A mycobacterial virulence gene cluster extending RD1 is required for cytolysis, bacterial spreading and ESAT-6 secretion
A mycobacterial virulence gene cluster extending RD1 is required for cytolysis, bacterial spreading and ESAT-6 secretion
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DOI：
10.1111/j.1365-2958.2004.04261.x
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发表时间：
2004-09-01
影响因子：
3.6
通讯作者：
Brown, EJ
中科院分区：
文献类型：
--
作者：
Gao, LY;Guo, S;Brown, EJ
在易感宿主中分枝杆菌感染的启动和维持尚不清楚。通过对海洋分枝杆菌转座子突变体库的筛选，分离到8个不能引起溶血的突变体，这些突变体都在结核分枝杆菌Rv 3866和Rv 3881 c之间的同源区域中具有转座子插入，该区域包括已知毒力基因簇RD 1（Rv 3871-Rv 3879 c）。分枝marinum突变体在体内毒力下降，不能分泌ESAT-6，与M.结核病RD 1突变体。M.与Rv 3866-Rv 3868同源的基因中的marinum突变体也不能积累细胞内ESAT-6，这表明这些基因在蛋白质的合成或稳定性中可能起作用。这些转座子突变体和ESAT-6/CFP-10缺失突变体均显示出对巨噬细胞的细胞溶解和细胞毒性降低，并且仅当细胞以低感染复数感染时才在感染的晚期阶段显著降低细胞内生长，这表明传播缺陷。野生型M细胞间传播的直接证据。在巨噬细胞和上皮细胞单层中通过显微镜检测获得marinum，但突变体在该测定中均为缺陷型。表达M.结核同源物与相应的M. marinum突变体，强调M.结核和M.我们将该区域中的marinum基因命名为extRD 1（extendedRD 1）。我们认为，减少膜溶解活性和有缺陷的传播是extRD 1突变体的衰减机制。这些结果扩展了最近关于M的基因组边界和功能的发现。结核病RD 1和建立extRD 1在分枝杆菌毒力中发挥作用的分子细胞基础。M的破坏。Rv 3881 c的marinum同源物，以前没有涉及毒力，导致巨噬细胞和体内的表型更加减毒，表明该基因在M.在宿主体内存活。
Initiation and maintenance of infection by mycobacteria in susceptible hosts are not well understood. A screen of Mycobacterium marinum transposon mutant library led to isolation of eight mutants that failed to cause haemolysis, all of which had transposon insertions in genes homologous to a region between Rv3866 and Rv3881c in Mycobacterium tuberculosis, which encompasses RD1 (Rv3871-Rv3879c), a known virulence gene cluster. The M. marinum mutants showed decreased virulence in vivo and failed to secrete ESAT-6, like M. tuberculosis RD1 mutants. M. marinum mutants in genes homologous to Rv3866-Rv3868 also failed to accumulate intracellular ESAT-6, suggesting a possible role for those genes in synthesis or stability of the protein. These transposon mutants and an ESAT-6/CFP-10 deletion mutant all showed reduced cytolysis and cytotoxicity to macrophages and significantly decreased intracellular growth at late stages of the infection only when the cells were infected at low multiplicity of infection, suggesting a defect in spreading. Direct evidence for cell-to-cell spread by wild-type M. marinum was obtained by microscopic detection in macrophage and epithelial monolayers, but the mutants all were defective in this assay. Expression of M. tuberculosis homologues complemented the corresponding M. marinum mutants, emphasizing the functional similarities between M. tuberculosis and M. marinum genes in this region that we designate extRD1 (extended RD1). We suggest that diminished membranolytic activity and defective spreading is a mechanism for the attenuation of the extRD1 mutants. These results extend recent findings on the genomic boundaries and functions of M. tuberculosis RD1 and establish a molecular cellular basis for the role that extRD1 plays in mycobacterial virulence. Disruption of the M. marinum homologue of Rv3881c, not previously implicated in virulence, led to a much more attenuated phenotype in macrophages and in vivo, suggesting that this gene plays additional roles in M. marinum survival in the host.