AHL Hydrolase Mediated Biofilm Inhibition

描述（由申请人提供）：细菌生物膜阻碍医院感染的控制，例如由医疗器械植入引起的感染。一种或多种群体感应（QS）信号水平的升高与细菌生物膜的形成有关，由于生物膜介导的抗生素抗性和宿主防御机制，这使得在种植体生长可适应表面定植的细菌难以根除。因此，需要开发控制群体感应的方法，从而控制铜绿假单胞菌和其他革兰氏阴性病原体的生物膜形成。与许多革兰氏阴性菌一样，N-酰基高丝氨酸内酯（AHL）是铜绿假单胞菌的主要QS信号，特别是N-丁醇- 1 -高丝氨酸内酯（C(4)- hsl）和N-（3-氧十二烷基）- 1 -高丝氨酸内酯（3-氧-C(12)- hsl）。目前已发现少数酶具有通过降解ahl来调节QS的潜力，但开发有限。我们已经从Geobacilli中分离出酶，这些酶对ahl具有明显的活性（作为内酯酶起作用），并且能够破坏液体培养中受试生物对ahl的依赖过程，包括铜绿假单胞菌生物膜的形成。我们最活跃的Geobacillus酶是一种非常稳定的可溶性酶，可以很容易地纯化，已经在商业上可行的系统中表达。我们建议进一步表征这些酶，相对于已知的AHL内酯酶，以判断它们是否适合我们的最终目标，将这些酶配制成医疗器械产品的涂层。通过抑制群体感应（quorum sensing， QS）依赖过程（如生物膜的形成）来预防与医疗器械相关的铜绿假单胞菌（Pseudomonas aeruginosa， PA）感染，使宿主防御机制和抗生素策略更有效，是一个具有巨大潜力的概念。为此，我们发现从地杆菌中分离的酶能够水解n -酰基- l-高丝氨酸内酯的内酯环，这是革兰氏阴性菌的细菌QS的分子触发器。此外，共培养纯化了这些Geobacillus酶，抑制了qs依赖性过程，如PA生物膜的形成。在第一阶段，我们将彻底表征我们的地杆菌酶的内酯酶活性。我们还将定量比较我们的地杆菌酶和其他新兴的内酯酶。

DESCRIPTION (provided by applicant): Bacterial biofilms thwart the control of nosocomial infections, such as those resulting from medical device implantation. Elevated levels of one or more quorum sensing (QS) signals have been linked to the formation of bacterial biofilms, which makes eradication of bacteria colonizing the growth-amenable surfaces of implants difficult due to their biofilm-mediated resistance to antibiotics and host defense mechanisms. Therefore, it is desirable to develop ways to control quorum sensing and thus biofilm formation of P. aeruginosa and other Gram-negative pathogens. As with many Gram-negative organisms, N-acyl homoserine lactones (AHL) function as major QS signals for P. aeruginosa, specifically N-butanoyl-l-homoserine lactone (C(4)-HSL) and N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C(12)-HSL). A small number of enzymes have been found that show potential for modulating QS by degrading AHLs, but there development has been limited. We have isolated enzymes from Geobacilli that possess demonstrable activity toward AHLs (functioning as lactonases) and are able to disrupt AHL-dependent processes of test organisms in liquid culture, including P. aeruginosa biofilm formation. Our most active Geobacillus enzyme, a very stable, soluble enzyme that can be readily purified, is already being expressed in a commercially viable system. We propose to further characterize these enzymes, relative to known AHL lactonases, to judge their suitability for our ultimate objective to formulate these enzymes into coatings for medical device products. PUBLIC HEALTH RELEVANCE Preventing medical device-related Pseudomonas aeruginosa (PA) infections by inhibiting quorum sensing (QS) dependent processes such as biofilm formation, allowing host defense mechanisms and antibiotic strategies to be more effective, is a concept that holds great potential. Towards these ends, we discovered enzymes isolated from species of Geobacillus are capable of hydrolyzing the lactone ring of N-acyl-L- homoserine lactones, the molecular trigger of bacterial QS for Gram-negative bacteria. Furthermore, co-culturing purified these Geobacillus enzymes inhibits QS-dependent processes, such as PA biofilm formation. In Phase I, we will thoroughly characterize the lactonase activities of our Geobacillus enzymes. We will also quantitatively compare our Geobacillus enzymes to other emerging lactonase enzymes.