BLRD Research Career Scientist Award Application

申请人的总体研究计划重点关注LOS和先天免疫的作用 系统并表明脂质 A 成分的磷酸化程度与 LOS 诱导先天免疫细胞因子介导的炎症的潜力，并且一般而言，其严重程度 奈瑟氏菌病。基于这些研究的结果，最近的工作已经进行了开发 针对淋球菌 LOS 的潜在治疗方法，因为淋病奈瑟菌感染是导致淋病的主要原因 据估计，美国每年有 85 万例，全世界每年有 8700 万例。在弗吉尼亚州内 卫生保健系统，2013 年至 2017 年期间淋病病例数有所增加 期间为 10,587。在美国军队中，军人被定义为淋病高危人群： 美国预防服务工作组指出，现役军人的高风险环境是 人们认为这会增加危险行为，从而增加包括淋病在内的性传播感染的发生率。一个 2007 至 2015 财年，现役人员共发现 27,658 例淋病病例（222.7 每 10 万名军人中有 179.3 人，而 2018 年每 10 万名非军人中有 179.3 人）。目前尚无针对 N 的疫苗。 由于多重耐药性（MDR）的惊人增加，对新抗生素的巨大需求， 这使得无法治疗的淋球菌感染的出现成为现实。因此，有一个令人信服的 需要新的抗菌药物来治疗淋球菌感染。 为此，在本次 SRCS 奖项的拟议资助期内要开展的项目如下： 如下：1）LpxC抑制剂和细胞穿透肽；我们最近报道了淋球菌的治疗 LpxC（催化脂质 A 生物合成第二步的酶）抑制剂具有杀菌作用 针对 MDR 和人类攻击淋球菌菌株，并减少细胞因子诱导，而无需明显的人类 细胞的细胞毒性。最近，我们评估了 12 种氨基酸细胞穿透性的杀菌潜力 肽（CPP），发现它能穿透细菌膜，对所有多药都有杀菌作用 测试了淋球菌的耐药菌株和人类攻击菌株，并减少了炎症细胞因子的诱导和 阻止细菌细胞侵入宫颈上皮细胞。这些新颖的数据突出了 LpxC 抑制剂和 CPP 作为治疗淋病奈瑟菌的有前景的抗菌剂，强烈支持本申请的假设： 用 LpxC 抑制剂抑制脂质 A 成分的生物合成并破坏外膜完整性 CPP 会影响细菌活力和宿主对体外和体内淋病奈瑟菌感染的反应，这 将对感染结果产生治疗影响。这两种潜在疗法的转化研究 淋病将是资助期内拟议调查的主要重点。 2) 乙二胺四乙酸 抑制剂；我们将继续与博士合作。夏琳·卡勒 (Charlene Kahler) 和爱丽丝·弗里林克 (Alice Vrielink) 大学 西澳大利亚州，我们的目标是淋球菌 EptA（脂质 A 的磷酸乙醇胺转移酶）， 含有小分子抑制剂，使淋病奈瑟菌对内部阳离子抗菌肽的杀灭敏感 中性粒细胞并减少炎症细胞因子的诱导。 3) 淋球菌在中性粒细胞中的存活；我们 我们将继续与弗吉尼亚大学的 Alison Criss 博士合作，以确定 LOS 的影响 PMN 中淋球菌存活的结构变异。 4）淋球菌疫苗；与博士合作。 加州大学旧金山分校的 Peter Beernink，我们将开发来自脑膜炎奈瑟菌菌株的天然外膜囊泡疫苗 被设计为表达潜在的保护性淋病奈瑟菌抗原。 总体而言，为下一个 SRCS 资助期提出的研究将继续支持 VA 的使命 通过研究促进潜在疗法的转化开发和医疗保健 针对淋病的疫苗，由于迅速流行，疾病预防控制中心将淋病列为紧急公共卫生威胁 MDR 菌株数量增加。

The overall research program of the applicant has focused on the role of LOS and the innate immune system and has shown that the degree of phosphorylation of the lipid A component is correlated with the potential of the LOS to induce innate immune cytokine-mediated inflammation and, in general, with the severity of Neisserial disease. Based on the results of these studies, recent work has undertaken the development of potential therapeutics targeting gonococcal LOS as 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. Within the U.S. military, service members are a defined high-risk group for gonorrhea as noted by the U.S. Preventive Services Task Force as the high-risk environment of active military service is thought to increase risky behavior and, thus, the rate of sexually transmitted infections including gonorrhea. A total of 27,658 cases of gonorrhea were identified among active duty personnel during FY 2007 to 2015 (222.7 per 100,000 military compared with 179.3 per 100,000 non-military in 2018). 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. Thus, there is a compelling need for new antimicrobials for gonococcal infections. To this end, the projects to be pursued during the proposed funding period of this SRCS award are as follows: 1) LpxC inhibitors and cell-penetrating peptides; we recently reported that treatment of gonococci with an inhibitor of LpxC, the enzyme that catalyzes the second step of lipid A biosynthesis, was bactericidal for MDR and human challenge strains of gonococci and reduced cytokine induction without apparent human cell cytotoxicity. Most recently, we evaluated the bactericidal potential of a 12 amino acid cell-penetrating peptide (CPP) and found that it penetrated the bacterial membrane and was bactericidal for all multi-drug resistant and human challenge strains of gonococci tested and reduced inflammatory cytokine induction and prevented bacterial cell invasion of cervical epithelial cells. These novel data highlight LpxC inhibitors 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. Translational studies of these two potential therapeutics for gonorrhea will be the primary focus of the investigations proposed for the award period. 2) EptA inhibitors; we will continue our collaboration with Drs. Charlene Kahler and Alice Vrielink of the University of Western Australia in which we are targeting gonococcal EptA, the phosphoethanolamine transferase for lipid A, with small molecule inhibitors to sensitize N. gonorrhoeae to killing by cationic antimicrobial peptides inside neutrophils and reduce the induction of inflammatory cytokines. 3) Gonococcal survival in neutrophils; we will continue our collaboration with Dr. Alison Criss of the University of Virginia to determine the impact of LOS structural variation on the survival of gonococci in PMNs. 4) Gonococcal vaccine; in collaboration with Dr. Peter Beernink of UCSF, we will develop native outer membrane vesicle vaccines from N. meningitidis strains engineered to express potentially protective N. gonorrhoeae antigens. Overall, the research proposed for the next SRCS funding period will continue to support the mission of VA Healthcare through studies that will facilitate the translational development of potential therapeutics and vaccines targeting gonorrhea, which is classified by CDC as an urgent public health threat due to the rapid increase in the number of MDR isolates.