Experimental evolution of Legionella pneumophila in mouse macrophages leads to strains with altered determinants of environmental survival

The Gram-negative bacterium, Legionella pneumophila, is a protozoan parasite and accidental intracellular pathogen of humans. We propose a model in which host cycling through multiple protozoan hosts in the environment holds L. pneumophila in a state of evolutionary stasis as a broad host-range pathogen. Using an experimental evolution approach, we tested this hypothesis by restricting L. pneumophila to growth within mouse macrophages for hundreds of generations. Whole-genome resequencing and high-throughput genotyping identified several parallel adaptive mutations and population dynamics that led to improved replication within macrophages. Based on these results, we provide a detailed view of the population dynamics of an experimentally evolving bacterial population, punctuated by frequent instances of transient clonal interference and selective sweeps. Non-synonymous point mutations in the flagellar regulator, fleN, resulted in increased uptake and broadly increased replication in both macrophages and amoebae. Mutations in multiple steps of the lysine biosynthesis pathway were also independently isolated, resulting in lysine auxotrophy and reduced replication in amoebae. These results demonstrate that under laboratory conditions, host restriction is sufficient to rapidly modify L. pneumophila fitness and host range. We hypothesize that, in the environment, host cycling prevents L. pneumophila host-specialization by maintaining pathways that are deleterious for growth in macrophages and other hosts.

革兰氏阴性菌嗜肺军团菌（Legionella pneumophila）是一种原生动物寄生虫，同时也是可偶然感染人类的胞内致病菌。我们提出如下模型：环境中宿主循环切换多种原生动物宿主的过程，会使嗜肺军团菌作为广谱宿主范围病原体维持进化停滞状态。本研究采用实验进化研究方法，通过将嗜肺军团菌限制在小鼠巨噬细胞内生长数百代，对该假说进行了验证。全基因组重测序（whole-genome resequencing）与高通量基因分型（high-throughput genotyping）技术鉴定出多组平行适应性突变与种群动态特征，这些突变与特征可提升嗜肺军团菌在巨噬细胞内的复制能力。基于上述结果，我们详细解析了实验进化细菌种群的种群动态：该过程频繁出现暂时性克隆干扰与选择性清除事件。鞭毛调控因子fleN发生的非同义点突变，可增强嗜肺军团菌对巨噬细胞与变形虫的摄取能力，并广谱提升其在这两类宿主中的复制效率。赖氨酸生物合成通路（lysine biosynthesis pathway）多个环节的突变亦被独立筛选得到，这类突变会导致嗜肺军团菌出现赖氨酸营养缺陷型（lysine auxotrophy），并降低其在变形虫内的复制能力。上述结果表明，在实验室条件下，宿主限制性培养足以快速改变嗜肺军团菌的适合度（fitness）与宿主范围（host range）。我们推测，在自然环境中，宿主循环切换的过程会保留那些在巨噬细胞及其他宿主内生长不利的通路，从而阻止嗜肺军团菌发生宿主特化。