Shankar Uma, Jain Neha, Majee Prativa, Kodgire Prashant, Sharma Tarun Kumar, Kumar Amit
Discipline of Biosciences and Biomedical Engineering, Indian Institute of Technology Indore, Indore, India.
Translational Health Science and Technology Institute, Faridabad, India.
Front Genet. 2020 Sep 25;11:935. doi: 10.3389/fgene.2020.00935. eCollection 2020.
, a gram-negative bacterium that causes cholera, has already caused seven major pandemics across the world and infects roughly 1.3-4 million people every year. Cholera treatment primarily involves oral rehydration therapy supplemented with antibiotics. But recently, multidrug-resistant strains of have emerged. High genomic plasticity further enhances the pathogenesis of this human pathogen. Guanines in DNA or RNA assemble to form G-quadruplex (GQ) structures which have begun to be seen as potential drug targeting sites for different pathogenic bacteria and viruses. In this perspective, we carried out a genome-wide hunt in using a bio-informatics approach and observed ∼85 G-quadruplex forming motifs (VC-PGQs) in chromosome I and ∼45 putative G-quadruplexs (PGQs) in chromosome II. Ten putative G-quadruplex forming motifs (VC-PGQs) were selected on the basis of conservation throughout the genus and functional analysis displayed their location in the essential genes encoding bacterial proteins, for example, methyl-accepting chemotaxis protein, orotate phosphoribosyl transferase protein, amidase proteins, etc. The predicted VC-PGQs were validated using different bio-physical techniques, including Nuclear Magnetic Resonance spectroscopy, Circular Dichroism spectroscopy, and electrophoretic mobility shift assay, which demonstrated the formation of highly stable GQ structures in the bacteria. The interaction of these VC-PGQs with the known specific GQ ligand, TMPyP4, was analyzed using ITC and molecular dynamics studies that displayed the stabilization of the VC-PGQs by the GQ ligands and thus represents a potential therapeutic strategy against this enteric pathogen by inhibiting the PGQ harboring gene expression, thereby inhibiting the bacterial growth and virulence. In summary, this study reveals the presence of conserved GQ forming motifs in the genome that has the potential to be used to treat the multi-drug resistance problem of the notorious enteric pathogen.
霍乱弧菌是一种引起霍乱的革兰氏阴性菌，已经在全球引发了七次重大疫情，每年感染约130万至400万人。霍乱治疗主要包括口服补液疗法并辅以抗生素。但最近，出现了多重耐药的霍乱弧菌菌株。高度的基因组可塑性进一步增强了这种人类病原体的致病性。DNA或RNA中的鸟嘌呤聚集形成G-四链体（GQ）结构，这些结构已开始被视为针对不同病原菌和病毒的潜在药物靶点。从这个角度来看，我们使用生物信息学方法在霍乱弧菌中进行了全基因组搜索，在染色体I中观察到约85个形成G-四链体的基序（VC-PGQs），在染色体II中观察到约45个推定的G-四链体（PGQs）。基于整个属的保守性选择了10个推定的形成G-四链体的基序（VC-PGQs），功能分析显示它们位于编码细菌蛋白的必需基因中，例如甲基接受趋化蛋白、乳清酸磷酸核糖转移酶蛋白、酰胺酶蛋白等。使用不同的生物物理技术对预测的VC-PGQs进行了验证，包括核磁共振光谱、圆二色光谱和电泳迁移率变动分析，这些分析证明了细菌中形成了高度稳定的GQ结构。使用等温滴定量热法（ITC）和分子动力学研究分析了这些VC-PGQs与已知的特异性GQ配体TMPyP4的相互作用，结果显示GQ配体可稳定VC-PGQs，因此代表了一种通过抑制携带PGQ的基因表达来对抗这种肠道病原体的潜在治疗策略，从而抑制细菌生长和毒力。总之，这项研究揭示了霍乱弧菌基因组中存在保守的形成GQ的基序，这些基序有可能用于解决这种臭名昭著的肠道病原体的多重耐药问题。
