Function and regulation of the intrinsic antibiotic resistome of Neisseria gonorrhoeae

淋病奈瑟菌（淋球菌; [Gc]）引起粘膜表面的局部，简单的感染和更具侵袭性的疾病，可能对男性和女性的生殖和一般健康产生严重的医疗后果。在美国，据估计淋病的发病率每年至少为85万例，而2012年全球发病率据报道为7800万例。除了疾病发病率和淋病对人类健康的影响的全球性问题之外，Gc菌株对目前用于双重治疗的一线抗生素（例如，头孢曲松和阿奇霉素）在美国和其他发达国家。我们的研究计划致力于了解Gc所拥有的内在系统，使其能够抵抗经典抗生素以及参与先天宿主防御的阳离子抗菌肽/蛋白（CAMP）。在没有新抗生素的情况下，特别是那些认识到未开发的目标的抗生素，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，但来自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，感觉双组分系统（TCS）的损失称为MisR/MisS导致Gc对庆大霉素（以及其他氨基糖苷类）和CAMP的超敏感性，但不是其他抗生素。最近的一份报告记录了MisR/MisS TCS的丢失赋予Gc生存缺陷，如在下生殖道感染的雌性小鼠模型中所评估的。我们推测，这种衰减的Gc是由于，在某种程度上，减少MisR阴性Gc的能力，以调节基因的重要性，以抵抗宿主衍生的CAMPs。MisR阴性Gc对氨基糖苷类和CAMP的选择性敏感性与这些抗菌剂如何进入铜绿假单胞菌并穿过细胞包膜的自我促进摄取模型一致。我们现在将在初步研究取得进展的基础上再接再厉。在特定目标1中，我们将使用遗传筛选技术、实验雌性小鼠感染模型和对表达降低的庆大霉素敏感性的临床分离株的分析，以进一步确定导致内在庆大霉素耐药的Gc基因库。我们假设，所鉴定的基因将编码参与细胞被膜结构、膜完整性和膜稳态的蛋白质，这些蛋白质协同作用以影响Gc对氨基糖苷类和CAMP的抗性水平。在具体目标2中，我们将确定Gc对亚致死水平庆大霉素的反应性，并确定相关基因。在具体目标3中，我们将定义影响Gc对庆大霉素敏感性的已鉴定基因突变的生理后果。这项工作的结果将提供关于人类病原体如Gc如何控制参与对抗菌剂的内在抗性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.