细胞穿透肽(CPPs)是有效转运反义寡核苷酸(ASO)的有前景的载体,包括肽核酸(PNA),进入细菌细胞以对抗多药耐药的细菌感染,显示出显著的治疗潜力。猪链球菌,革兰氏阳性细菌,是猪的主要细菌病原体和新兴的人畜共患病原体。在这项研究中,通过超分辨率结构照明显微镜(SR-SIM)的组合,流式细胞术分析,和毒性分析试验,我们研究了4种CPPs将PNA递送到猪链球菌细胞中的适用性:HIV-1TAT有效地穿透猪链球菌细胞,对猪链球菌具有低毒性;(RXR)4XB具有高穿透效率,对猪链球菌具有固有毒性;(KFF)3K的穿透效率低于HIV-1TAT和(RXR)4XB;K8未能穿透猪链球菌细胞。对必需促旋酶A亚基基因具有特异性的HIV-1TAT缀合的PNA(TAT-anti-gyrAPNA)有效抑制猪链球菌的生长。TAT-anti-gyrAPNA对猪链球菌的血清型2、4、5、7和9株具有显着的杀菌作用,已知会导致人类感染。我们的研究证明了CPP-ASO缀合物作为新的抗微生物化合物用于对抗猪链球菌感染的潜力。此外,我们的研究结果表明,应用SR-SIM和流式细胞术分析提供了一种方便的,直观,和具有成本效益的方法来鉴定合适的CPP用于将货物分子递送到细菌细胞中。
Cell-penetrating peptides (CPPs) are promising carriers to effectively transport antisense oligonucleotides (ASOs), including peptide nucleic acids (PNAs), into bacterial cells to combat multidrug-resistant bacterial infections, demonstrating significant therapeutic potential. Streptococcus suis, a Gram-positive bacterium, is a major bacterial pathogen in pigs and an emerging zoonotic pathogen. In this study, through the combination of super-resolution structured illumination microscopy (SR-SIM), flow cytometry analysis, and toxicity analysis assays, we investigated the suitability of four CPPs for delivering PNAs into S. suis cells: HIV-1 TAT efficiently penetrated S. suis cells with low toxicity against S. suis; (RXR)4XB had high penetration efficiency with inherent toxicity against S. suis; (KFF)3K showed lower penetration efficiency than HIV-1 TAT and (RXR)4XB; K8 failed to penetrate S. suis cells. HIV-1 TAT-conjugated PNA specific for the essential gyrase A subunit gene (TAT-anti-gyrA PNA) effectively inhibited the growth of S. suis. TAT-anti-gyrA PNA exhibited a significant bactericidal effect on serotypes 2, 4, 5, 7, and 9 strains of S. suis, which are known to cause human infections. Our study demonstrates the potential of CPP-ASO conjugates as new antimicrobial compounds for combating S. suis infections. Furthermore, our findings demonstrate that applying SR-SIM and flow cytometry analysis provides a convenient, intuitive, and cost-effective approach to identifying suitable CPPs for delivering cargo molecules into bacterial cells.