Characterization of the role of the ToxR-modulated outer membrane porins OmpU and OmpT in Vibrio cholerae virulence
Characterization of the role of the ToxR-modulated outer membrane porins OmpU and OmpT in Vibrio cholerae virulence
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DOI：
10.1128/jb.183.12.3652-3662.2001
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发表时间：
2001-06-01
影响因子：
3.2
通讯作者：
Klose, KE
中科院分区：
文献类型：
--
作者：
Provenzano, D;Lauriano, CM;Klose, KE
ToxR是霍乱弧菌表达毒力因子所需的跨膜调节蛋白，它直接激活和抑制两种外膜蛋白OmpU和OmpT的转录。在霍乱弧菌ompU和ompT的编码序列中构建了框内染色体缺失，在ompU中引入两个不同的缺失，以探讨ompU和ompT蛋白在霍乱弧菌存活和毒力因子表达中的作用；第一个(小)缺失的Delta ompU1去除了84个内部氨基酸(AA)的编码序列，而第二个(大)缺失Delta ompU2去除了完整的氨基端274aa的编码序列，Delta ompU1的生长缺陷不能被全长ompU的表型表达所补充，相反，Delta ompU2的菌株在丰富的培养基中表现出野生型的生长动力学，这表明这是缺乏OmpU的菌株的真实表型，并且截短的OmpU蛋白而不是OmpU的缺失可能是Delta ompU1表型的原因。在野生型以及Delta toxR和Delta ompU2菌株中也构建了一个删除了OmpT(Delta OmpT)全长273aa编码序列的大片段，这些菌株在富培养基中表现出野生型生长动力学，但Delta ompU2菌株与野生型、Delta ompT和Delta ompU2 Delta ompT菌株相比，在脱氧胆酸盐中缺乏生长，从而加强了对OmpU孔蛋白的正面作用，而对对阴离子洗涤剂的耐药性具有负面作用。Delta ompU2、Delta ompT和Delta ompU2 Delta ompT菌株在体外显示出野生型毒力因子表达水平，对多粘菌素B和血清以及体内定植水平与野生型菌株相似。我们的结果表明：(I)OmpU和OmpT不是先前认为的必需蛋白质；(Ii)这些孔蛋白有助于霍乱弧菌对阴离子洗涤剂的抵抗力；(Iii)OmpU和OmpT不是毒力因子体外表达或体内肠道定植所必需的。
ToxR, the transmembrane regulatory protein required for expression of virulence factors in the human diarrheal pathogen Vibrio cholerae, directly activates and represses the transcription of two outer membrane porins, OmpU and OmpT, respectively. In an attempt to dissect the role of the OmpU and OmpT porins in viability and virulence factor expression, in-frame chramosomal deletions were constructed in the coding sequences of ompU and ompT of V. cholerae, Two separate deletions were introduced into ompU; the first (small) deletion, Delta ompU1, removed the coding sequence for 84 internal amino acids (aa), while the second (large) deletion, Delta ompU2, removed the coding sequence for the entire amino-terminal 274 aa, The Delta ompU1 strain had a growth defect that could not be complemented by episomal expression of full-length ompU, In contrast, a strain with Delta ompU2 displayed wild-type growth kinetics in rich media, suggesting that this is the true phenotype of a strain lacking OmpU and that the truncated OmpU protein, rather than the absence of OmpU, may be the cause for the Delta ompU1 phenotype, A large deletion removing the coding sequence for the entire N-terminal 273 aa of OmpT (Delta ompT was also constructed in wild-type as well as Delta toxR and Delta ompU2 strains, and these strains displayed wild-type growth kinetics in rich media, However, the Delta ompU2 strain was deficient for growth in deoxycholate compared to wild-type, Delta ompT, and Delta ompU2 Delta ompT strains, reinforcing a positive role far the OmpU porin and a negative role for the OmpT porin in V. cholerae resistance to anionic detergents. The Delta ompU2, Delta ompT, and Delta ompU2 Delta ompT strains exhibited wild-type levels of in vitro virulence factor expression and resistance to polymyxin B and serum and in vivo colonization levels similar to a wild-type strain in the infant mouse intestine. Our results demonstrate that (i) OmpU and OmpT are not essential proteins, as was previously thought; (ii) these porins contribute to V. cholerae resistance to anionic detergents; and (iii) OmpU and OmpT are not essential for virulence factor expression in vitro or intestinal colonization in vivo.