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An integrated model system to gain mechanistic insights into biofilm-associated antimicrobial resistance in Pseudomonas aeruginosa MPAO1
npj Biofilms and Microbiomes ( IF 9.2 ) Pub Date : 2020-10-30 , DOI: 10.1038/s41522-020-00154-8
Adithi R. Varadarajan , Raymond N. Allan , Jules D. P. Valentin , Olga E. Castañeda Ocampo , Vincent Somerville , Franziska Pietsch , Matthias T. Buhmann , Jonathan West , Paul J. Skipp , Henny C. van der Mei , Qun Ren , Frank Schreiber , Jeremy S. Webb , Christian H. Ahrens

Pseudomonas aeruginosa MPAO1 is the parental strain of the widely utilized transposon mutant collection for this important clinical pathogen. Here, we validate a model system to identify genes involved in biofilm growth and biofilm-associated antibiotic resistance. Our model employs a genomics-driven workflow to assemble the complete MPAO1 genome, identify unique and conserved genes by comparative genomics with the PAO1 reference strain and genes missed within existing assemblies by proteogenomics. Among over 200 unique MPAO1 genes, we identified six general essential genes that were overlooked when mapping public Tn-seq data sets against PAO1, including an antitoxin. Genomic data were integrated with phenotypic data from an experimental workflow using a user-friendly, soft lithography-based microfluidic flow chamber for biofilm growth and a screen with the Tn-mutant library in microtiter plates. The screen identified hitherto unknown genes involved in biofilm growth and antibiotic resistance. Experiments conducted with the flow chamber across three laboratories delivered reproducible data on P. aeruginosa biofilms and validated the function of both known genes and genes identified in the Tn-mutant screens. Differential protein abundance data from planktonic cells versus biofilm confirmed the upregulation of candidates known to affect biofilm formation, of structural and secreted proteins of type VI secretion systems, and provided proteogenomic evidence for some missed MPAO1 genes. This integrated, broadly applicable model promises to improve the mechanistic understanding of biofilm formation, antimicrobial tolerance, and resistance evolution in biofilms.



中文翻译:

一个综合的模型系统,可深入了解铜绿假单胞菌MPAO1中与生物膜相关的抗药性

铜绿假单胞菌MPAO1是该重要临床病原体广泛使用的转座子突变体集合的亲本菌株。在这里,我们验证一个模型系统,以识别参与生物膜生长和生物膜相关抗生素抗性的基因。我们的模型采用基因组学驱动的工作流程来组装完整的MPAO1基因组,通过与PAO1参考菌株的比较基因组学和蛋白质组学在现有组件中遗漏的基因来鉴定独特且保守的基因。在200多个独特的MPAO1基因中,我们鉴定了在绘制针对PAO1的公共Tn-seq数据集(包括抗毒素)时被忽略的六个通用必需基因。使用方便,易用,用于生物膜生长的基于软光刻技术的微流控室,以及在微量滴定板中带有Tn突变体库的屏幕。该筛查确定了迄今为止未知的基因,这些基因参与了生物膜的生长和抗生素的耐药性。在三个实验室中对流动室进行的实验提供了可重复的数据铜绿假单胞菌生物膜,并验证了已知基因和在Tn突变体筛选中鉴定的基因的功能。来自浮游细胞与生物膜的蛋白质丰度数据的差异证实了已知影响生物膜形成的候选蛋白的上调,VI型分泌系统的结构蛋白和分泌蛋白的上调,并为某些MPAO1基因缺失提供了蛋白质组学证据。这种集成的,广泛适用的模型有望改善对生物膜形成,抗微生物耐受性以及生物膜阻力演变的机理的理解。

更新日期:2020-10-30
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