Roles of Cysteine Proteases Cwp84 and Cwp13 in Biogenesis of the Cell Wall of Clostridium difficile
Roles of Cysteine Proteases Cwp84 and Cwp13 in Biogenesis of the Cell Wall of Clostridium difficile
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DOI：
10.1128/jb.00248-11
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发表时间：
2011-07-01
影响因子：
3.2
通讯作者：
Fairweather, Neil F.
中科院分区：
文献类型：
--
作者：
de la Riva, Luca;Willing, Stephanie E.;Fairweather, Neil F.
艰难梭菌表达许多细胞壁蛋白，包括丰富的高分子量和低分子量S层蛋白（SLP）。这些蛋白质通过半胱氨酸蛋白酶Cwp84翻译后切割前体SlpA而产生。我们比较了C.艰难梭菌菌株，其在cwp84或其同源物cwp13中含有插入突变，并与表达其基因的野生型或突变形式的质粒互补。我们表明，在cwp84突变体的细胞壁中的未切割的SlpA的存在下，在细胞表面的其他细胞壁蛋白质的异常保留的结果，所证明的分泌的蛋白质Cwp66和Cwp2到生长培养基中。这些表型通过与表达野生型Cwp84酶的质粒互补而恢复，但不与在活性位点中编码Cys116Ala取代的质粒互补。cwp13突变体正常切割SlpA前体，并具有野生型样菌落表型。Cwp84和Cwp13均作为酶原产生，其通过切割加工以产生成熟酶。在Cwp84的情况下，这种切割似乎不是自催化的，而在Cwp13中，自催化被证明为Cys109Ala突变体没有进行加工。Cwp13似乎在Cwp84的加工中起作用，但对Cwp84的活性不是必需的。Cwp13在HMW SLP结构域中切割SlpA，我们认为这可能反映了在细胞表面错误折叠蛋白的切割和降解中的作用。
Clostridium difficile expresses a number of cell wall proteins, including the abundant high-molecular-weight and low-molecular-weight S-layer proteins (SLPs). These proteins are generated by posttranslational cleavage of the precursor SlpA by the cysteine protease Cwp84. We compared the phenotypes of C. difficile strains containing insertional mutations in either cwp84 or its paralog cwp13 and complemented with plasmids expressing wild-type or mutant forms of their genes. We show that the presence of uncleaved SlpA in the cell wall of the cwp84 mutant results in aberrant retention of other cell wall proteins at the cell surface, as demonstrated by secretion of the proteins Cwp66 and Cwp2 into the growth medium. These phenotypes are restored by complementation with a plasmid expressing wild-type Cwp84 enzyme but not with one encoding a Cys116Ala substitution in the active site. The cwp13 mutant cleaved the SlpA precursor normally and had a wild-type-like colony phenotype. Both Cwp84 and Cwp13 are produced as proenzymes which are processed by cleavage to produce mature enzymes. In the case of Cwp84, this cleavage does not appear to be autocatalytic, whereas in Cwp13 autocatalysis was demonstrated as a Cys109Ala mutant did not undergo processing. Cwp13 appears to have a role in processing of Cwp84 but is not essential for Cwp84 activity. Cwp13 cleaves SlpA in the HMW SLP domain, which we suggest may reflect a role in cleavage and degradation of misfolded proteins at the cell surface.