High extracellular levels of Mycobacterium tuberculosis glutamine synthetase and superoxide dismutase in actively growing cultures are due to high expression and extracellular stability rather than to a protein-specific export mechanism
High extracellular levels of Mycobacterium tuberculosis glutamine synthetase and superoxide dismutase in actively growing cultures are due to high expression and extracellular stability rather than to a protein-specific export mechanism
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DOI：
10.1128/iai.69.10.6348-6363.2001
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发表时间：
2001-10-01
影响因子：
3.1
通讯作者：
Horwitz, MA
中科院分区：
文献类型：
--
作者：
Tullius, MV;Harth, G;Horwitz, MA
谷氨酰胺合成酶（GS）和超氧化物歧化酶（SOD）是被认为在结核分枝杆菌的致病性中起重要作用的大的多聚体酶，是活跃生长的培养物中细菌的主要培养物滤液蛋白。虽然这些蛋白质缺乏前导肽，但它们在生长的早期阶段存在于细胞外培养基中表明它们可能是主动分泌的。为了了解它们的输出机制，我们从快速生长的非致病性耻垢分枝杆菌中克隆了同源基因（glnA 1和sodA），产生了耻垢分枝杆菌的glnA 1和sodA突变体。通过等位基因交换，以及在这些突变体中定量表达和输出分枝杆菌和非分枝杆菌GS和SOD。我们还定量表达和输出同源和异源SOD从M。结核当每一个基因都从多拷贝质粒中表达时，嗜酸粒细胞白血病输出的结核分枝杆菌和结核分枝杆菌比例相当。与先前在野生型菌株中的观察结果相反，smeglobulin GS（在glnA 1菌株中）或SOD（在sodA菌株中）。令人惊讶的是，重组的Al. smegalovirus和M.结核菌株甚至输出非分枝杆菌SOD。为了确定这些大的无前导蛋白的输出是表达依赖性的程度，我们构建了重组M。高水平表达绿色荧光蛋白（GFP）的结核分枝杆菌菌株和重组M.共表达M. smegalgus GS，M. smeegalyptus SOD和M.结核病BfrB（细菌铁蛋白）高水平。重组M.结核菌株甚至在生长的早期阶段输出GFP，并且其比例与内源性M.结核病GS和SOD。类似地，重组耻垢酸铝菌株输出细菌铁蛋白，一种大的（类似于500-kDa）、无前导的多聚体蛋白，其比例与GS和SOD相当。相比之下，高水平表达的大型，无领导，多聚体蛋白质苹果酸脱氢酶不会导致细胞外积累，因为蛋白质是高度不稳定的细胞外。这些结果表明，与预期相反，M。在活跃生长的培养物中，结核病GS和SOD的增加不是由于蛋白质特异性输出机制，而是由于细菌渗漏或自溶，并且这些酶的细胞外丰度仅仅是由于它们的高水平表达和细胞外稳定性。相同的决定因素可能解释了在活跃生长的M的细胞外介质中存在其他无前导蛋白。结核菌培养
Glutamine synthetase (GS) and superoxide dismutase (SOD), large multimeric enzymes that are thought to play important roles in the pathogenicity of Mycobacterium tuberculosis, are among the bacterium's major culture filtrate proteins in actively growing cultures. Although these proteins lack a leader peptide, their presence in the extracellular medium during early stages of growth suggested that they might be actively secreted. To understand their mechanism of export, we cloned the homologous genes (glnA1 and sodA) from the rapid-growing, nonpathogenic Mycobacterium smegmatis, generated glnA1 and sodA mutants of M. smegmatis by allelic exchange, and quantitated expression and export of both mycobacterial and nonmycobacterial GSs and SODs in these mutants. We also quantitated expression and export of homologous and heterologous SODs from M. tuberculosis. When each of the genes was expressed from a multicopy plasmid, AL smegmatis exported comparable proportions of both the Al. tuberculosis and M. smegmatis GSs (in the glnA1 strain) or SODs (in the sodA strain), in contrast to previous observations in wild-type strains. Surprisingly, recombinant Al. smegmatis and M. tuberculosis strains even exported nonmycobacterial SODs. To determine the extent to which,export of these large, leaderless proteins is expression dependent, we constructed a recombinant M. tuberculosis strain expressing green fluorescent protein (GFP) at high levels and a recombinant M. smegmatis strain coexpressing the M. smegmatis GS, M. smegmatis SOD, and M. tuberculosis BfrB (bacterioferritin) at high levels. The recombinant M. tuberculosis strain exported GFP even in early stages of growth and at proportions very similar to those of the endogenous M. tuberculosis GS and SOD. Similarly, the recombinant Al. smegmatis strain exported bacterioferritin, a large (similar to 500-kDa), leaderless, multimeric protein, in proportions comparable to GS and SOD. In contrast, high-level expression of the large, leaderless, multimeric protein malate dehydrogenase did not lead to extracellular accumulation because the protein was highly unstable extracellularly. These findings indicate that, contrary to expectations, export of M. tuberculosis GS and SOD in actively growing cultures is not due to a protein-specific export mechanism, but rather to bacterial leakage or autolysis, and that the extracellular abundance of these enzymes is simply due to their high level of expression and extracellular stability. The same determinants likely explain the presence of other leaderless proteins in the extracellular medium of actively growing M. tuberculosis cultures.