Gene-specific effects of antisense phosphorodiamidate morpholino oligomer-peptide conjugates on Escherichia coli and Salmonella enterica serovar typhimurium in pure culture and in tissue culture
Gene-specific effects of antisense phosphorodiamidate morpholino oligomer-peptide conjugates on Escherichia coli and Salmonella enterica serovar typhimurium in pure culture and in tissue culture
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DOI：
10.1128/aac.01286-05
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发表时间：
2006-08-01
影响因子：
4.9
通讯作者：
Geller, Bruce L.
中科院分区：
文献类型：
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作者：
Tilley, Lucas D.;Hine, Orion S.;Geller, Bruce L.
目的是通过提高细菌细胞对反义磷酸二酯morpholino寡聚物（PMOs）的摄取来提高其功效。四种不同的细菌渗透肽，PFFRFFRFFXB， RTRTRFLRRTXB， RXXRXXRXXB和kffkffkxb （X是6-氨基已酸，B是β -丙氨酸），分别偶联到两个不同的PMOs上，这两个PMOs与荧光素酶报告基因（Inc）和生存所需基因（acpP）的起始密码子附近区域互补。在大肠杆菌的生长培养中，针对Inc的Luc肽- pmos抑制了23%至80%的荧光素酶活性。在无细胞翻译反应中，Luc RTRTRFLRRTXB-PMO对荧光素酶合成的抑制作用明显高于其他Luc肽-PMO或未偶联于肽的Luc PMO。以AcpP为靶点的肽- pmos对大肠杆菌或肠炎沙门氏菌血清型鼠伤寒杆菌的生长有不同程度的抑制作用，这取决于菌株。AcpP RFFRFFRFFXB-PMO、AcpP RTRTRFLRRTXB-PMO、AcpP KFFKFFKFFKXB-PMO和氨苄西林在大肠杆菌W3110中，生长8it后使CFU/ml降低50%的浓度（50%抑制浓度[IC50]）分别为3.6、10.8、9.5和7.5 μ M。通过挽救在AcpP肽- pmo靶向区域沉默突变的表达AcpP的mero二倍体菌株的生长，显示了AcpP肽- pmo的序列特异性效应。在感染肠致病性大肠杆菌（EPEC）的Caco-2培养物中，10 μ M AcpP RTRTRFLRR TXB-PMO或AcpP RFFRFFRFFXB-PMO基本上清除了感染。AcpP RTRTR FLRRTXB-PMO或AcpP RFFRFFRFFXB-PMO在epec感染的Caco-2培养物中的IC50均为3 μ m。综上所述，RFFRFFRFFXB、RTRTRFLRRTXB或KFFKFFKFFXB与PMO共价结合时，与未附着肽的PMOs相比，显著增强了靶基因的抑制作用。
The objective was to improve efficacy of antisense phosphorodiamidate morpholino oligomers (PMOs) by improving their uptake into bacterial cells. Four different bacterium-permeating peptides, PFFRFFRFFXB, RTRTRFLRRTXB, RXXRXXRXXB, and KFFKFFKFFKXB (X is 6-aminohexanoic acid and B is beta-alanine), were separately coupled to two different PMOs that are complementary to regions near the start codons of a luciferase reporter gene (Inc) and a gene required for viability (acpP). Luc peptide-PMOs targeted to Inc inhibited luciferase activity 23 to 80% in growing cultures of Escherichia coli. In cell-free translation reactions, Luc RTRTRFLRRTXB-PMO inhibited luciferase synthesis significantly more than the other Luc peptide-PMOs or the Luc PMO not coupled to peptide. AcpP peptide-PMOs targeted to acpP inhibited growth of E. coli or Salmonella enterica serovar Typhimurium to various extents, depending on the strain. The concentrations of AcpP RFFRFFRFFXB-PMO, AcpP RTRTRFLRRTXB-PMO, AcpP KFFKFFKFFKXB-PMO, and ampicillin that reduced CFU/ml by 50% after 8 It of growth (50% inhibitory concentration [IC50]) were 3.6, 10.8, 9.5, and 7.5 mu M, respectively, in E. coli W3110. Sequence-specific effects of AcpP peptide-PMOs were shown by rescuing growth of a merodiploid strain that expressed acpP with silent mutations in the region targeted by AcpP peptide-PMO. In Caco-2 cultures infected with enteropathogenic E. coli (EPEC), 10 mu M AcpP RTRTRFLRR TXB-PMO or AcpP RFFRFFRFFXB-PMO essentially cleared the infection. The IC50 of either AcpP RTRTR FLRRTXB-PMO or AcpP RFFRFFRFFXB-PMO in EPEC-infected Caco-2 culture was 3 mu M. In summary, RFFRFFRFFXB, RTRTRFLRRTXB, or KFFKFFKFFXB, when covalently bonded to PMO, significantly increased inhibition of expression of targeted genes compared to PMOs without attached peptide.