Structure-function analysis reveals that the Pseudomonas aeruginosa Tps4 two-partner secretion system is involved in CupB5 translocation.
Structure-function analysis reveals that the Pseudomonas aeruginosa Tps4 two-partner secretion system is involved in CupB5 translocation.
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DOI：
10.1002/pro.2640
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发表时间：
2015-05
期刊：
影响因子：
8
通讯作者：
Filloux, Alain
中科院分区：
文献类型：
--
作者：
Garnett, James A.;Muhl, Daniela;Douse, Christopher H.;Hui, Kailyn;Busch, Andreas;Omisore, Ayodele;Yang, Yi;Simpson, Peter;Marchant, Jan;Waksman, Gabriel;Matthews, Steve;Filloux, Alain
铜绿假单胞菌是一种革兰氏阴性机会性细菌，与囊性纤维化患者同义，可引起肺部慢性感染。这种病原体是研究生物膜的一种模式生物：一种嵌入细胞外基质的细菌种群，它提供保护，免受环境压力并导致持久性。一些Chaperone-Usher通路，即CupA-CupE，通过在细菌表面组装粘连菌毛在这些过程中发挥关键作用。其中一个由cupB操纵子编码，它是独特的，因为它包含一个非伴侣引导基因产物CupB5。双伴侣分泌（TPS）系统由一个c端完整的膜β-桶状孔与位于外质中的串联n端POTRA（多肽运输相关）结构域（TpsB）和一个分泌底物（TpsA）组成。通过核磁共振，我们发现TpsB4 （LepB）与CupB5及其预测的同源伙伴TpsA4 （LepA）相互作用，这是一种细胞外蛋白酶。此外，通过细胞研究，我们证实TpsB4可以在铜绿假单胞菌外膜上转运CupB5，这与之前的观察结果形成了对比，即CupB3 P-usher分泌CupB5。为了支持我们的研究结果，我们还证明tps4/cupB操作子受RocS1传感器的协同调节，这表明铜绿假单胞菌在这些系统之间发展了协同作用。此外，我们已经确定了TpsB4- potra1结构域的溶液结构，并结合核磁共振化学位移定位和体内突变分析，我们计算了整个TpsB4周围质区与TpsA4和CupB5分泌基元复合物的模型。这些数据突出了TpsB4在外质中识别TpsA4/CupB5的特定残基，并表明每个POTRA结构域具有不同的作用。
Pseudomonas aeruginosa is a Gram-negative opportunistic bacterium, synonymous with cystic fibrosis patients, which can cause chronic infection of the lungs. This pathogen is a model organism to study biofilms: a bacterial population embedded in an extracellular matrix that provide protection from environmental pressures and lead to persistence. A number of Chaperone-Usher Pathways, namely CupA-CupE, play key roles in these processes by assembling adhesive pili on the bacterial surface. One of these, encoded by the cupB operon, is unique as it contains a nonchaperone-usher gene product, CupB5. Two-partner secretion (TPS) systems are comprised of a C-terminal integral membrane β-barrel pore with tandem N-terminal POTRA (POlypeptide TRansport Associated) domains located in the periplasm (TpsB) and a secreted substrate (TpsA). Using NMR we show that TpsB4 (LepB) interacts with CupB5 and its predicted cognate partner TpsA4 (LepA), an extracellular protease. Moreover, using cellular studies we confirm that TpsB4 can translocate CupB5 across the P. aeruginosa outer membrane, which contrasts a previous observation that suggested the CupB3 P-usher secretes CupB5. In support of our findings we also demonstrate that tps4/cupB operons are coregulated by the RocS1 sensor suggesting P. aeruginosa has developed synergy between these systems. Furthermore, we have determined the solution-structure of the TpsB4-POTRA1 domain and together with restraints from NMR chemical shift mapping and in vivo mutational analysis we have calculated models for the entire TpsB4 periplasmic region in complex with both TpsA4 and CupB5 secretion motifs. The data highlight specific residues for TpsA4/CupB5 recognition by TpsB4 in the periplasm and suggest distinct roles for each POTRA domain.
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