Establishing an invertebrate <i>Galleria mellonella</i> greater wax moth larval model of <i>Neisseria gonorrhoeae</i> infection

<i>Neisseria gonorrhoeae</i> (gonococcus) causes the human sexually transmitted disease gonorrhea. Studying gonococcal pathogenesis and developing new vaccines and therapies to combat the increasing prevalence of multi-antibiotic resistant bacteria has made use of many <i>ex vivo</i> models based on human cells and tissues, and <i>in vivo</i> vertebrate models, for example, rodent, pig and human. The focus of the current study was to examine the utility of the invertebrate greater wax moth <i>Galleria mellonella</i> as an <i>in vivo</i> model of gonococcal infection. We observed that a threshold of ~10⁶ – 10⁷ gonococci/larva was required to kill &gt;50% of larvae (P &lt; 0.05), and increased toxicity correlated with reduced health index scores and pronounced histopathological changes such as increases in the total lesion grade, melanized nodules, hemocyte reaction, and multifocal adipose body degeneration. Larval death was independent of the expression of pilus or Opa protein or LOS sialylation within a single gonococcal species studied, but the model could demonstrate relative toxicity of different isolates. <i>N. meningitidis, N. lacatamica</i> and gonococci all killed larvae equally, but were significantly less toxic (P &gt; 0.05) than <i>Pseudomonas aeruginosa</i>. Larvae primed with nontoxic doses of gonococci were more susceptible to subsequent challenge with homologous and heterologous bacteria, and larval survival was significantly reduced (P &lt; 0.05) in infected larvae after depletion of their hemocytes with clodronate-liposomes. The model was used to test the anti-gonococcal properties of antibiotics and novel antimicrobials. Ceftriaxone (P &lt; 0.05) protected larvae from infection with different gonococcal isolates, but not azithromycin or monocaprin or ligand-coated silver nanoclusters (P &gt; 0.05).淋病奈瑟菌（*Neisseria gonorrhoeae*，俗称淋球菌）可引发人类性传播疾病淋病。针对淋球菌致病机制的研究，以及为应对多重抗生素耐药菌感染率持续上升而开发新型疫苗与治疗手段的工作，已广泛采用基于人体细胞与组织的体外（ex vivo）模型，以及体内（in vivo）脊椎动物模型，例如啮齿类、猪及人类模型。本研究的核心目标，是探究无脊椎动物大蜡螟（*Galleria mellonella*）作为淋球菌感染体内模型的应用价值。研究结果显示，需达到约10⁶–10⁷个淋球菌/幼虫的感染阈值，方可导致超过50%的幼虫死亡（P < 0.05）；且毒素毒性的增强与幼虫健康指数评分降低、显著的组织病理学变化密切相关，这些变化包括总病变等级升高、黑色素化结节形成、血细胞反应以及多灶性脂肪体变性。幼虫的死亡与所测试的单一淋球菌菌株的菌毛（pilus）、Opa蛋白（Opa protein）或脂多糖（LOS）唾液酸化表达无关，但该模型能够区分不同淋球菌分离株的相对毒性。脑膜炎奈瑟菌（*Neisseria meningitidis*）、乳糖奈瑟菌（*Neisseria lacatamica*）与淋球菌对幼虫的致死能力相当，但三者的毒性均显著低于铜绿假单胞菌（*Pseudomonas aeruginosa*，P > 0.05）。经非毒性剂量淋球菌预致敏的幼虫，对后续同源或异源细菌攻击的易感性显著提升；而使用氯膦酸盐脂质体（clodronate-liposomes）耗竭血细胞的感染幼虫，其存活率亦显著降低（P < 0.05）。该模型被用于评估抗生素及新型抗微生物制剂的抗淋球菌活性。头孢曲松（P < 0.05）可有效保护幼虫免受不同淋球菌分离株的感染，但阿奇霉素、单癸酸甘油酯（monocaprin）以及配体包被的银纳米团簇（silver nanoclusters）并未展现出类似的保护效果（P > 0.05）。