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Abstract：
Acinetobacter baumannii, a strictly aerobic, non-lactose fermented Gram-negative bacteria, is one of the important pathogens of nosocomial infection. Major facilitator superfamily (MFS) transporter membrane proteins are a class of proteins that widely exists in microbial genomes and have been revealed to be related to biofilm formation in a variety of microorganisms. However, as one of the MFS transporter membrane proteins, little is known about the role of BIT33_RS14560 in A. baumannii. To explore the effects of BIT33_RS14560 on biofilm formation of A. baumannii, the biofilm formation abilities of 62 isolates were firstly investigated and compared with their transcript levels of BIT33_RS14560. Then, this specific gene was over-expressed in a standard A. baumannii strain (ATCC 19606) and two isolates of extensively drug-resistant A. baumannii (XDR-Ab). Bacterial virulence was observed using a Galleria mellonella infection model. High-throughput transcriptome sequencing (RNA seq) was performed on ATCC 19606 over-expressed strain and its corresponding empty plasmid control strain. Spearman\'s correlation analysis indicated a significant negative correlation (R = -0.569, p = 0.000) between the △CT levels of BIT33_RS1456 and biofilm grading of A. baumannii isolates. The amount of A. baumannii biofilm was relatively high within 12-48 h. Regardless of standard or clinical strains; the biofilm biomass in the BIT33_RS14560 overexpression group was significantly higher than that in the control group ( p < 0.0001). Kaplan-Meier survival curve analysis showed that the mortality of G. mellonella was significantly higher when infected with the BIT33_RS14560 overexpression strain (χ2 = 8.462, p = 0.004). RNA-Seq showed that the mRNA expression levels of three genes annotated as OprD family outer membrane porin, glycosyltransferase family 39 protein, and glycosyltransferase family 2 protein, which were related to bacterial adhesion, biofilm formation, and virulence, were significantly upregulated when BIT33_RS14560 was over-expressed. Our findings provided new insights in identifying potential drug targets for the inhibition of biofilm formation. We also developed a practical method to construct an over-expressed vector that can stably replicate in XDR-Ab isolates.
摘要：
鲍曼不动杆菌,严格的有氧运动，非乳糖发酵革兰氏阴性菌，是医院感染的重要病原体之一。主要促进剂超家族（MFS）转运蛋白是一类广泛存在于微生物基因组中的蛋白质，已被发现与多种微生物中的生物膜形成有关。然而,作为MFS转运膜蛋白之一，对BIT33_RS14560在鲍曼不动杆菌中的作用知之甚少。探讨BIT33_RS14560对鲍曼不动杆菌生物膜形成的影响,首先研究了62株分离株的生物膜形成能力，并将其与BIT33_RS14560的转录水平进行了比较。然后，该特定基因在标准鲍曼不动杆菌菌株(ATCC19606)和两个广泛耐药鲍曼不动杆菌(XDR-Ab)分离株中过表达。使用GalleriaMellonella感染模型观察细菌毒力。在ATCC19606过表达的菌株及其相应的空质粒对照菌株上进行高通量转录组测序(RNAseq)。Spearman相关分析表明BIT33_RS1456的△CT水平与鲍曼不动杆菌的生物膜分级之间存在显着负相关（R=-0.569，p=0.000）。鲍曼不动杆菌生物膜的量在12-48小时内相对较高。无论标准菌株还是临床菌株;BIT33_RS14560过表达组中的生物膜生物量显著高于对照组(p<0.0001)。Kaplan-Meier存活曲线分析显示，当感染BIT33_RS14560过表达菌株时，梅洛氏菌的死亡率显着升高（χ2=8.462，p=0.004）。RNA-Seq显示三个基因的mRNA表达水平注释为OprD家族外膜孔蛋白,糖基转移酶家族39蛋白，和糖基转移酶家族2蛋白，与细菌粘附有关，生物膜的形成，和毒力，当BIT33_RS14560过表达时显著上调。我们的发现为识别抑制生物膜形成的潜在药物靶标提供了新的见解。我们还开发了一种实用的方法来构建可以在XDR-Ab分离物中稳定复制的过表达载体。
