我们先前报道了线性阳离子12个氨基酸的细胞穿透肽(CPP)对淋病奈瑟菌具有杀菌作用。在这项研究中,我们的目标是确定线性CPP的环化对其对淋病奈瑟菌的抗菌活性和对人细胞的细胞毒性的影响。我们比较了用线性CPP处理4小时与通过硫醚或二硫键环化的CPPs对人类攻击和在细胞培养基中生长的淋病奈瑟菌的多药耐药(MDR)菌株的杀菌效果。10%胎牛血清(FBS)。分析了脂寡糖(LOS)唾液酸化对杀菌活性的影响。我们确定了CPPs治疗体外感染淋病奈瑟菌的人细胞的能力,减少人类单核细胞对淋球菌的炎症反应,杀死三种共生奈瑟菌,并抑制淋球菌生物膜。环化的CPPs在100µM时杀死所有菌株的淋球菌100%,在20µM时杀死>90%,并且比线性形式更有效。与线性CPP相比,硫醚连接而不是二硫键连接的CPP对人宫颈细胞的细胞毒性较小。LOS唾液酸化对杀菌活性的影响很小。在治疗受感染的人类细胞时,20µM的硫醚连接的CPP杀死>60%的细胞外和细胞内细菌,并降低THP-1细胞的TNF-α表达。CPPs对致病性和共生奈瑟菌的效力相似。硫醚连接的CPP部分根除了淋球菌生物膜。未来的研究将集中在确定淋病雌性小鼠模型中的功效。淋病奈瑟菌仍然是性传播感染的主要原因,2020年全球有8200万例,2021年美国有710,151例确诊病例,比2017年增长25%。淋病奈瑟菌可感染多种组织,包括尿道,子宫颈,直肠,咽部,和结膜。最严重的后遗症是感染妇女,因为淋球菌上升到上生殖道并引起盆腔炎,慢性盆腔疼痛,10%-20%的女性不孕症。由于缺乏任何疫苗,控制淋球菌感染被广泛认为是越来越具有挑战性的。淋病奈瑟菌对除一类抗生素外的所有抗生素迅速产生耐药性,多重耐药菌株的出现可能导致无法治愈的感染。因此,淋病被疾病控制中心(CDC)列为紧急公共卫生威胁。本文提出的关于淋病新疗法的研究已经鉴定了环状细胞穿透肽(CPP)作为靶向淋病奈瑟菌的有效分子。
We previously reported that a linear cationic 12-amino acid cell-penetrating peptide (CPP) was bactericidal for Neisseria gonorrhoeae. In this study, our objectives were to determine the effect of cyclization of the linear CPP on its antibacterial activity for N. gonorrhoeae and cytotoxicity for human cells. We compared the bactericidal effect of 4-hour treatment with the linear CPP to that of CPPs cyclized by a thioether or a disulfide bond on human challenge and multi-drug resistant (MDR) strains of N. gonorrhoeae grown in cell culture media with 10% fetal bovine serum (FBS). The effect of lipooligosaccharide (LOS) sialylation on bactericidal activity was analyzed. We determined the ability of the CPPs to treat human cells infected in vitro with N. gonorrhoeae, to reduce the inflammatory response of human monocytic cells to gonococci, to kill strains of three commensal Neisseria species, and to inhibit gonococcal biofilms. The cyclized CPPs killed 100% of gonococci from all strains at 100 µM and >90% at 20 µM and were more potent than the linear form. The thioether-linked but not the disulfide-linked CPP was less cytotoxic for human cervical cells compared to the linear CPP. LOS sialylation had minimal effect on bactericidal activity. In treating infected human cells, the thioether-linked CPP at 20 µM killed >60% of extra- and intracellular bacteria and reduced TNF-α expression by THP-1 cells. The potency of the CPPs for the pathogenic and the commensal Neisseria was similar. The thioether-linked CPP partially eradicated gonococcal biofilms. Future studies will focus on determining efficacy in the female mouse model of gonorrhea.IMPORTANCENeisseria gonorrhoeae remains a major cause of sexually transmitted infections with 82 million cases worldwide in 2020, and 710,151 confirmed cases in the US in 2021, up 25% from 2017. N. gonorrhoeae can infect multiple tissues including the urethra, cervix, rectum, pharynx, and conjunctiva. The most serious sequelae are suffered by infected women as gonococci ascend to the upper reproductive tract and cause pelvic inflammatory disease, chronic pelvic pain, and infertility in 10%-20% of women. Control of gonococcal infection is widely recognized as increasingly challenging due to the lack of any vaccine. N. gonorrhoeae has quickly developed resistance to all but one class of antibiotics and the emergence of multidrug-resistant strains could result in untreatable infections. As such, gonorrhea is classified by the Center for Disease Control (CDC) as an urgent public health threat. The research presented herein on new therapeutics for gonorrhea has identified a cyclic cell-penetrating peptide (CPP) as a potent molecule targeting N. gonorrhoeae.