BosR: a novel biofilm-specific regulator in Pseudomonas aeruginosa. BosR: a novel biofilm-specific regulator in Pseudomonas aeruginosa

Biofilms are the most common cause of bacterial infections in humans and notoriously hard to treat due to their ability to withstand antibiotics and host immune defenses. To overcome the current lack of effective antibiofilm therapies and guide future design, the identification of novel gene targets is crucial. In this regard, transcriptional regulators have been proposed as promising targets for antimicrobial drug design, since they simultaneously affect multiple genes, typically lack human orthologs, and can be inactivated by small molecules that prevent dimerization. Therefore, a Transposon insertion sequencing approach was employed to systematically identify regulatory genes phenotypically affecting biofilm growth in Pseudomonas aeruginosa PA14. A screen of a pool of 300,000 transposon insertion mutants identified 349 genes involved in biofilm growth on hydroxy apatite, including 47 regulators. Detection of 19 regulatory genes participating in well-established biofilm pathways validated the results. An additional 28 novel prospective biofilm regulators suggested the requirement of multiple one-component transcriptional regulators. Defect phenotypes were confirmed for five one-component transcriptional regulators PA14_43720, PA14_56430, PA14_36180, arsR and merD as well as the protein kinase yeaG, which have not been implicated in biofilm growth before. Promisingly, the transcriptional regulator PA14_43720 displayed a conserved role in biofilm growth since its ortholog in P. aeruginosa strain PAO1 was also required for biofilm growth. Overall, our results highlighted that the gene network driving biofilm growth is complex and involves regulators beyond the primarily studied groups of two-component systems and cyclic diguanylate signaling proteins. Overall design: TnSeq experiments on PA14 grown under different conditions, namely planktonic and biofilm, for 96 hours. Additionally, the TnSeq pool was characterized at T0 as a reference condition. Each condition included three biological replicates.

生物膜（biofilm）是人类细菌性感染最常见的诱因，因其可耐受抗生素与宿主免疫防御，向来以难以治疗著称。为解决当前有效抗生物膜治疗手段匮乏的困境，并为未来疗法开发提供指引，鉴定新型基因靶点至关重要。据此，转录调控因子（transcriptional regulators）被认为是抗菌药物开发的极具潜力的靶点——因其可同时调控多基因表达、通常不存在人类同源基因，且可被阻断二聚化的小分子物质灭活。因此，本研究采用转座子插入测序（transposon insertion sequencing, TnSeq）方法，系统鉴定铜绿假单胞菌（Pseudomonas aeruginosa）PA14中表型层面影响生物膜生长的调控基因。通过对30万个转座子插入突变体库的筛选，本研究共鉴定出349个参与羟基磷灰石表面生物膜生长的基因，其中包含47个调控基因。对参与经典生物膜通路的19个调控基因的验证实验，证实了本次筛选结果的可靠性。另有28个新型潜在生物膜调控基因的发现，提示多类单组分转录调控因子（one-component transcriptional regulators）参与了生物膜生长过程。针对5个此前未被报道参与生物膜生长的单组分转录调控因子PA14_43720、PA14_56430、PA14_36180、arsR、merD以及蛋白激酶yeaG，本研究证实了其缺陷表型。值得关注的是，转录调控因子PA14_43720在生物膜生长中发挥了保守功能——其在铜绿假单胞菌PAO1菌株中的同源基因同样参与了生物膜生长过程。综上，本研究结果表明，驱动生物膜生长的基因网络极为复杂，其所涉及的调控因子不仅包括主流研究的双组分系统（two-component systems）与环二鸟苷酸（cyclic diguanylate）信号蛋白，还包含其他类型的调控基因。
实验整体设计：对PA14菌株分别在浮游态与生物膜态两种条件下培养96小时，开展转座子插入测序实验；同时以T0时刻的转座子突变体库作为参考对照。每组条件均设置3次生物学重复（biological replicates）。