Institute of Biochemistry, Heinrich-Heine-Universität, Düsseldorf, Germany.
Center for Advanced Imaging (CAi), Heinrich-Heine-Universität, Düsseldorf, Germany.
Appl Environ Microbiol. 2022 Feb 8;88(3):e0189621. doi: 10.1128/AEM.01896-21. Epub 2021 Dec 1.
Secretion systems are essential for Gram-negative bacteria, as these nanomachineries allow communication with the outside world by exporting proteins into the extracellular space or directly into the cytosol of a host cell. For example, type I secretion systems (T1SS) secrete a broad range of substrates across both membranes into the extracellular space. One well-known example is the hemolysin A (HlyA) T1SS from Escherichia coli, which consists of an ABC transporter (HlyB), a membrane fusion protein (HlyD), the outer membrane protein TolC, and the substrate HlyA, a member of the family of repeats in toxins (RTX) toxins. Here, we determined the amount of TolC at the endogenous level (parental strain, UTI89) and under conditions of overexpression [T7 expression system, BL21(DE3)-BD]. The overall amount of TolC was not influenced by the overexpression of the HlyBD complex. Moving one step further, we determined the localization of the HlyA T1SS by superresolution microscopy. In contrast to other bacterial secretion systems, no polarization was observed with respect to endogenous or overexpression levels. Additionally, the cell growth and division cycle did not influence polarization. Most importantly, the size of the observed T1SS clusters did not correlate with the recently proposed outer membrane islands. These data indicate that T1SS clusters at the outer membrane, generating domains of so-far-undescribed identity. Uropathogenic Escherichia coli (UPEC) strains cause about 110 million urinary tract infections each year worldwide, representing a global burden to the health care system. UPEC strains secrete many virulence factors, among these, the TX toxin hemolysin A via a cognate T1SS into the extracellular space. In this study, we determined the endogenous copy number of the HlyA T1SS in UTI89 and analyzed the surface localization in BL21(DE3)-BD and UTI89, respectively. With approximately 800 copies of the T1SS in UTI89, this is one of the highest expressed bacterial secretion systems. Furthermore, and in clear contrast to other secretion systems, no polarized surface localization was detected. Finally, quantitative analysis of the superresolution data revealed that clusters of the HlyA T1SS are not related to the recently identified outer membrane protein islands. These data provide insights into the quantitative molecular architecture of the HlyA T1SS.
分泌系统对于革兰氏阴性菌至关重要，因为这些纳米机器通过将蛋白质输出到细胞外空间或直接输出到宿主细胞的细胞质中来与外界进行通讯。例如，I 型分泌系统 (T1SS) 将广泛的底物穿过两个膜分泌到细胞外空间。一个众所周知的例子是大肠杆菌的溶血素 A (HlyA) T1SS，它由 ABC 转运体 (HlyB)、膜融合蛋白 (HlyD)、外膜蛋白 TolC 和底物 HlyA 组成，HlyA 是毒素重复家族的成员 (RTX) 毒素。在这里，我们确定了内源性水平（亲本菌株，UTI89）和过表达条件下（T7 表达系统，BL21(DE3)-BD）TolC 的数量。HlyBD 复合物的过表达并不影响 TolC 的总量。更进一步，我们通过超分辨率显微镜确定了 HlyA T1SS 的定位。与其他细菌分泌系统不同，无论是内源性水平还是过表达水平，都没有观察到极化。此外，细胞生长和分裂周期不影响极化。最重要的是，观察到的 T1SS 簇的大小与最近提出的外膜岛无关。这些数据表明，T1SS 簇位于外膜上，形成了迄今为止尚未描述的身份领域。 尿路致病性大肠杆菌 (UPEC) 菌株每年在全球范围内导致约 1.1 亿例尿路感染，给医疗保健系统带来了全球性负担。UPEC 菌株分泌许多毒力因子，其中溶血素 A 通过同源 T1SS 分泌到细胞外空间。在这项研究中，我们确定了 UTI89 中 HlyA T1SS 的内源性拷贝数，并分别分析了 BL21(DE3)-BD 和 UTI89 中的表面定位。UTI89 中的 T1SS 约有 800 个拷贝，这是表达水平最高的细菌分泌系统之一。此外，与其他分泌系统形成鲜明对比的是，没有检测到极化的表面定位。最后，对超分辨率数据的定量分析表明，HlyA T1SS 的簇与最近发现的外膜蛋白岛无关。这些数据为 HlyA T1SS 的定量分子结构提供了新的见解。
