The amoebae plate test implicates a paralogue of LpxB in the interaction of Legionella pneumophila with Acanthamoeba castellanii
The amoebae plate test implicates a paralogue of LpxB in the interaction of Legionella pneumophila with Acanthamoeba castellanii
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DOI：
10.1099/mic.0.27563-0
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发表时间：
2005-01-01
期刊：
影响因子：
2.8
通讯作者：
Hilbi, H
中科院分区：
文献类型：
--
作者：
Albers, U;Reus, K;Hilbi, H
嗜肺军团菌是一种淡水变形虫的细菌寄生虫，它也生长在肺泡巨噬细胞中，从而导致可能致命的肺炎军团病。阿米巴和巨噬细胞的细胞内生长机制相似，需要1cm/Dot型IV分泌系统。本文报道了一种阿米巴平板试验（APT）的发展，以分析在鹿角棘阿米巴存在的琼脂平板上发现的嗜肺乳杆菌野生型和icm/dot突变株的生长情况。在APT中，野生型嗜肺乳杆菌即使在高稀释条件下也能形成健壮的菌落，icmT、-R、-P或dotB突变体不能生长，icmS或-G突变体部分生长缺陷。利用icmS或icmG突变株在嗜肺乳杆菌染色体文库中筛选在变形虫存在时抑制生长缺陷的基因。一个含有icmS和两侧icm基因的icmS抑制质粒被分离出来，表明该质粒弥补了突变体的细胞内生长缺陷。相比之下，不同的icmG抑制质粒使icmG突变体对A. castellanii具有更强的细胞毒性，而不增强变形虫或RAW264.7巨噬细胞的细胞内增殖。在抑制质粒插入物中删除单个基因，鉴定出军团菌细胞毒性抑制因子ic（军团菌细胞毒性抑制因子）-A、-B、-C和-D是增强icmG突变株细胞毒性所必需的。相应的蛋白分别与水解酶、nlp相关金属蛋白酶、脂质A双糖合成酶和ABC转运蛋白序列相似。LcsC是脂质a双糖合成酶LpxB的一种推测的类似物，其过表达增加了icmG突变株对castellanii的细胞毒性，但其他icm/dot或rpoS突变株对castellanii的细胞毒性没有增加。基于序列比较和染色体定位，IcsB和IcsC可能编码参与细胞壁维持和肽聚糖代谢的酶。本文建立的APT可能有助于识别与阿米巴原虫宿主相互作用相关的其他细菌因子。
Legionella pneumophila is a bacterial parasite of freshwater amoebae which also grows in alveolar macrophages and thus causes the potentially fatal pneumonia Legionnaires' disease. Intracellular growth within amoebae and macrophages is mechanistically similar and requires the 1cm/Dot type IV secretion system. This paper reports the development of an assay, the amoebae plate test (APT), to analyse growth of L. pneumophila wild-type and icm/dot mutant strains spotted on agar plates in the presence of Acanthamoeba castellanii. In the APT, wild-type L. pneumophila formed robust colonies even at high dilutions, icmT, -R, -P or dotB mutants failed to grow, and icmS or -G mutants were partially growth defective. The icmS or icmG mutant strains were used to screen an L. pneumophila chromosomal library for genes that suppress the growth defect in the presence of the amoebae. An icmS suppressor plasmid was isolated that harboured the icmS and flanking icm genes, indicating that this plasmid complements the intracellular growth defect of the mutant. In contrast, different icmG suppressor plasmids rendered the icmG mutant more cytotoxic for A. castellanii without enhancing intracellular multiplication in amoebae or RAW264.7 macrophages. Deletion of individual genes in the suppressor plasmids inserts identified Ics (Legionella cytotoxic suppressor) -A, -B, -C and -D as being required for enhanced cytotoxicity of an icmG mutant strain. The corresponding proteins show sequence similarity to hydrolases, NlpD-related metalloproteases, lipid A disaccharide synthases and ABC transporters, respectively. Overexpression of LcsC, a putative paralogue of the lipid A disaccharide synthase LpxB, increased cytotoxicity of an icmG mutant but not that of other icm/dot or rpoS mutant strains against A. castellanii. Based on sequence comparison and chromosomal location, IcsB and IcsC probably encode enzymes involved in cell wall maintenance and peptidoglycan metabolism. The APT established here may prove useful to identify other bacterial factors relevant for interactions with amoeba hosts.