Background

Sporulation is a complex cell differentiation programme shared by many members of the Firmicutes, the end result of which is a highly resistant, metabolically inert spore that can survive harsh environmental insults. Clostridioides difficile spores are essential for transmission of disease and are also required for recurrent infection. However, the molecular basis of sporulation is poorly understood, despite parallels with the well-studied Bacillus subtilis system. The spore envelope consists of multiple protective layers, one of which is a specialised layer of peptidoglycan, called the cortex, that is essential for the resistant properties of the spore. We set out to identify the enzymes required for synthesis of cortex peptidoglycan in C. difficile.

Methods

Bioinformatic analysis of the C. difficile genome to identify putative homologues of Bacillus subtilis spoVD was combined with directed mutagenesis and microscopy to identify and characterise cortex-specific PBP activity.

Results

Deletion of CDR20291_2544 (SpoVD_Cd) abrogated spore formation and this phenotype was completely restored by complementation in cis. Analysis of SpoVD_Cd revealed a three domain structure, consisting of dimerization, transpeptidase and PASTA domains, very similar to B. subtilis SpoVD. Complementation with SpoVD_Cd domain mutants demonstrated that the PASTA domain was dispensable for formation of morphologically normal spores. SpoVD_Cd was also seen to localise to the developing spore by super-resolution confocal microscopy.

Conclusions

We have identified and characterised a cortex specific PBP in C. difficile. This is the first characterisation of a cortex-specific PBP in C. difficile and begins the process of unravelling cortex biogenesis in this important pathogen.

中文翻译：

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皮质特异性青霉素结合蛋白有助于艰难梭菌孢子的耐热性

背景

孢子形成是厚壁菌门的许多成员共享的复杂细胞分化程序，其最终结果是一种高度抗性、代谢惰性的孢子，可以在严酷的环境侵害中存活下来。艰难梭菌孢子是疾病传播所必需的，也是反复感染所必需的。然而，尽管与经过充分研究的枯草芽孢杆菌系统相似，但人们对孢子形成的分子基础知之甚少。孢子包膜由多个保护层组成，其中一层是特殊的肽聚糖层，称为皮层，对孢子的抗性至关重要。我们着手鉴定在艰难梭菌中合成皮质肽聚糖所需的酶。

方法

艰难梭菌基因组的生物信息学分析以确定枯草芽孢杆菌 spoVD 的假定同源物，并结合定向诱变和显微镜来鉴定和表征皮质特异性 PBP 活性。

结果

CDR20291_2544 (SpoVD _Cd ) 的缺失消除了孢子的形成，这种表型通过顺式互补完全恢复。SpoVD _{Cd 的}分析揭示了三个域结构，由二聚化、转肽酶和 PASTA 域组成，与枯草芽孢杆菌SpoVD非常相似。与 SpoVD _Cd域突变体的互补表明 PASTA 域对于形态正常的孢子的形成是可有可无的。通过超分辨率共聚焦显微镜还观察到SpoVD _Cd定位于发育中的孢子。

结论

我们已经鉴定并表征了艰难梭菌中的皮质特异性 PBP 。这是艰难梭菌中皮质特异性 PBP 的首次表征，并开始了在这一重要病原体中解开皮质生物发生的过程。