Srivastava Vishal, Bandhu Sheetal, Mishra Shivam, Chaudhuri Tapan K
Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India.
Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India.
Biochim Biophys Acta Proteins Proteom. 2025 Jan 1;1873(1):141046. doi: 10.1016/j.bbapap.2024.141046. Epub 2024 Sep 4.
Serratiopeptidase, a serine protease traditionally used as an oral anti-inflammatory drug has been found to show antibiofilm action. Structurally, it comprises of two distinct domains; viz-the N-terminal catalytic domain (Ncat) and a C-terminal RTX (Repeat-In-Toxin) domain (Crtx). Understanding the antibiofilm action of the serratiopeptidase molecule, as well as the antibiofilm action of each of its two domains, was the objective of this study.
Separate clones to express the complete recombinant serratiopeptidase protein and its variant containing a mutation in the catalytic site, the N-terminal catalytic domain and its mutant, and the C-terminal Repeat-In-Toxin domain were prepared, and the proteins were purified. The impact of these proteins on pre-existing biofilms, as well as their effect upon addition of these proteins during biofilm formation was investigated.
In our investigation, we have been able to analyze the antibiofilm action of serratiopeptidase in detail. Obtained results conclude that while N-terminally located proteolytic domain of serratiopeptidase conventionally acts against biofilms by hydrolytic activity, the C-terminal domain regulates or prevents biofilm formation by yet unknown mechanism in addition to its known function as an C-terminal located calcium modulated internal chaperone ensuring the proper folding and secretion of the molecule. The study's findings give new evidence that the Crtx domain plays a significant role in antibiofilm action. The proteolytic Ncat domain breaks down pre-formed biofilms. The C-terminal domain, on the other hand, acts as an inhibitor of biofilm formation by regulating or preventing biofilm development.
沙雷氏菌肽酶是一种传统上用作口服抗炎药物的丝氨酸蛋白酶，已被发现具有抗生物膜作用。从结构上看，它由两个不同的结构域组成；即N端催化结构域（Ncat）和C端RTX（毒素重复序列）结构域（Crtx）。本研究的目的是了解沙雷氏菌肽酶分子的抗生物膜作用及其两个结构域各自的抗生物膜作用。
制备了单独的克隆，以表达完整的重组沙雷氏菌肽酶蛋白及其在催化位点含有突变的变体、N端催化结构域及其突变体以及C端毒素重复序列结构域，并对这些蛋白进行了纯化。研究了这些蛋白对预先形成的生物膜的影响，以及在生物膜形成过程中添加这些蛋白后的作用。
在我们的研究中，我们能够详细分析沙雷氏菌肽酶的抗生物膜作用。获得的结果表明，虽然沙雷氏菌肽酶位于N端的蛋白水解结构域通常通过水解活性对抗生物膜，但C端结构域除了作为位于C端的钙调节内部伴侣确保分子的正确折叠和分泌这一已知功能外，还通过未知机制调节或阻止生物膜形成。该研究结果提供了新的证据，表明Crtx结构域在抗生物膜作用中发挥着重要作用。蛋白水解性的Ncat结构域分解预先形成的生物膜。另一方面，C端结构域通过调节或阻止生物膜形成而作为生物膜形成的抑制剂。
