Transfer of R388 Derivatives by a Pathogenesis-Associated Type IV Secretion System into both Bacteria and Human Cells
Transfer of R388 Derivatives by a Pathogenesis-Associated Type IV Secretion System into both Bacteria and Human Cells
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DOI：
10.1128/jb.05905-11
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发表时间：
2011-11-01
影响因子：
3.2
通讯作者：
Llosa, Matxalen
中科院分区：
文献类型：
--
作者：
Fernandez-Gonzalez, Esther;de Paz, Hector D.;Llosa, Matxalen
细菌 IV 型分泌系统 (T4SS) 参与细菌结合和蛋白质易位至动物细胞等过程。在这项工作中，我们改变了参与 DNA 转移致病性的 T4SS 的底物。含有质粒 R388 的部分接合机制的质粒通过人兼性细胞内病原体汉赛巴尔通体的 T4SS 转移至受体细菌和人血管内皮细胞。大约 2% 的人类细胞表达来自该质粒的绿色荧光蛋白 (GFP) 基因。不同大小的质粒以相似的效率转移。 B. henselae 编码两个 T4SS：VirB/VirD4 和 Trw。 Delta virB 突变株存在转移缺陷，而 Delta trwE 突变株的 DNA 转移仅受到轻微损害。在所有情况下，DNA 转移都依赖于 R388 的蛋白质 TrwC（接合松弛酶），这意味着它是通过类似接合的机制发生的。 TrwC 的 DNA 解旋酶缺陷突变体不能促进 DNA 转移。在缺少 R388 的偶联蛋白 TrwB 的情况下，DNA 转移效率下降 1 log。在涉及与 T4SS 相互作用的区域中的 TrwB 点突变也获得了相同的低效率。 TrwB 在细菌双杂交测定中与 VirB10 相互作用，表明它可能充当 VirB/VirD4 T4SS 的 R388 底物的招募者。 TrwB ATPase 突变体表现为显性失活，将 DNA 转移效率降至几乎为零的水平。从受感染的人类细胞中回收的 B. henselae 细菌在某些条件下可以将可移动质粒转移到受体大肠杆菌中，这强调了 T4SS 的多功能性。
Bacterial type IV secretion systems (T4SSs) are involved in processes such as bacterial conjugation and protein translocation to animal cells. In this work, we have switched the substrates of T4SSs involved in pathogenicity for DNA transfer. Plasmids containing part of the conjugative machinery of plasmid R388 were transferred by the T4SS of human facultative intracellular pathogen Bartonella henselae to both recipient bacteria and human vascular endothelial cells. About 2% of the human cells expressed a green fluorescent protein (GFP) gene from the plasmid. Plasmids of different sizes were transferred with similar efficiencies. B. henselae codes for two T4SSs: VirB/VirD4 and Trw. A Delta virB mutant strain was transfer deficient, while a Delta trwE mutant was only slightly impaired in DNA transfer. DNA transfer was in all cases dependent on protein TrwC of R388, the conjugative relaxase, implying that it occurs by a conjugation-like mechanism. A DNA helicase-deficient mutant of TrwC could not promote DNA transfer. In the absence of TrwB, the coupling protein of R388, DNA transfer efficiency dropped 1 log. The same low efficiency was obtained with a TrwB point mutation in the region involved in interaction with the T4SS. TrwB interacted with VirB10 in a bacterial two-hybrid assay, suggesting that it may act as the recruiter of the R388 substrate for the VirB/VirD4 T4SS. A TrwB ATPase mutant behaved as dominant negative, dropping DNA transfer efficiency to almost null levels. B. henselae bacteria recovered from infected human cells could transfer the mobilizable plasmid into recipient Escherichia coli under certain conditions, underscoring the versatility of T4SSs.