Research Department, Health Protection Agency, Porton Down, Salisbury, Wiltshire SP4 0JG, UK.
J Biol Chem. 2009 Dec 11;284(50):34666-73. doi: 10.1074/jbc.M109.051177. Epub 2009 Oct 6.
Clostridium difficile is a major and growing problem as a hospital-associated infection that can cause severe, recurrent diarrhea. The mechanism by which the bacterium colonizes the gut during infection is poorly understood but undoubtedly involves protein components within the surface layer (S-layer), which play a role in adhesion. In C. difficile, the S-layer is composed of two principal components, the high and low molecular weight S-layer proteins, which are formed from the post-translational cleavage of a single precursor, SlpA. In the present study, we demonstrate that a recently characterized cysteine protease, Cwp84 plays a role in maturation of SlpA. Using a gene knock-out approach, we show that inactivation of the Cwp84 gene in C. difficile 630DeltaErm results in a bacterial phenotype in which only immature, single chain SlpA comprises the S-layer. The Cwp84 knock-out mutants (CDDeltaCwp84) displayed significantly different colony morphology compared with the wild-type strain and grew more slowly in liquid medium. SlpA extracted from CDDeltaCwp84 was readily cleaved into its mature subunits by trypsin treatment. Addition of trypsin to the growth medium also cleaved SlpA on CDDeltaCwp84 and increased the growth rate of the bacterium in a dose-dependent manner. Using the hamster model for C. difficile infection, CDDeltaCwp84 was found to be competent at causing disease with a similar pathology to the wild-type strain. The data show that whereas Cwp84 plays a role in the cleavage of SlpA, it is not an essential virulence factor and that bacteria expressing immature SlpA are able to cause disease.
艰难梭菌是一种主要且日益严重的医院相关感染，可导致严重、反复的腹泻。该细菌在感染期间定殖肠道的机制尚未完全了解，但无疑涉及表面层 (S-层) 内的蛋白质成分，这些成分在黏附中起作用。在艰难梭菌中，S-层由两个主要成分组成，即高分子量和低分子量 S-层蛋白，它们由单个前体 SlpA 的翻译后切割形成。在本研究中，我们证明了最近表征的一种半胱氨酸蛋白酶 Cwp84 在 SlpA 的成熟中起作用。使用基因敲除方法，我们表明在 C. difficile 630DeltaErm 中 Cwp84 基因的失活导致细菌表型，其中仅不成熟的单链 SlpA 构成 S-层。与野生型菌株相比，Cwp84 敲除突变体 (CDDeltaCwp84) 的菌落形态明显不同，并且在液体培养基中生长得更慢。从 CDDeltaCwp84 中提取的 SlpA 很容易被胰蛋白酶切割成其成熟亚基。向生长培养基中添加胰蛋白酶也可切割 CDDeltaCwp84 上的 SlpA，并以剂量依赖的方式增加细菌的生长速度。在艰难梭菌感染的仓鼠模型中，CDDeltaCwp84 有能力引起疾病，其病理学与野生型菌株相似。数据表明，尽管 Cwp84 在 SlpA 的切割中起作用，但它不是必需的毒力因子，并且表达不成熟 SlpA 的细菌能够引起疾病。
