogenic ΔdotA mutant strain, which is unable to replicate intracellularly. We showed that A. castellanii underwent clear morphological and transcriptional rewiring over the course of L. pneumophila infection. Through improved annotation of the A. castellanii genome, we determined that these transcriptional changes primarily involved biological processes utilizing small GTPases, including cellular transport, signaling, metabolism and replication. In addition, a number of sirtuin-encoding genes in A. castellanii were found to be conserved and upregulated during L. pneumophila infection. Silencing of sirtuin gene, sir6f (ACA1_153540) resulted in the inhibition of A. castellanii cell proliferation during infection and reduced L. pneumophila replication. Overall our findings identified several biological pathways in amoebae that may support L. pneumophila replication and A. castellanii proliferation in environmental conditions.

嗜肺军团菌（Legionella pneumophila）是一类环境源性细菌，其演化出了在土壤与水生变形虫（如卡氏棘阿米巴（Acanthamoeba castellanii））的捕食压力下存活的能力，这一过程无意间赋予了该菌在人类细胞内存活与增殖的能力。嗜肺军团菌可引发军团菌病（Legnaire's Disease），人类通过吸入携带变形虫与该菌的水气溶胶而发生暴露。这类气溶胶源自人工水源中的水生生物膜，例如空调冷却塔与加湿器。在这些人造环境中，卡氏棘阿米巴会支持嗜肺军团菌的胞内增殖，进而促进该菌的存续与传播，并为其抵御外界应激提供庇护。尽管二者存在紧密的演化关联，目前学界对卡氏棘阿米巴如何响应嗜肺军团菌感染的分子机制仍知之甚少。本研究针对卡氏棘阿米巴在嗜肺军团菌感染后的全局转录响应展开了系统性分析。我们将感染野生型嗜肺军团菌的卡氏棘阿米巴，与感染同基因ΔdotA突变菌株（该菌株无法进行胞内增殖）的卡氏棘阿米巴进行了对照实验。结果显示，在嗜肺军团菌感染进程中，卡氏棘阿米巴出现了显著的形态学与转录组重编程现象。通过对卡氏棘阿米巴基因组进行优化注释，我们确定这些转录变化主要涉及依赖小GTP酶的生物学过程，包括细胞运输、信号转导、代谢与细胞增殖。此外，研究发现卡氏棘阿米巴中的多个编码去乙酰化酶（sirtuin）的基因在嗜肺军团菌感染期间呈现保守表达并发生上调。对去乙酰化酶基因sir6f（ACA1_153540）进行基因沉默后，会抑制感染过程中卡氏棘阿米巴的细胞增殖，并降低嗜肺军团菌的增殖水平。综上，本研究的发现明确了变形虫中若干可能在环境条件下支持嗜肺军团菌增殖与卡氏棘阿米巴自身增殖的生物学通路。