Abstract：
Many proteins of the Repeats in Toxins (RTX) protein family are toxins of Gram-negative pathogens including hemolysin A (HlyA) of uropathogenic E. coli. RTX proteins are secreted via Type I secretion systems (T1SS) and adopt their native conformation in the Ca2+-rich extracellular environment. Here we employed the E. coli HlyA T1SS as a heterologous surrogate system for the RTX toxin MbxA from the bovine pathogen Moraxella bovis. In E. coli the HlyA system successfully activates the heterologous MbxA substrate by acylation and secretes the precursor proMbxA and active MbxA allowing purification of both species in quantities sufficient for a variety of investigations. The activating E. coli acyltransferase HlyC recognizes the acylation sites in MbxA, but unexpectedly in a different acylation pattern as for its endogenous substrate HlyA. HlyC-activated MbxA shows host species-independent activity including a so-far unknown toxicity against human lymphocytes and epithelial cells. Using live-cell imaging, we show an immediate MbxA-mediated permeabilization and a rapidly developing blebbing of the plasma membrane in epithelial cells, which is associated with immediate cell death.
摘要：
毒素重复序列(RTX)蛋白质家族的许多蛋白质是革兰氏阴性病原体的毒素,包括泌尿致病性大肠杆菌的溶血素A(HlyA)。RTX蛋白通过I型分泌系统（T1SS）分泌，并在富含Ca2的细胞外环境中采用其天然构象。在这里，我们采用大肠杆菌HlyAT1SS作为来自牛病原体牛莫拉氏菌的RTX毒素MbxA的异源替代系统。在大肠杆菌中,HlyA系统通过酰化成功地激活异源MbxA底物,并分泌前体proMbxA和活性MbxA,从而允许以足以进行各种研究的量纯化两种物种。活化的大肠杆菌酰基转移酶HlyC识别MbxA中的酰化位点，但出乎意料的是其内源性底物HlyA的酰化模式不同。HlyC激活的MbxA显示出宿主物种无关的活性，包括对人淋巴细胞和上皮细胞的迄今未知的毒性。使用活细胞成像，我们显示了立即MbxA介导的透化和上皮细胞质膜的快速发展的起泡，这与细胞立即死亡有关。
