Abstract

Paraoxonase 2 (PON2) is considered as a potential anti-biofilm agent due to the highest lactonase activity among the PON family members implicating quorum quenching in gram-negative bacteria. However, PON2 is expressed mostly in insoluble fractions in the bacterial expression host which limits its application as an anti-biofilm agent. Therefore, obtaining the native human PON2 (HuPON2) protein in soluble form, better protein yield, stability, and enzymatic activities is essential. In this study, procedures for obtaining a high yield of the native form of HuPON2 in soluble and active forms were optimized. Guanidinium hydrochloride solubilized the HuPON2 protein, however, it is lethal for several bacteria, and thus a major problem for studying the various downstream application of the protein. Therefore, another refolding process for native HuPON2 was optimized. Owing to the promiscuous nature of HuPON2, we hypothesized that it could inhibit the biofilm formation in Mycobacterium smegmatis also. Interestingly, we observed a significant inhibition of the biofilm formation by HuPON2_Rf. However, the primary target of HuPON2 and the probable mechanism behind the quorum quenching in M. smegmatis need to be further explored, which would help widen the scope of HuPON2 as a potential anti-biofilm agent beyond the gram-negative bacteria.

摘要

对氧磷酶 2 (PON2) 被认为是一种潜在的抗生物膜剂，因为 PON 家族成员中内酯酶活性最高，这意味着革兰氏阴性菌中的群体猝灭。然而，PON2 在细菌表达宿主中主要以不溶性组分表达，这限制了它作为抗生物膜剂的应用。因此，获得可溶形式的天然人 PON2 (HuPON2) 蛋白、更好的蛋白质产量、稳定性和酶活性至关重要。在这项研究中，优化了以可溶性和活性形式获得高产量 HuPON2 天然形式的程序。Guanidinium hydrochloride 可溶解 HuPON2 蛋白，但它对多种细菌具有致死性，因此成为研究该蛋白各种下游应用的主要问题。所以，优化了原生 HuPON2 的另一个重折叠过程。由于 HuPON2 的混杂性，我们假设它可以抑制生物膜的形成耻垢分枝杆菌也。有趣的是，我们观察到 HuPON2_Rf 对生物膜形成的显着抑制。然而，HuPON2 的主要靶标和耻垢分枝杆菌群体猝灭背后的可能机制需要进一步探索，这将有助于扩大 HuPON2 作为革兰氏阴性菌以外的潜在抗生物膜剂的范围。