Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, New Delhi, India.
Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India.
mSphere. 2024 Jan 30;9(1):e0046423. doi: 10.1128/msphere.00464-23. Epub 2023 Dec 19.
Emergence and spread of the hypervirulent pathotype of have significantly increased infection rates in community as well as healthcare settings. There is an increasing interest to identify discriminating features between classical (cKp) and hypervirulent (hvKp) to facilitate our understanding of the rapid emergence and dissemination of the hypervirulent pathotype. Here, we sought to identify unique epigenetic signatures of hvKp pathotype that differ from its classical counterpart using single-base resolution methylome analysis of native DNA sequencing on the Oxford Nanopore Technologies platform. The overall global adenine methylation in GATC motifs (i.e., Dam methylation motif) and cytosine methylation in CCWGG motifs (i.e., Dcm methylation motif) were significantly higher in hvKp isolates compared to that in cKp isolates, irrespective of their position in chromosomes or putative extra-chromosomal genetic elements. Notably, we observed significant enrichment of hypermethylated GATC and CCWGG motifs in the virulome of hvKp compared to hvKp genes not directly associated with virulence. We also observed increased methylation of GATC and CCWGG motifs in the capsule synthesis locus of hvKp isolates compared to cKp isolates. Furthermore, we identified several differentially methylated genes (DMGs) between the two pathotypes; interestingly, these DMGs include metal ion transporters, multidrug efflux pumps, transcriptional regulators of stress response, and genes associated with biofilm formation. Our results highlight hypermethylation of GATC and CCWGG motifs as unique epigenetic signatures of hvKp isolates.IMPORTANCEHypervirulent (hvKp) is a more virulent and rapidly evolving hypermucoviscous pathotype of classical (cKp). The hypervirulent pathotype is a major public health concern and is associated with high infection rates in community as well as hospital settings. With the recent emergence of multidrug-resistant hvKp, it has become imperative to investigate non-classical mechanisms such as epigenetics in addition to canonical biochemical and genetic mechanisms that delineate and differentiate the hypervirulent pathotype from its classical counterpart. Here, we identify genome-wide differences in adenine and cytosine methylation marks at well-characterized motifs between the two pathotypes. Overall, significantly higher levels of methylation were observed across chromosomal DNA and extrachromosomal elements in hvKp compared to cKp. Among hvKp isolates, the genes associated with virulence are particularly enriched for methylation marks. Our findings shed light on how epigenetic signatures may help distinguish the pathogenic potential of bacteria.
产酸克雷伯菌的高毒力表型的出现和传播显著增加了社区和医疗机构中的感染率。人们越来越有兴趣识别经典产酸克雷伯菌（cKp）和高毒力产酸克雷伯菌（hvKp）之间的区别特征，以帮助我们理解高毒力表型的快速出现和传播。在这里，我们试图使用牛津纳米孔技术平台上的原生 DNA 测序的单碱基分辨率甲基组分析，来鉴定 hvKp 表型的独特表观遗传特征，这些特征与经典表型不同。总体而言，hvKp 分离株中的 GATC 基序（即 Dam 甲基化基序）中的腺嘌呤整体全局甲基化和 CCWGG 基序（即 Dcm 甲基化基序）中的胞嘧啶甲基化显著高于 cKp 分离株，无论其在染色体中的位置还是假定的染色体外遗传元件如何。值得注意的是，与与毒力直接相关的 hvKp 基因相比，我们在 hvKp 的毒力组中观察到高度甲基化的 GATC 和 CCWGG 基序显著富集。我们还观察到 hvKp 分离株中荚膜合成基因座中的 GATC 和 CCWGG 基序的甲基化程度高于 cKp 分离株。此外，我们在两种表型之间鉴定了几个差异甲基化基因（DMG）；有趣的是，这些 DMG 包括金属离子转运蛋白、多药外排泵、应激反应的转录调节剂以及与生物膜形成相关的基因。我们的结果强调了 GATC 和 CCWGG 基序的高甲基化是 hvKp 分离株的独特表观遗传特征。
高毒力（hvKp）是经典产酸克雷伯菌（cKp）更具毒力和快速进化的高粘液性表型。高毒力表型是一个主要的公共卫生问题，与社区和医院环境中的高感染率有关。随着最近多药耐药性 hvKp 的出现，除了区分和区分高毒力表型与其经典对应物的典型生化和遗传机制之外，研究非经典机制（如表观遗传学）变得至关重要。在这里，我们在两种表型之间的特征明确的基序中鉴定了全基因组范围内腺嘌呤和胞嘧啶甲基化标记的差异。总体而言，hvKp 中染色体 DNA 和染色体外元件上的甲基化水平明显高于 cKp。在 hvKp 分离株中，与毒力相关的基因特别富含甲基化标记。我们的研究结果揭示了表观遗传特征如何帮助区分细菌的致病潜力。
