Zuberi Azna, Misba Lama, Khan Asad U
Medical Microbiology and Molecular Biology Lab., Interdisciplinary Biotechnology Unit, Aligarh Muslim UniversityAligarh, India.
Front Cell Infect Microbiol. 2017 May 26;7:214. doi: 10.3389/fcimb.2017.00214. eCollection 2017.
Biofilm is a sessile bacterial accretion embedded in self-producing matrix. It is the root cause of about 80% microbial infections in human. Among them, biofilms are most prevalent in medical devices associated nosocomial infections. The objective of this study was to inhibit biofilm formation by targeting gene involved in quorum sensing, one of the main mechanisms of biofilm formation. Hence we have introduced the CRISPRi, first time to target luxS gene. luxS is a synthase, involved in the synthesis of Autoinducer-2(AI-2), which in turn guides the initial stage of biofilm formation. To implement CRISPRi system for luxS gene suppression, we have synthesized complementary sgRNA to target gene sequence and co-expressed with dCas9, a mutated form of an endonuclease. Suppression of luxS expression was confirmed through qRT-PCR. The effect of luxS gene on biofilm inhibition was studied through crystal violet assay, XTT reduction assay and scanning electron microscopy. We conclude that CRISPRi system could be a potential strategy to inhibit bacterial biofilm through mechanism base approach.
生物膜是一种嵌入自身产生的基质中的固着细菌聚集体。它是人类约80%微生物感染的根本原因。其中，生物膜在与医疗设备相关的医院感染中最为普遍。本研究的目的是通过靶向参与群体感应（生物膜形成的主要机制之一）的基因来抑制生物膜形成。因此，我们首次引入了CRISPRi来靶向luxS基因。luxS是一种合成酶，参与自诱导物-2（AI-2）的合成，而AI-2又指导生物膜形成的初始阶段。为了实施用于抑制luxS基因的CRISPRi系统，我们合成了与靶基因序列互补的sgRNA，并与一种突变形式的内切核酸酶dCas9共表达。通过qRT-PCR证实了luxS表达的抑制。通过结晶紫测定、XTT还原测定和扫描电子显微镜研究了luxS基因对生物膜抑制的作用。我们得出结论，CRISPRi系统可能是一种通过基于机制的方法抑制细菌生物膜的潜在策略。
