School of Infection and Immunity, College of Medical, Veterinary & Life Sciences, University of Glasgow, Scotland, United Kingdom.
Molecular Microbiology, School of Biosciences, University of Sheffield, England, United Kingdom.
PLoS Pathog. 2023 Jun 29;19(6):e1011015. doi: 10.1371/journal.ppat.1011015. eCollection 2023 Jun.
Clostridioides difficile is responsible for substantial morbidity and mortality in antibiotically-treated, hospitalised, elderly patients, in which toxin production correlates with diarrhoeal disease. While the function of these toxins has been studied in detail, the contribution of other factors, including the paracrystalline surface layer (S-layer), to disease is less well understood. Here, we highlight the essentiality of the S-layer in vivo by reporting the recovery of S-layer variants, following infection with the S-layer-null strain, FM2.5. These variants carry either correction of the original point mutation, or sequence modifications which restored the reading frame, and translation of slpA. Selection of these variant clones was rapid in vivo, and independent of toxin production, with up to 90% of the recovered C. difficile population encoding modified slpA sequence within 24 h post infection. Two variants, subsequently named FM2.5varA and FM2.5varB, were selected for study in greater detail. Structural determination of SlpA from FM2.5varB indicated an alteration in the orientation of protein domains, resulting in a reorganisation of the lattice assembly, and changes in interacting interfaces, which might alter function. Interestingly, variant FM2.5varB displayed an attenuated, FM2.5-like phenotype in vivo compared to FM2.5varA, which caused disease severity more comparable to that of R20291. Comparative RNA sequencing (RNA-Seq) analysis of in vitro grown isolates revealed large changes in gene expression between R20291 and FM2.5. Downregulation of tcdA/tcdB and several genes associated with sporulation and cell wall integrity may account for the reported attenuated phenotype of FM2.5 in vivo. RNA-seq data correlated well with disease severity with the more virulent variant, FM2.5varA, showing s similar profile of gene expression to R20291 in vitro, while the attenuated FM2.5varB showed downregulation of many of the same virulence associated traits as FM2.5. Cumulatively, these data add to a growing body of evidence that the S-layer contributes to C. difficile pathogenesis and disease severity.
艰难梭菌会导致大量接受抗生素治疗的住院老年患者出现发病率和死亡率，其产毒与腹泻病相关。尽管这些毒素的功能已被详细研究，但其他因素（包括副晶层（S 层））对疾病的贡献则了解较少。在这里，我们通过报告感染 S 层缺失株 FM2.5 后 S 层变体的恢复情况，强调了 S 层在体内的重要性。这些变体要么纠正了原始点突变，要么恢复了阅读框并翻译 slpA 的序列修饰。这些变体克隆在体内的选择非常迅速，与产毒无关，在感染后 24 小时内，高达 90%的恢复艰难梭菌种群编码修饰后的 slpA 序列。随后选择了两个变体 FM2.5varA 和 FM2.5varB 进行更详细的研究。FM2.5varB 的 SlpA 结构测定表明蛋白结构域的取向发生了改变，导致晶格组装重新组织，并改变了相互作用界面，这可能会改变功能。有趣的是，与 FM2.5varA 相比，变体 FM2.5varB 在体内表现出减弱的、类似于 FM2.5 的表型，而 FM2.5varA 导致的疾病严重程度更类似于 R20291。对体外生长分离株的比较 RNA 测序（RNA-Seq）分析显示，R20291 和 FM2.5 之间的基因表达发生了巨大变化。tcdA/tcdB 和几个与孢子形成和细胞壁完整性相关的基因下调可能是 FM2.5 在体内表现出减弱表型的原因。RNA-seq 数据与疾病严重程度相关性良好，毒力更强的变体 FM2.5varA 在体外与 R20291 具有相似的基因表达谱，而减弱的 FM2.5varB 则下调了许多与 FM2.5 相同的毒力相关特征。总之，这些数据增加了越来越多的证据，表明 S 层有助于艰难梭菌的发病机制和疾病严重程度。
