Targeting of LOS for Treatment of Antibiotic-Resistant Neisseria gonorrhoeae

由淋病奈瑟菌引起的感染是发病率的主要原因，据估计， 美国和全球每年有8700万例。在退伍军人管理局卫生保健系统内，淋病病例 2013至2017年间增加，在此期间总数为10,587人。最严重的 当淋球菌上升到生殖道上部时，感染的妇女会出现后遗症 盆腔炎在10%-20%的女性感染中，包括广泛的 炎症状态，通常会导致慢性盆腔疼痛、不孕和异位妊娠。没有 由于多重耐药性的惊人上升，淋病奈瑟菌的疫苗和对新抗生素的巨大需求 (MDR)，这使得出现无法治愈的淋球菌感染成为现实。目前仅限 推荐头孢曲松和阿奇霉素作为一线治疗药物，临床分离株对这两种药物都有耐药性。 据报道，这些抗生素在丹麦、加拿大和日本等国都有报道。因此，有一个 迫切需要新的抗菌剂来治疗淋球菌感染。 淋球菌脂低聚糖(LOS)与人类先天免疫系统的研究 已表明，LOS的脂质A部分是细胞因子介导的炎症的主要诱导者，并 其他人的研究表明，类脂A也有助于淋球菌感染。这些数据让我们找到了 靶向脂质A生物合成将是对抗淋病奈瑟菌的有效方法 感染。我们最近报道，抑制LpxC，一种催化脂质A第二步的酶 生物合成，对九种多重耐药和人类挑战的淋球菌和 减少了细胞因子的诱导，没有明显的人类细胞毒性。 根据LpxC抑制剂的数据，我们推测LOS的抑制导致了膜的破坏 生物合成对淋球菌是致命的。为了研究这一点，我们最近评估了一种细菌的杀菌潜力 12个氨基酸细胞穿透肽(CPP)对淋球菌多药耐药株和人攻毒株的作用 发现CPP穿透细菌膜，对所有9种MDR和人类都有杀菌作用 被测试的淋球菌的挑战菌株。重要的是，在存活过程中没有对CPP产生明显的抵抗 菌落中的细菌对CPP的敏感性与接触CPP后再撤退的细菌相同。 此外，CPP减少炎性细胞因子的诱导，防止宫颈细菌细胞的侵袭。 上皮细胞在没有可测量的细胞毒性的情况下。 这些新的数据突出了LpxC和CPP作为有前景的淋病奈瑟菌的抗菌剂，并强烈 支持LpxC抑制脂质A组分生物合成这一应用假说 抑制剂和用CPP破坏外膜完整性将影响细菌活力和宿主对 淋球菌在体外和体内的感染，这将对感染的结果产生治疗影响。 本项目致力于优化和测试CPP和LpxC抑制剂在相关疾病中的效果 体外杀菌活性、细胞因子诱导、溶血和细胞毒性测定。机械论研究将 包括DNA结合、细胞通透性、蛋白质分解抗性、蛋白质结合和 MtrCDE、MacAB和Normal淋球菌外排泵的作用。主要候选CPP和LpxC抑制剂 在体外确定的将被测试的体内疗效，药代动力学和心血管毒性 已建立的淋球菌生殖道感染的雌性小鼠模型已越来越多地用于 淋病治疗候选抗菌药的评价。 我们希望我们的研究结果将证明这两种抗菌剂作为新的抗菌剂的有效性 治疗淋球菌感染，这是由于耐多药淋球菌菌株的增加而迫切需要的。 这将是第一次测试这两类抗菌剂对淋病疗效的同类研究。

Infections due to N. gonorrhoeae are a major cause of morbidity with an estimated 850,000 cases in the U.S. and 87 million cases worldwide annually. Within the VA Health Care System, cases of gonorrhea increased between 2013 and 2017 with the total number in that time period at 10,587. The most serious sequelae are suffered by infected women as gonococci ascend to the upper reproductive tract and cause pelvic inflammatory disease in 10-20% of women with infections, which encompasses a wide range of inflammatory conditions and often leads to chronic pelvic pain, infertility, and ectopic pregnancy. There is no vaccine to N. gonorrhoeae and a great need for new antibiotics due to the alarming rise in multidrug-resistance (MDR), which is making emergence of untreatable gonococcal infections a real prospect. Currently only ceftriaxone and azithromycin are recommended for first-line therapy, and clinical isolates resistant to both of those antibiotics have been reported in countries including Denmark, Canada, and Japan. Thus, there is a compelling need for new antimicrobials for gonococcal infections. Our studies to date of N. gonorrhoeae lipooligosaccharide (LOS) and the human innate immune system have shown that the lipid A portion of LOS is the primary inducer of cytokine-mediated inflammation and investigations by others have shown that the lipid A also facilitates gonococcal infection. These data led us to the concept that targeting lipid A biosynthesis would be an effective approach to combating N. gonorrhoeae infections. We recently reported that inhibition of LpxC, the enzyme that catalyzes the second step of lipid A biosynthesis, was bactericidal for nine multidrug-resistant and human challenge strains of gonococci and reduced cytokine induction without apparent human cell cytotoxicity. From the LpxC inhibitor data, we postulated that membrane disruption due to the inhibition of LOS biosynthesis was lethal for gonococci. To investigate this, we recently evaluated the bactericidal potential of a 12 amino acid cell-penetrating peptide (CPP) for MDR and human challenge strains of N. gonorrhoeae and found that the CPP penetrated the bacterial membrane and was bactericidal for all nine MDR and human challenge strains of gonococci tested. Importantly, no apparent resistance to the CPP developed in surviving bacteria as susceptibility was the same in bacteria from colonies after exposure to CPP and then retreated. Further, the CPP reduced inflammatory cytokine induction and prevented bacterial cell invasion of cervical epithelial cells in the absence of measurable cell cytotoxicity. These novel data highlight LpxC and CPP as promising antimicrobials for N. gonorrhoeae and strongly support the hypothesis of this application that inhibiting the biosynthesis of lipid A components with LpxC inhibitors and disrupting outer membrane integrity with CPP will impact bacterial viability and host response to N. gonorrhoeae infection in vitro and in vivo, which will have a therapeutic impact on infection outcomes. This project is focused on optimizing and testing the efficacy of the CPP and LpxC inhibitor in relevant in vitro assays of bactericidal activity, cytokine induction, hemolysis, and cell cytotoxicity. Mechanistic studies will include investigations of DNA binding, cell permeabilization, proteolysis resistance, protein binding and the effect of the MtrCDE, MacAB and NorM gonococcal efflux pumps. The lead candidate CPP and LpxC inhibitor identified in vitro will be tested for in vivo efficacy, pharmacokinetics and cardiovascular toxicity in an established female mouse model of gonococcal genital tract infection that has been increasingly used for evaluation of candidate antimicrobials for treatment of gonorrhea. We expect that the results from our studies will demonstrate the efficacy of these two antimicrobials as new therapeutics for N. gonorrhoeae infection, which are urgently needed given the rise in MDR gonococcal strains. This will be the first study of its kind to test these two classes of antimicrobials for efficacy against gonorrhea.