Department of Microbiology and Immunology, University of Iowagrid.214572.7, Iowa City, Iowa, USA.
J Bacteriol. 2022 Jun 21;204(6):e0012122. doi: 10.1128/jb.00121-22. Epub 2022 May 16.
The WalR-WalK two-component regulatory system (TCS) is found in all , in which it regulates the expression of multiple genes required for remodeling the cell envelope during growth and division. Unlike most TCSs, WalRK is essential for viability, so it has attracted interest as a potential antibiotic target. In this study, we used overexpression of WalR and CRISPR interference to investigate the Wal system of Clostridioides difficile, a major cause of hospital-associated diarrhea in high-income countries. We confirmed that the operon is essential and identified morphological defects and cell lysis as the major terminal phenotypes of altered expression. We also used transcriptome sequencing (RNA-seq) to identify over 150 genes whose expression changes in response to WalR levels. This gene set is enriched in cell envelope genes and includes genes encoding several predicted PG hydrolases and proteins that could regulate PG hydrolase activity. A distinct feature of the C. difficile cell envelope is the presence of an S-layer, and we found that WalR affects expression of several genes which encode S-layer proteins. An unexpected finding was that some Wal-associated phenotypic defects were inverted in comparison to what has been reported for other . For example, downregulation of Wal signaling caused C. difficile cells to become longer rather than shorter, as in Bacillus subtilis. Likewise, downregulation of Wal rendered C. difficile more sensitive to vancomycin, whereas reduced Wal activity is linked to increased vancomycin resistance in Staphylococcus aureus. The WalRK two-component system (TCS) is essential for coordinating synthesis and turnover of peptidoglycan in . We investigated the WalRK TCS in Clostridioides difficile, an important bacterial pathogen with an atypical cell envelope. We confirmed that WalRK is essential and regulates cell envelope biogenesis, although several of the phenotypic changes we observed were opposite to what has been reported for other . We also identified over 150 genes whose expression is controlled either directly or indirectly by WalR. Overall, our findings provide a foundation for future investigations of an important regulatory system and potential antibiotic target in C. difficile.
沃尔 R-沃尔 K 双组分调控系统 (TCS) 存在于所有中，它调控着在生长和分裂过程中重塑细胞包膜所需的多种基因的表达。与大多数 TCS 不同，WalRK 对生存至关重要，因此它作为一种潜在的抗生素靶标引起了人们的兴趣。在这项研究中，我们使用 WalR 的过表达和 CRISPR 干扰来研究艰难梭菌的 Wal 系统，艰难梭菌是高收入国家医院相关腹泻的主要原因。我们证实了 operon 是必需的，并确定形态缺陷和细胞裂解是改变表达的主要终末表型。我们还使用转录组测序 (RNA-seq) 来鉴定超过 150 个基因，这些基因的表达变化对 WalR 水平有响应。这个基因集富含细胞包膜基因，包括编码几种预测 PG 水解酶和可调节 PG 水解酶活性的蛋白质的基因。艰难梭菌细胞包膜的一个显著特征是存在 S-层，我们发现 WalR 影响编码 S-层蛋白的几个基因的表达。一个意外的发现是，与其他相比，一些与 Wal 相关的表型缺陷被颠倒了。例如，Wal 信号的下调导致艰难梭菌细胞变得更长，而不是像枯草芽孢杆菌那样更短。同样，Wal 的下调使艰难梭菌对万古霉素更敏感，而 Wal 活性的降低与金黄色葡萄球菌中万古霉素耐药性的增加有关。WalRK 双组分系统 (TCS) 对协调的合成和周转至关重要。我们研究了艰难梭菌的 WalRK TCS，艰难梭菌是一种具有非典型细胞包膜的重要细菌病原体。我们证实 WalRK 是必需的，并调节细胞包膜生物发生，尽管我们观察到的一些表型变化与其他报道的不同。我们还鉴定了超过 150 个基因，它们的表达要么直接要么间接受到 WalR 的控制。总的来说，我们的研究结果为未来研究艰难梭菌中的重要调控系统和潜在抗生素靶标提供了基础。
