Raafat Marwa M, Ali-Tammam Marwa, Ali Amal E
Department of Microbiology & Immunology, Faculty of Pharmaceutical Sciences & Pharmaceutical Industries, Future University in Egypt (FUE), New Cairo, Egypt.
Infect Drug Resist. 2019 Jun 7;12:1583-1596. doi: 10.2147/IDR.S182889. eCollection 2019.
Quorum quenching, the interference of a Quorum sensing (QS) system that contributes to the pathogenesis through triggering the production of various virulence determinants, is among the newly suggested antivirulence strategies. This study aimed at screening of N-Acyl homoserine lactonase activity from local bacterial isolate, and investigating its effect on () virulence and biofilm formation. Soil bacteria were screened for gene coding for lactonase enzyme by Polymerase Chain reaction and sequencing of gene homologs. Lactonase activity and spectrum were assessed in the cell-free lysate by well diffusion assay using KYC55. A bacterial isolate showing the highest N-acyl-homoserine lactones degradation percentage was identified by gene amplification and sequencing of the 16S rRNA gene and its gene homolog. High performance liquid chromatography was used to confirm N-acyl-homoserine lactone degradation. The effect of cell-free lysate on the biofilm formation ability and cytotoxicity of PAO1 and clinical isolates from different clinical sources were assessed by static microtiter plate and viability assay, respectively Lactonase gene and activity were identified in three spp. isolates. They showed broad catalytic activities against tested N-acyl-homoserine lactones. However, The lactonase activity in the cell- free lysate of isolate 30b showed the highest significant degradation percentage on all tested signals; N-butanoyl-L-homoserine lactone (71%), N-hexanoyl-l-homoserine lactone (100%), N-decanoyl-homoserine lactone (100%), N-(3-oxohexanoyl)-L-homoserine lactone (37.5%), N-(oxodecanoyl)-L-homoserine lactone (100%), and N-(3-oxododecanoyl)-L-homoserine lactone (100%). Alignment of the amino acid sequences of AiiA protein of isolate 30b showed 96% identity with () homologous lactonases in the GenBank database, and the isolate was designated as isolate 30b. Cell-free lysate of isolate 30b reduced biofilm formation significantly in 93% of isolates. The highest mean percentage of reduction in the biofilm was 86%. Moreover, the viability percentage of human lung carcinoma A549 cells infected by and treated with cell-free lysate of isolate 30b increased up to 15%. The results of this study highlight the potential of lactonases as a promising strategy to combat virulence.
群体感应淬灭是一种新提出的抗毒力策略，它是对群体感应（QS）系统的干扰，该系统通过触发各种毒力决定因素的产生而促进发病机制。本研究旨在从本地细菌分离物中筛选N-酰基高丝氨酸内酯酶活性，并研究其对（）毒力和生物膜形成的影响。通过聚合酶链反应和基因同源物测序，筛选土壤细菌中编码内酯酶的基因。使用KYC55通过孔扩散法在无细胞裂解物中评估内酯酶活性和谱。通过16S rRNA基因及其基因同源物的基因扩增和测序，鉴定出显示最高N-酰基高丝氨酸内酯降解百分比的细菌分离物。使用高效液相色谱法确认N-酰基高丝氨酸内酯的降解。分别通过静态微量滴定板和活力测定评估无细胞裂解物对铜绿假单胞菌PAO1和来自不同临床来源的临床分离株的生物膜形成能力和细胞毒性的影响。在三个假单胞菌属分离物中鉴定出内酯酶基因和活性。它们对测试的N-酰基高丝氨酸内酯显示出广泛的催化活性。然而，分离株30b的无细胞裂解物中的内酯酶活性在所有测试信号上显示出最高的显著降解百分比；N-丁酰-L-高丝氨酸内酯（71%），N-己酰-L-高丝氨酸内酯（100%），N-癸酰高丝氨酸内酯（100%），N-（3-氧代己酰基）-L-高丝氨酸内酯（37.5%），N-（氧代癸酰基）-L-高丝氨酸内酯（100%），和N-（3-氧代十二酰基）-L-高丝氨酸内酯（100%）。分离株30b的AiiA蛋白的氨基酸序列与GenBank数据库中的（）同源内酯酶显示96%的同一性，该分离株被命名为假单胞菌分离株30b。分离株30b的无细胞裂解物在93%的分离株中显著降低了生物膜形成。生物膜减少的最高平均百分比为86%。此外，被铜绿假单胞菌感染并用分离株30b的无细胞裂解物处理的人肺癌A549细胞的活力百分比提高到15%。本研究结果突出了内酯酶作为对抗铜绿假单胞菌毒力的一种有前景策略的潜力。
