The Legionella pneumophila icmGCDJBF Genes Are Required for Killing of Human Macrophages

Previously, a collection of mutants of Legionella pneumophila that had lost the ability to multiply within and kill human macrophages was generated by Tn903dIIlacZ transposon mutagenesis and classified into DNA hybridization groups. A subset of these mutants was complemented by a plasmid, pMW100, containing a 13.5-kb genomic DNA insert. This plasmid restored the ability to multiply within and produce cytopathic effects on human macrophages to members of DNA hybridization groups II, IV, VI, and XVII. A region of the genomic insert of pMW100 was sequenced, and eight potential genes were identified and named icmE, icmG, icmC, icmD, icmJ, icmB, icmF, and tphA. None of the genes encode potential protein products with significant homology to previously characterized proteins, except for tphA, whose product has significant homology to a family of metabolite/H(+) symport proteins from gram-negative bacteria. The positions of the Tn903dIIlacZ insertions within the genes were determined by nucleotide sequencing. No Tn903dIIlacZ insertions mapped to icmG, icmJ, or tphA; therefore, these loci were mutated to test whether they were required for macrophage killing. Complementation analysis was used to evaluate the roles of the potential gene products and provide information on the organization of transcriptional units within the region. The results indicate that all identified open reading frames except tphA are required for killing of human macrophages.

此前，研究人员通过Tn903dIIlacZ转座子诱变技术构建了一批丧失在人类巨噬细胞内增殖并杀伤巨噬细胞能力的嗜肺军团菌（Legionella pneumophila）突变体菌株，并将这些突变体划分为不同的DNA杂交群。其中部分突变体可被携带13.5kb基因组DNA插入片段的质粒pMW100实现功能互补。该质粒可恢复DNA杂交群II、IV、VI及XVII组突变体在人类巨噬细胞内增殖并引发细胞病变效应的能力。研究人员对pMW100的基因组插入片段区域进行测序，共鉴定出8个潜在基因，并将其命名为icmE、icmG、icmC、icmD、icmJ、icmB、icmF及tphA。除tphA外，其余基因编码的潜在蛋白产物均未与已表征的蛋白存在显著同源性；tphA的编码产物与革兰氏阴性菌中的一类代谢物/H+同向转运蛋白家族具有显著同源性。研究人员通过核苷酸测序确定了Tn903dIIlacZ转座子在各基因内的插入位点。未在icmG、icmJ及tphA基因中检测到Tn903dIIlacZ转座子插入，因此研究人员对这三个基因座进行定向突变，以验证其是否参与巨噬细胞杀伤过程。研究人员通过互补分析评估了各潜在基因产物的功能，并解析了该区域内转录单元的组织模式。实验结果表明，除tphA外，所有鉴定出的开放阅读框均为人类巨噬细胞杀伤过程所必需。