Function and regulation of the intrinsic antibiotic resistome of Neisseria gonorrhoeae

淋病奈瑟菌(淋球菌；[GC])既可引起粘膜表面的局部、非复杂感染，也可引起更多侵袭性疾病，对男性和女性的生殖和一般健康造成严重的医学后果。据估计，美国每年的淋病发病率至少为850,000例，而2012年全球淋病发病率据报道为7,800万例。除了疾病发病率和淋病对人类健康的影响等全球性问题外，GC菌株对美国和其他发达国家目前用于双重疗法的一线抗生素(如头孢曲松和阿奇霉素)的敏感性或临床抗药性也有所降低。我们的研究项目致力于了解GC所具有的内在系统，使其能够抵抗经典抗生素以及参与天然宿主防御的阳离子抗菌肽/蛋白(CAMP)。在缺乏新的抗生素，特别是那些识别未开发的靶标的抗生素的情况下，关于可能进入临床实践的现有抗生素的耐药性发展的信息至关重要。例如，庆大霉素在非洲被广泛用于治疗淋病，但世界这一地区的耐药性数据很少。这是不幸的，因为在发达国家，庆大霉素已被建议作为治疗淋病的替代抗生素。我们有证据表明，失去一种称为MISR/MISH的感觉双组分系统(TCS)会导致GC对庆大霉素(以及其他氨基糖苷类)和cAMP高度敏感，但对其他抗生素不敏感。最近的一份报告证明，在雌性小鼠下生殖道感染模型中，MISR/MISTCS的缺失导致GC存在生存缺陷。我们推测，GC的这种衰减部分是由于MISR阴性的GC调节对宿主衍生cAMP的抗性重要的基因的能力降低。MISR阴性GC对氨基糖苷类和cAMP的选择性敏感性与这些抗菌素如何进入铜绿假单胞菌并穿过细胞膜的自我促进摄取模型一致。我们现在将在初步研究的基础上再接再厉。在具体目标1中，我们将利用基因筛选技术、实验性雌性小鼠感染模型和对庆大霉素敏感性降低的临床分离株的分析，进一步确定与庆大霉素固有耐药性有关的GC基因谱系。我们假设已识别的基因将编码参与细胞包膜结构、膜完整性和膜稳态的蛋白质，这些蛋白质协同工作，影响GC对氨基糖苷类和cAMP的耐药性水平。在特定的目标2中，我们将确定GC对亚致死水平的庆大霉素的反应性，并确定相关基因。在具体目标3中，我们将定义影响GC对庆大霉素易感性的已识别基因突变的生理后果。这项工作的结果将提供关于像GC这样的人类病原体如何控制与抗菌素内在耐药性有关的基因表达的基本知识。

Neisseria gonorrhoeae (the gonococcus; [Gc]) causes both localized, uncomplicated infections at mucosal surfaces and more invasive forms of disease that can have severe medical consequences for the reproductive and general health of men and women. In the United States it is estimated that the incidence of gonorrhea is at least 850,000 cases per year while the worldwide incidence in 2012 was reported to be 78 million cases. In addition to the global problems of disease incidence and impact of gonorrhea on human health, Gc strains have developed decreased susceptibility or clinical resistance to front-line antibiotics currently used in dual therapy (e.g., ceftriaxone and azithromycin) in the USA and other developed countries. Our research program is dedicated to understanding the intrinsic systems possessed by Gc that allow it to resist classical antibiotics as well as cationic antimicrobial peptides/proteins (CAMPs) that participate in innate host defense. In the absence of new antibiotics, especially those recognizing unexploited targets, information regarding the development of resistance to existing antibiotics likely to enter clinical practice is critical. For instance, gentamicin has been used extensively in Africa to treat gonorrhea but resistance data from this part of the world is scant. This is unfortunate because gentamicin has been suggested to be an alternative antibiotic for treatment of gonorrhea in developed countries. We have evidence that loss of a sensory two component system (TCS) termed MisR/MisS results in Gc hyper-susceptibility to both gentamicin (as well as other aminoglycosides) and CAMPs, but not other antibiotics. A recent report documented that loss of the MisR/MisS TCS conferred a survival defect on Gc as assessed in a female mouse model of lower genital tract infection. We hypothesize that such attenuation of Gc was due, in part, to a reduced capacity of MisR-negative Gc to regulate genes important for resistance to host-derived CAMPs. The selective sensitivity of MisR-negative Gc to aminoglycosides and CAMPs is consistent with the self-promoted uptake model invoked for how these antimicrobials enter Pseudomonas aeruginosa and traverse the cell envelope. We will now build on the progress made in our initial studies. In Specific Aim 1 we will use genetic screening techniques, an experimental female mouse infection model and analysis of clinical isolates expressing decreased gentamicin susceptibility to further define the repertoire of Gc genes that contribute to intrinsic gentamicin resistance. We hypothesize that the identified genes will encode proteins that participate in cell envelope structure, membrane integrity and membrane homeostasis that work in unison to impact levels of Gc resistance to aminoglycosides and CAMPs. In Specific Aim 2 we will ascertain the responsiveness of Gc to sub-lethal levels of gentamicin and determine the responsible genes. In Specific Aim 3 we will define the physiological consequences of mutations in the identified genes that influence Gc susceptibility to gentamicin. The results from this work will provide basic knowledge as to how human pathogens like Gc control expression of genes involved in intrinsic resistance to antimicrobials.