Tear Protein Microbial Regulation

泪液杀菌活性保护眼睛表面免受环境病原体的侵袭，并可能调节水平。 共生细菌。Lacritin是一种富含于人类基础泪液和反射性泪液中的前分泌有丝分裂原， 裂解增强的C末端蛋白形式的释放，包括那些对大肠杆菌、铜绿假单胞菌具有杀菌作用的蛋白形式 (Migula和PA14)、单产链球菌、金黄色葡萄球菌和表皮葡萄球菌。脱除C末端蛋白形式的研究 人的泪水通过反复通过抗Lacritin C-(但不是N-)末端抗体柱来耗尽所有的泪水 抗菌活性，包被在合成肽AQKLKKFSLLKPWA‘N-104’中的一种活性，也称为 蛋白质形式。作为一种阳离子，主要是a-螺旋两亲性多肽，膜破裂造成的死亡 预期--这一假设在很大程度上被模型细菌膜的表面等离子共振排除了，并且 与亲本‘N-65’片段的代谢分析表明细胞死亡机制受调控。非常 从3985个非必需的完整E.ColiKeio集合中筛选出N-104耐药突变株是很有意义的 基因敲除。FeoB、POTH、ybaE、yhfZ或ybdM基因的敲除足以产生抗性，但不能 等摩尔量的氨苄西林。FeoB和Poth转座子插入突变体也是如此 条件致病菌铜绿假单胞菌PA14。YbaE和YbdM在大肠杆菌和铜绿假单胞菌中没有特征 其中的功能可能不同。铜绿假单胞菌不含YhfZ。FeoB是一种众所周知的致病因子 呼吸道铜绿假单胞菌、图拉氏杆菌和嗜肺乳杆菌、肠道空肠弯曲菌和幽门螺杆菌； 大肠埃希氏菌，但以前没有与眼部感染有关。FeoB和Poth促成或形成 各自的亚铁和腐胺和摄取通道，YbaE(建议命名为细菌细胞外 溶质结合蛋白‘)似乎是一个ABC转运蛋白亚基，与外膜有遗传相互作用 蛋白A，YbdM是一个含有核酸酶的PARB结构域。亚铁和腐胺都是 细菌细胞生长，但宿主细胞也是必需的，尤其是无血管角膜。媒体 补充10倍摩尔过量的腐胺，但不完全补充其他多胺亚精胺 废除依赖N-104的细菌死亡(泪液腐烂是N-104蛋白形式的十分之一)。 POTH基因在大肠杆菌中的互补作用与此相反。之前的代谢组学研究(那没有 检测亚铁)显示，N-104亲本‘N-65’能迅速抑制细胞内的大肠杆菌腐胺。我们的 眼下的焦点是FeoB和Poth。我们的工作假设是FeoB和/或POTH是毒力 眼表面病原体的机制与N-104是泪液杀菌活性的主要来源。我们的直接客户 目的是阐明N-104是如何通过FeoB或POTH触发杀戮的。 弗吉尼亚大学夏洛茨维尔分校 马萨诸塞州波士顿哈佛大学

Tear microbicidal activity protects the surface of the eye from environmental pathogens and may regulate levels of commensal bacteria. Lacritin, a prosecretory mitogen enriched in human basal and reflex tears, is subject to cleavage-potentiated release of C-terminal proteoforms, including those bactericidal for E. coli, P. aeruginosa (Migula and PA14), L. monoctyogenes, S. aureus and S. epidermidis. Removal of C-terminal proteoforms from human tears by repeated passage over anti-lacritin C- (but not N-) terminal antibody columns depletes all tear antimicrobial activity, an activity encapsulated in the synthetic peptide AQKLLKKFSLLKPWA 'N-104', also a proteoform. As a cationic, largely a-helical amphipathic peptide, death by membrane disruption would be expected - a hypothesis largely ruled out by surface plasmon resonance with model bacterial membranes, and metabolomic analysis with parent 'N-65' fragment that suggests a regulated cell death mechanism. Very revealing were screens for N-104 resistant mutants out of the full E. coli Keio collection of 3,985 nonessential gene knockouts. Knockout of feoB, potH, ybaE, yhfZ or ybdM was sufficient to confer resistance, but not to equimolar amounts of ampicillin. The same is true for feoB and potH transposon insertion mutants of opportunistic pathogen P. aeruginosa PA14. YbaE and YbdM are uncharacterized in E. coli and P. aeruginosa where functions may differ. YhfZ is absent from P. aeruginosa. FeoB is a well-known virulence factor of respiratory P. aeruginosa, F. tularensis and L. pneumophila, gut C. jejuni and H. pylori, and uropathogenic E. coli, but has not previously been associated with ocular infections. FeoB and PotH contribute to or form respective ferrous iron and putrescine and uptake channels, YbaE (with proposed name 'bacterial extracellular solute-binding protein') appears to be an ABC transporter subunit with a genetic interaction to outer membrane protein A, and YbdM is a ParB domain containing nuclease. Ferrous iron and putrescine are each essential for bacterial cell growth, and yet are also required by host cells, especially the avascular cornea. Media supplementation with a 10-fold molar excess of putrescine, but not fellow polyamine spermidine, completely abrogates N-104 dependent bacterial death (tear putrescine is 1/10th that of N-104 proteoform). Complementation of potH- E. coli by potH+ cDNA does the opposite. Prior metabolomic studies (that did not detect ferrous iron) revealed that N-104 parent 'N-65' rapidly suppresses intracellular E. coli putrescine. Our immediate focus is on FeoB and PotH. Our working hypothesis is that FeoB and/or PotH are virulence mechanisms for ocular surface pathogens with N-104 a main source of tear bactericidal activity. Our immediate goal is to elucidate how N-104, through FeoB or PotH triggers killing. University of Virginia Charlottesville Virginia Harvard University Boston Massachusetts