An intact S-layer is advantageous to Clostridioides difficile within the host.
An intact S-layer is advantageous to Clostridioides difficile within the host.
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DOI：
10.1371/journal.ppat.1011015
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发表时间：
2023-06
期刊：
影响因子：
6.7
通讯作者：
中科院分区：
文献类型：
--
作者：
艰难梭菌是抗生素治疗的住院老年患者大量发病和死亡的原因，其中毒素的产生与腹泻病有关。虽然已详细研究了这些毒素的功能，但包括晶旁表面层（s层）在内的其他因素对疾病的影响尚不清楚。在这里，我们通过报道感染s层缺失菌株FM2.5后s层变异的恢复，强调了s层在体内的重要性。这些变异要么对原点突变进行了修正，要么对序列进行了修改，恢复了阅读框，并翻译了slpA。这些变异克隆在体内的选择是快速的，并且不依赖于毒素的产生，在感染后24小时内，高达90%的恢复艰难梭菌群体编码修改的slpA序列。两个变体，后来被命名为FM2.5varA和FM2.5varB，被选中进行更详细的研究。FM2.5varB中SlpA的结构测定表明，蛋白质结构域的取向发生了改变，导致晶格组装的重组和相互作用界面的改变，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fm2.5样表型，其导致的疾病严重程度与R20291相似。体外培养菌株的比较RNA测序（RNA- seq）分析显示，R20291和FM2.5之间的基因表达发生了很大变化。tcdA/tcdB和几个与产孢和细胞壁完整性相关的基因的下调可能是FM2.5在体内表型减弱的原因。RNA-seq数据与疾病严重程度密切相关，毒性更强的变体FM2.5 vara在体外显示出与R20291相似的基因表达谱，而减毒的FM2.5 varb显示出许多与FM2.5相同的毒力相关性状的下调。累积起来，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的严重程度。艰难梭菌的s层是一种覆盖细菌细胞外表面的副晶阵列，但其对整体疾病的贡献尚不清楚。如前所述，自发性slpA-null突变FM2.5， slpA点突变为研究s层在疾病中的作用提供了机会。在这里，我们证实了这种菌株在体内的毒性较低，尽管它能有效地定植宿主并产生毒素。我们还展示了恢复slpA翻译并产生s层的体内序列修饰选择。虽然这种修饰不会影响单个SlpA（子）结构域的整体3D结构，但它们会导致结构域的方向改变和随后的s层组装。重要的是，体外RNA-Seq分析显示，FM2.5和R20291之间的基因表达存在很大差异。检测到的与毒素表达和产孢有关的基因转录差异表明，s层在宿主内提供了选择性生存优势，这有助于疾病的严重程度。
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. The S-layer of C. difficile is a paracrystalline array that covers the outer surface of the bacterial cell but its contribution to overall disease remains unclear. As previously described, spontaneous slpA-null mutant, FM2.5, with a point mutation in slpA offered an opportunity to study the role of the S-layer in disease. Here, we confirm that this strain is less virulent in vivo despite effectively colonising the host and producing toxin. We also show in vivo selection for sequence modifications that restore slpA translation and produce an S-layer. While such modifications do not affect the overall 3D structure of individual SlpA (sub)domains, they can lead to altered orientation of the structural domains and subsequent S-layer assembly. Importantly, RNA-Seq analysis in vitro showed large differences in gene expression between FM2.5 and R20291. Detected differences in transcription of genes involved in toxin expression and sporulation suggests that the S-layer provides a selective survival advantage within the host, which contributes to disease severity.
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影响因子：
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作者：
Cowardin CA;Kuehne SA;Buonomo EL;Marie CS;Minton NP;Petri WA Jr
通讯作者：
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DOI：
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