Comprehensive Mutational Analysis of the Lasso Peptide Klebsidin

Antibiotic resistance is a growing threat to public health, making
the development of antibiotics of critical importance. One promising
class of potential new antibiotics are ribosomally synthesized and
post-translationally modified peptides (RiPPs), which include klebsidin,
a lasso peptide from Klebsiella pneumoniae that inhibits certain bacterial RNA polymerases. We develop a high-throughput
assay based on growth inhibition of Escherichia coli to analyze the mutational tolerance of klebsidin. We transform a
library of klebsidin variants into E. coli and use next-generation DNA sequencing to count the frequency of
each variant before and after its expression, thereby generating functional
scores for 320 of 361 single amino acid changes. We identify multiple
positions in the macrocyclic ring and the C-terminal tail region of
klebsidin that are intolerant to mutation, as well as positions in
the loop region that are highly tolerant to mutation. Characterization
of selected peptide variants scored as active reveals that each adopts
a threaded lasso conformation; active loop variants applied extracellularly
as peptides slow the growth of E. coli and K. pneumoniae. We generate an E. coli strain with a mutation in RNA polymerase
that confers resistance to klebsidin and similarly carry out a selection
with the klebsidin library. We identify a single variant, klebsidin
F9Y, that maintains activity against the resistant E. coli when expressed intracellularly. This finding
supports the utility of this method and suggests that comprehensive
mutational analysis of lasso peptides can identify unique and potentially
improved variants.

抗菌耐药性日益成为对公共健康的严重威胁，因此抗生素的研发显得尤为关键。一类具有潜力的新型抗生素为核糖体合成和翻译后修饰的肽类（RiPPs），其中klebsidin便是来源于肺炎克雷伯菌的一种环状肽，它能抑制某些细菌的RNA聚合酶。本研究开发了一种基于大肠杆菌生长抑制的高通量检测方法，用以分析klebsidin的突变耐受性。我们将klebsidin的各种变体库转入大肠杆菌中，并通过下一代DNA测序技术，在表达前后对每个变体的频率进行计数，从而为361个氨基酸变化中的320个生成功能评分。我们确定了klebsidin大环结构和C端尾巴区域中多个对突变不耐受的位置，以及环状区域中高度耐受突变的位置。对评分较高的具有活性的肽类变体进行分析发现，它们均呈现为螺旋状环状结构；将具有活性的环状肽变体作为外源肽应用于细胞外，能够减缓大肠杆菌和肺炎克雷伯菌的生长。我们通过RNA聚合酶突变构建了一种对klebsidin具有耐药性的大肠杆菌菌株，并使用klebsidin库进行筛选。我们发现了一个单一的变体klebsidin F9Y，在细胞内表达时仍能维持对耐药性大肠杆菌的活性。这一发现证实了该方法的有效性，并表明对环状肽进行全面突变分析能够识别独特的、可能得到改进的变体。