Ramalhete Sara, Roseiro Isabel, Cassona Carolina P, Feliciano Carolina Alves, Serrano Mónica, Henriques Adriano O
Instituto de Tecnologia Química e Biológica António Xavier, Oeiras, Portugal.
mSphere. 2025 Sep 30;10(9):e0018625. doi: 10.1128/msphere.00186-25. Epub 2025 Sep 3.
Most strains of produce two toxins, TcdA and TcdB, which are mainly responsible for the disease symptoms. TcdA and TcdB are coded for by genes in the pathogenicity locus (PaLoc). Some epidemic strains, however, such as R20291, of ribotype 027, additionally produce a binary toxin, CDT, coded for by genes in the CDT locus (CdtLoc). We analyzed the relationship between transcription of the TcdA and CDT encoding genes in strain R20291 using single-cell analysis with orthogonal fluorescence reporters. We found that most of the cells express whereas a much smaller fraction expresses and most of those also express . Expression of begins during exponential growth and persists during the stationary phase of growth, while the main period of transcription occurs as cells enter the stationary phase. This translates into early synthesis and release of CDT from the cells, whereas production of TcdA is detected mainly from the onset of the stationary phase. Both the PaLoc and CdtLoc also code for regulatory proteins, TcdR, a sigma factor, and CdtR, a response regulator of the LytR family, required for expression of the toxin-encoding genes, respectively. While CdtR contributes to the expression of , as shown before, we found reduced expression of in the absence of TcdR. Hence, TcdR and CdtR cross-regulate the PaLoc and the CdtLoc. Finally, and unlike the case for TcdA, we found no evidence for the association of CDT with mature spores.IMPORTANCEThe enteropathogen causes a spectrum of intestinal diseases, ranging from mild diarrhea to severe conditions such as intestinal inflammation, perforation, and sepsis, that may lead to death, primarily through the production of two cytotoxins, TcdA and TcdB. Certain strains, however, such as those of ribotypes 027 and 078, additionally produce a binary toxin, CDT. Here, we employed single-cell analysis to investigate toxin gene expression in epidemic strain R20297 (RT027), commonly associated with severe infections. We found that CDT is synthesized early during growth, while TcdA is produced at the onset of the stationary phase, and that the two populations partially overlap. We also identify cross-regulation between two key regulatory proteins, TcdR and CdtR, which control TcdA/TcdB and CDT production. These insights into the mechanisms of toxin production at the population level may contribute to the development of targeted therapies for managing infections and the resulting complications.
大多数 菌株会产生两种毒素，即TcdA和TcdB，它们是导致疾病症状的主要原因。TcdA和TcdB由致病位点（PaLoc）中的基因编码。然而，一些流行菌株，如核糖体分型027的R20291，还会产生一种二元毒素CDT，由CDT位点（CdtLoc）中的基因编码。我们使用正交荧光报告基因的单细胞分析方法，分析了R20291菌株中TcdA和CDT编码基因转录之间的关系。我们发现，大多数细胞表达 ，而表达 的细胞比例要小得多，并且大多数表达 的细胞也表达 。 的表达在指数生长期开始，并在生长稳定期持续，而 转录的主要时期发生在细胞进入稳定期时。这导致CDT从细胞中早期合成并释放，而TcdA的产生主要在稳定期开始时被检测到。PaLoc和CdtLoc还分别编码调节蛋白，即TcdR（一种sigma因子）和CdtR（LytR家族的一种反应调节因子），它们是毒素编码基因表达所必需的。如之前所示，CdtR有助于 的表达，但我们发现在没有TcdR的情况下 的表达会降低。因此，TcdR和CdtR相互交叉调节PaLoc和CdtLoc。最后，与TcdA的情况不同，我们没有发现CDT与成熟孢子相关的证据。
肠道病原体 会引发一系列肠道疾病，从轻度腹泻到严重的病症，如肠道炎症、穿孔和败血症，这些病症可能导致死亡，主要是通过产生两种细胞毒素TcdA和TcdB。然而，某些菌株，如核糖体分型027和078的菌株，还会产生一种二元毒素CDT。在这里，我们采用单细胞分析方法来研究通常与严重感染相关的流行菌株R20297（RT027）中的毒素基因表达。我们发现CDT在生长早期合成，而TcdA在稳定期开始时产生，并且这两个群体部分重叠。我们还确定了两种关键调节蛋白TcdR和CdtR之间的交叉调节，它们控制TcdA/TcdB和CDT的产生。这些在群体水平上对毒素产生机制的见解可能有助于开发针对性疗法，以管理 感染及其引发的并发症。
