Gonococcal Infection and Gene Expression in Female Mice

描述(申请人提供)：淋病奈瑟菌(GC)引起强烈的炎症反应，其特征是细胞内存在大量多形核白细胞(PMN)。PMN在体外杀死GC，但不是通过氧气依赖防御。纯化的组织蛋白酶G(CG)和中性粒细胞中发现的两种抗菌肽(APs)LL37和杀菌/通透性增加蛋白(BPI)在体外对GC具有杀伤作用。这些因素是否挑战GC还没有在感染模型中进行测试。在目前的资助期间，我们发现GC唾液酸化转移酶(LST)通过阻断吞噬细胞的吞噬作用在逃避小鼠PMN的杀伤中起到很强的作用，并且唾液酸化增加了GC在PMN中的存活率。重要的是，在雌性小鼠的实验生殖道感染过程中，第一个突变株显著减弱。我们还证明了GC mtrC-mtrD-mtrE主动外排泵系统在抵抗大环内酯类抗生素、青霉素和宿主固有防御的抗微生物因素中起着关键作用，并且由于mtrR阻遏基因突变而过表达mtrCDE操纵子的突变体比野生型GC更适合体内。与MtrC-MtrD-mtrE外排系统促进宿主防御的逃逸一致，我们表明MtrCDE缺陷突变株对与中肠毒素相关的抗菌蛋白痉挛更敏感，该蛋白是人类LL37的小鼠同源物。我们假设GC利用两种截然不同的机制来逃避PMN的杀伤，即细菌表面的唾液酸化和AP的主动外流。在这里，我们将明确确定杀死GC的中性粒细胞因子(S)，并确定LST如何保护内化GC免受中性粒细胞杀伤(目标1)。为此，纯化的CG、LL37、JRAMP和BPI的活性将与唾液酸化和非唾液酸化的GC进行比较。将进行结合研究，以确定唾液酸化保护GC的机制。来自人类和CG或抽筋缺陷小鼠CG的PMN将被测试其对GC的杀伤活性，并将正常小鼠与CG或抽筋敲除小鼠的野生型和同基因第一个突变的感染进行比较。BPI缺陷的小鼠将用于类似的研究。在目标2中，我们将确定mtrCDE突变体在小鼠模型中减弱的基础，并在mtrCDE表达或LST活性不同的菌株中测量mtrCDE和LST在逃避AP和PMN方面的相对贡献。将测试人类中性粒细胞和来自正常和抽筋缺陷小鼠的中性粒细胞，并将进行小鼠感染实验，以确定抽筋是否在MtrCDE缺陷GC或MTRR位点突变的体内表型中起作用。还将确定mtre和mtrr基因座突变体对目标1中测试的因素的敏感性。AP和PMN调节mtrCDE操纵子和lst表达的能力将被检测。我们还将构建突变菌株，以测试mtrC-mtrD-mtre外排泵系统和LST在逃避抑制宿主因子方面的相对贡献。最后，针对mtrC转录本的硫代修饰反义寡核苷酸(PS-ODN)将被测试为增加GC对AP、抗生素和PMN杀伤的潜在治疗方法。 公共卫生相关性：淋病是美国第二大可报告感染，也是盆腔炎以及宫外孕和不孕症相关并发症的主要原因。淋病也是艾滋病毒传播的一个辅助因素。目前还没有淋病疫苗，抗生素耐药菌株的迅速出现是一个真正令人担忧的问题。这个项目将导致更好地理解淋病奈瑟菌是如何逃避宿主的先天性反应的，这可能是逃避适应性免疫反应的关键的第一步。确定这种病原体是如何利用或逃避宿主免疫系统的，可能会导致新的治疗或预防策略，从而预防或减少淋病的发病率和相关的发病率和死亡率。

DESCRIPTION (provided by applicant): Neisseria gonorrhoeae (Gc) induces an intense inflammatory response that is characterized by the presence of numerous polymorphonuclear leukocytes (PMNs) with intracellular Gc. PMNs kill Gc in vitro, but not by oxygen-dependent defenses. Purified cathepsin G (CG) and two antimicrobial peptides (APs) found within PMNs, LL37 and the bactericidal/permeability- increasing protein (BPI), kill Gc in vitro. Whether these factors challenge Gc has not been tested in an infection model. In the current funding period we showed that Gc sialyltransferase (Lst) plays a strong a role in evasion of killing by murine PMNs by blocking opsonophagocytic uptake and that sialylation increases survival of Gc within PMNs. Importantly, an lst mutant was significantly attenuated during experimental genital tract infection of female mice. We also demonstrated that the Gc MtrC-MtrD-MtrE active efflux pump system, which plays a role in resistance to macrolide antibiotics, penicillin, and antimicrobial factors of the host innate defense, is critical for murine infection, and mutants that over-express the mtrCDE operon due to mutations in the mtrR repressor locus are more fit than wild type Gc in vivo. Consistent with the MtrC-MtrD-MtrE efflux system facilitating evasion of host defenses, we showed MtrCDE-deficient mutants are more sensitive to the cathelicidin-related antimicrobial protein CRAMP, which is the murine homologue of human LL37. We hypothesize that Gc utilizes two dramatically different mechanisms for evasion of PMN killing, sialylation of the bacterial surface and active efflux of APs. Here we will definitively identify the PMN factor(s) that kill Gc and determine how Lst protects internalized Gc from PMN killing (Aim 1). To this end, the activity of purified CG, LL37, CRAMP, and BPI will be measured against sialylated and nonsialylated Gc. Binding studies will be performed to define the mechanism by which sialylation protects Gc. PMNs from humans and CG- or CRAMP-deficient mice CG will be tested for their killing activity against Gc, and infections by wild type and isogenic lst mutant will be compared in normal mice versus CG or CRAMP knock out mice. BPI-deficient mice will be made for similar studies. In Aim 2, we will identify the basis for the attenuation of the mtrCDE mutants in the murine model and measure the relative contribution of MtrCDE and Lst in evasion of APs and PMNs in strains that differ in mtrCDE expression or Lst activity. Human PMNs and PMNs from normal and CRAMP-deficient mice will be tested and mouse infection experiments will be performed to determine if CRAMP plays a role in the in vivo phenotypes of MtrCDE-deficient Gc or mtrR locus mutants. The sensitivity of mtrE and mtrR locus mutants to the factors tested in aim 1 will also be determined. The capacity of APs and PMNs to modulate the expression of mtrCDE operon and lst will be examined. We will also construct mutant strains that are designed to test the relative contribution of the MtrC-MtrD- MtrE efflux pump system and Lst in evasion of inhibitory host factors. Finally, phosphorothiorate- modified antisense oligo-deoxynucleotides (PS-ODNs) that target the mtrC transcript will be tested as a potential therapy for increasing Gc susceptibility to APs, antibiotics, and PMN killing. PUBLIC HEALTH RELEVANCE: Gonorrhea is the second leading reportable infection in the U.S. and a major cause of pelvic inflammatory disease and the associated complications of ectopic pregnancy and infertility. Gonorrhea is also a co-factor for HIV transmission. There is no gonorrhea vaccine and the rapid emergence of antibiotic resistant strains is a real concern. This project will lead to a better understanding of how N. gonorrhoeae establishes evades the host innate response, which is likely a critical first step in evading the adaptive immune response. Identifying how this pathogen capitalizes or evades the host immune system may lead to new therapeutic or prophylactic strategies, which could prevent or reduce the incidence of gonorrhea and associated morbidity and mortality.