Shannon Austin, Johnson Tanya, Roberts Cameron S, Chaton Catherine T, Korotkov Konstantin V, Sandkvist Maria
Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, USA.
Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky, USA.
J Bacteriol. 2025 Aug 21;207(8):e0014425. doi: 10.1128/jb.00144-25. Epub 2025 Jul 14.
The Type II Secretion System (T2SS) of the Gram-negative pathogen is considered a virulence factor primarily because it facilitates the secretion of cholera toxin upon arrival of this pathogen in the human intestinal lumen, resulting in the profuse watery diarrhea associated with infection. Results from transposon mutagenesis screens suggest that the genes that code for the T2SS are essential. The deletion of these genes impairs the secretion of the T2SS's nearly two dozen protein substrates, leading to the selection of suppressor mutations, extracytoplasmic stress, and poor growth of the mutant bacteria. Here, we explore the role of the C-terminal PDZ domain of the T2SS component EpsC, and why transposon insertions in do not affect bacterial viability unlike insertions in most other genes. Our data show that the deletion of does not affect the growth of and we demonstrate via mass spectrometry that the serine protease VesB is the only known T2SS substrate fully reliant on PDZ for secretion. We confirm this finding with immuno- and enzyme assays and show that the immunoglobulin (Ig)-like fold of VesB contains PDZ-dependent secretion information that can be grafted onto β-lactamase, causing this normally periplasmic protein to be secreted in a PDZ-dependent manner. This work adds to a growing body of evidence about substrate selection by the T2SS and reinforces the view that the C-terminal domain of EpsC and its homologs are required for the secretion of only a subset of T2SS substrates within a given species.
The T2SS is common in Gram-negative pathogens, facilitating the secretion of various toxins and enzymes; however, the mechanisms of substrate selection and secretion remain poorly understood. Here, we demonstrate that the C-terminal PDZ domain of the T2SS "clamp" protein EpsC is only required for secretion of VesB in and that VesB's Ig-fold contains PDZ-dependent secretion information that can be functionally grafted onto a non-secreted periplasmic protein.
革兰氏阴性病原体的II型分泌系统（T2SS）被认为是一种毒力因子，主要是因为当这种病原体到达人类肠腔时，它有助于霍乱毒素的分泌，导致与感染相关的大量水样腹泻。转座子诱变筛选结果表明，编码T2SS的基因是必需的。这些基因的缺失会损害T2SS近二十多种蛋白质底物的分泌，导致抑制突变的选择、胞外应激以及突变细菌的生长不良。在这里，我们探讨了T2SS组分EpsC的C端PDZ结构域的作用，以及为什么在该结构域中的转座子插入不像在大多数其他基因中的插入那样影响细菌活力。我们的数据表明，该结构域的缺失不影响细菌的生长，并且我们通过质谱证明丝氨酸蛋白酶VesB是唯一已知的完全依赖PDZ进行分泌的T2SS底物。我们通过免疫和酶测定证实了这一发现，并表明VesB的免疫球蛋白（Ig）样折叠包含PDZ依赖性分泌信息，该信息可以嫁接到β-内酰胺酶上，使这种通常位于周质的蛋白质以PDZ依赖性方式分泌。这项工作增加了关于T2SS底物选择的越来越多的证据，并强化了这样一种观点，即EpsC及其同源物的C端结构域对于给定物种中仅一部分T2SS底物的分泌是必需的。
T2SS在革兰氏阴性病原体中很常见，有助于各种毒素和酶的分泌；然而，底物选择和分泌的机制仍知之甚少。在这里，我们证明T2SS“夹子”蛋白EpsC的C端PDZ结构域仅对VesB在该细菌中的分泌是必需的，并且VesB的Ig折叠包含PDZ依赖性分泌信息，该信息可以在功能上嫁接到非分泌性周质蛋白上。
