Identification of novel genes in genornic islands that contribute to Salmonella typhimurium replication in macrophages
Identification of novel genes in genornic islands that contribute to Salmonella typhimurium replication in macrophages
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DOI：
10.1099/mic.0.2006/004747-0
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发表时间：
2007-04-01
期刊：
影响因子：
2.8
通讯作者：
Fuchs, Thilo M.
中科院分区：
文献类型：
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作者：
Klumpp, Jochen;Fuchs, Thilo M.
鼠伤寒沙门氏菌（S. typhimurium）在巨噬细胞内生存和增殖。为了鉴定野生型样细胞内复制所必需的基因，对基于同源重组基因破坏的ATCC 14028菌株的突变体文库进行了筛选。从鼠伤寒沙门氏菌基因组随机产生的染色体片段被克隆到一个温度敏感型载体中，并且通过在非许可温度下选择的插入 - 重复诱变（IDM）获得了大约8000个单独的突变体克隆。在小鼠巨噬细胞中对复制缺陷进行大规模筛选（但在丰富或基本培养基中生长时没有），揭示了一组减毒突变体，通过对诱变片段进行PCR扩增和测序进一步对其进行了表征。在对带有载体插入注释位置的沙门氏菌基因组图谱进行分析之后，发现10个非共线区域的基因内有33个减毒插入的聚集。virK、gipA和5个SPI - 2基因以及7个非极性缺失中的插入验证了该筛选。未观察到突变体的入侵缺陷。含有钴胺素合成和1,2 - 丙二醇降解基因的cob - cbi - pdu簇被证明是沙门氏菌在巨噬细胞内复制所必需的。这些数据产生了一个真核糖缀合物和磷脂作为细胞内复制细菌的替代碳、氮和能量来源的模型。报道了SPI - 6以及Gifsy - 1和Gifsy - 2原噬菌体岛尚未知晓的成分对细胞内复制的贡献，以及缺失STM1677（可能编码一种LysR样转录调节因子）的突变体细胞内生长速率降低五倍的情况。三个双突变体（每个都缺失cob - cbi - pdu簇或Gifsy - 1原噬菌体的两种基因产物）的细胞内复制速率被证明低于相应的单突变体，这表明细微的细胞内优势的累加效应有助于沙门氏菌在体内的适应性。
Salmonella enterica serovar Typhimurium (S. typhimurium) survives and proliferates within macrophage cells. A mutant library of strain ATCC 14028 based on gene disruption by homologous recombination was screened in order to identify genes that are required for wild-type-like intracellular replication. Randomly generated chromosomal fragments from the genome of S. typhimurium were cloned into a temperature-sensitive vector, and approximately 8000 individual mutant clones were obtained by insertional-duplication mutagenesis (IDM) upon selection at non-permissive temperature. Large-scale screening for replication defects in mouse macrophages, but not during growth in rich or minimal medium, revealed a set of attenuated mutants that were further characterized by PCR amplification and sequencing of the mutagenic fragments. Following analysis of a Salmonella genome map with the annotated positions of vector insertions, an accumulation of 33 attenuating insertions within genes of ten non-collinear regions was found. Insertions in virK, gipA and five SPI-2 genes as well as seven non-polar deletions validated the screen. No invasion deficiencies of the mutants were observed. The cob-cbi-pdu cluster containing the genes for cobalamin synthesis and 1,2-propanediol degradation was shown to be required for Salmonella replication within macrophages. These data gave rise to a model of eukaryotic glycoconjugates and phospholipids as alternative carbon, nitrogen and energy sources for intracellularly replicating bacteria. The contribution of as yet unknown components of SPI-6 and the Gifsy-1 and Gifsy-2 prophage islands to intracellular replication is reported, as well as the fivefold reduced intracellular growth rate of a mutant with a deletion of STM1677, which probably encodes a LysR-like transcriptional regulator. The intracellular replication rate of three double mutants, each lacking two gene products of the cob-cbi-pdu cluster or the Gifsy-1 prophage, was shown to be lower than that of the respective single mutants, suggesting that additive effects of subtle intracellular advantages contribute to Salmonella fitness in vivo.