Jin Bo, Newton Salete M C, Shao Yi, Jiang Xiaoxu, Charbit Alain, Klebba Phillip E
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019, USA.
Mol Microbiol. 2006 Feb;59(4):1185-98. doi: 10.1111/j.1365-2958.2005.05015.x.
Listeria monocytogenes is a Gram-positive bacterium that causes severe opportunistic infections in humans and animals. We biochemically characterized, for the first time, the iron uptake processes of this facultative intracellular pathogen, and identified the genetic loci encoding two of its membrane iron transporters. Strain EGD-e used iron complexes of hydroxamates (ferrichrome and ferrichrome A, ferrioxamine B), catecholates (ferric enterobactin, ferric corynebactin) and eukaryotic binding proteins (transferrin, lactoferrin, ferritin, haemoglobin). Quantitative determinations showed 10-100-fold lower affinity for ferric siderophores (Km approximately 1-10 nM) than Gram-negative bacteria, and generally lower uptake rates. Vmax for [59Fe]-enterobactin (0.15 pMol per 10(9) cells per minute) was 400-fold lower than that of Escherichia coli. For [59Fe]-corynebactin, Vmax was also low (1.2 pMol per 10(9) cells per minute), but EGD-e transported [59Fe]-apoferrichrome similarly to E. coli (Vmax=24 pMol per 10(9) cells per minute). L. monocytogenes encodes potential Fur-regulated iron transporters at 2.031 Mb (the fur-fhu region), 2.184 Mb (the feo region), 2.27 Mb (the srtB region) and 2.499 Mb (designated hupDGC region). Chromosomal deletions in the fur-fhu and hupDGC regions diminished iron uptake from ferric hydroxamates and haemin/haemoglobin respectively. In the former locus, deletion of fhuD (lmo1959) or fhuC (lmo1960) strongly reduced [59Fe]-apoferrichrome uptake. Deletion of hupC (lmo2429) eliminated the uptake of haemin and haemoglobin, and decreased the virulence of L. monocytogenes 50-fold in mice. Elimination of srtB region genes (Deltalmo2185, Deltalmo2186, Deltalmo2183), both sortase structural genes (DeltasrtB, DeltasrtA, DeltasrtAB), fur and feoB did not impair iron transport. However, deletion of bacterioferritin (Deltafri, lmo943; 0.97 Mb) decreased growth and altered iron uptake: Vmax of [59Fe]-corynebactin transport tripled in this strain, whereas that of [59Fe]-apoferrichrome decreased 20-fold.
单核细胞增生李斯特菌是一种革兰氏阳性细菌，可在人类和动物中引发严重的机会性感染。我们首次对这种兼性胞内病原体的铁摄取过程进行了生化特性分析，并鉴定了编码其两种膜铁转运蛋白的基因位点。菌株EGD-e利用异羟肟酸铁络合物（铁载体蛋白、铁载体蛋白A、去铁胺B）、儿茶酚酸铁络合物（肠杆菌素铁、棒杆菌素铁）以及真核结合蛋白（转铁蛋白、乳铁蛋白、铁蛋白、血红蛋白）。定量测定表明，与革兰氏阴性细菌相比，它对铁载体铁的亲和力低10 - 100倍（Km约为1 - 10 nM），且摄取速率通常较低。[59Fe] - 肠杆菌素的Vmax（每分钟每10^9个细胞0.15皮摩尔）比大肠杆菌低400倍。对于[59Fe] - 棒杆菌素，Vmax也较低（每分钟每10^9个细胞1.2皮摩尔），但EGD-e转运[59Fe] - 脱铁铁载体蛋白的情况与大肠杆菌相似（Vmax = 每分钟每10^9个细胞24皮摩尔）。单核细胞增生李斯特菌在2.031 Mb（fur - fhu区域）、2.184 Mb（feo区域）、2.27 Mb（srtB区域）和2.499 Mb（指定为hupDGC区域）编码潜在的Fur调节铁转运蛋白。fur - fhu区域和hupDGC区域的染色体缺失分别减少了从异羟肟酸铁和血红素/血红蛋白的铁摄取。在前一个基因位点，fhuD（lmo1959）或fhuC（lmo1960）的缺失强烈降低了[59Fe] - 脱铁铁载体蛋白的摄取。hupC（lmo2429）的缺失消除了血红素和血红蛋白的摄取，并使单核细胞增生李斯特菌在小鼠中的毒力降低了50倍。srtB区域基因（Deltalmo2185、Deltalmo2186、Deltalmo2183）、两种分选酶结构基因（DeltasrtB、DeltasrtA、DeltasrtAB）、fur和feoB的缺失均未损害铁转运。然而，细菌铁蛋白（Deltafri，lmo943；0.97 Mb）的缺失降低了生长并改变了铁摄取：该菌株中[59Fe] - 棒杆菌素转运的Vmax增加了两倍，而[59Fe] - 脱铁铁载体蛋白的Vmax降低了20倍。
