Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London, London, SW7 2AZ, UK.
Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, London, WC1E 7HT, UK.
Sci Rep. 2017 Aug 23;7(1):9204. doi: 10.1038/s41598-017-08322-1.
Clostridium difficile is a gastrointestinal pathogen but how the bacterium colonises this niche is still little understood. Sortase enzymes covalently attach specific bacterial proteins to the peptidoglycan cell wall and are often involved in colonisation by pathogens. Here we show C. difficile proteins CD2537 and CD3392 are functional substrates of sortase SrtB. Through manipulation of the C-terminal regions of these proteins we show the SPKTG motif is essential for covalent attachment to the cell wall. Two additional putative substrates, CD0183 which contains an SPSTG motif, and CD2768 which contains an SPQTG motif, are not cleaved or anchored to the cell wall by sortase. Finally, using an in vivo asymmetric cleavage assay, we show that despite containing a conserved SPKTG motif, in the absence of SrtB these proteins are localised to disparate cellular compartments.
艰难梭菌是一种胃肠道病原体，但该细菌如何定殖于这个生态位仍知之甚少。天冬酰胺基内肽酶将特定的细菌蛋白共价连接到肽聚糖细胞壁上，并且经常参与病原体的定植。在这里，我们证明艰难梭菌蛋白 CD2537 和 CD3392 是天冬酰胺基内肽酶 SrtB 的功能性底物。通过对这些蛋白质的 C 末端区域的操作，我们表明 SPKTG 基序对于共价连接到细胞壁是必需的。另外两个假定的底物 CD0183 含有 SPSTG 基序，CD2768 含有 SPQTG 基序，它们不能被天冬酰胺基内肽酶切割或锚定到细胞壁上。最后，使用体内不对称切割测定法，我们表明，尽管含有保守的 SPKTG 基序，但在没有 SrtB 的情况下，这些蛋白质被定位到不同的细胞区室。
