Gonococcal Infection and Gene Expression in Female Mice

描述（由申请方提供）：淋病奈瑟菌（Gc）诱导强烈的炎症反应，其特征在于存在大量具有细胞内Gc的多形核白细胞（PMN）。PMNs在体外杀死Gc，但不是通过氧依赖性防御。纯化的组织蛋白酶G（CG）和在PMN中发现的两种抗微生物肽（AP）LL 37和杀菌/通透性增加蛋白（BPI）在体外杀死Gc。这些因素是否挑战Gc尚未在感染模型中进行测试。在当前的资助期内，我们发现Gc唾液酸转移酶（Lst）通过阻断调理吞噬细胞的摄取在逃避小鼠PMN的杀伤中起着重要作用，并且唾液酸化增加了Gc在PMN中的存活。重要的是，在雌性小鼠的实验性生殖道感染期间，lst突变体显著减弱。我们还证明了Gc MtrC-MtrD-MtrE主动外排泵系统，其在对大环内酯类抗生素、青霉素和宿主先天防御的抗微生物因子的抗性中起作用，对于小鼠感染是至关重要的，并且由于mtrR阻遏物位点中的突变而过表达mtrCDE操纵子的突变体在体内比野生型Gc更合适。与促进逃避宿主防御的MtrC-MtrD-MtrE外排系统一致，我们表明MtrCDE缺陷突变体对凯萨林菌素相关的抗菌蛋白CRAMP更敏感，CRAMP是人LL 37的鼠同源物。我们假设，GC利用两个显着不同的机制，逃避PMN的杀伤，唾液酸化的细菌表面和主动流出的AP。在这里，我们将明确识别PMN因子（S）杀死Gc，并确定Lst如何保护内化的Gc免受PMN杀伤（目的1）。为此，将测量纯化的CG、LL 37、CRAMP和BPI对唾液酸化和非唾液酸化Gc的活性。将进行结合研究以确定唾液酸化保护Gc的机制。将测试来自人和CG-或CRAMP-缺陷小鼠CG的PMN对Gc的杀伤活性，并且将在正常小鼠与CG或CRAMP敲除小鼠中比较野生型和同基因lst突变体的感染。将制备BPI缺陷小鼠用于类似研究。在目标2中，我们将确定小鼠模型中mtrCDE突变体减弱的基础，并测量mtrCDE和Lst在mtrCDE表达或Lst活性不同的菌株中逃避AP和PMN的相对贡献。将测试人PMN和来自正常和CRAMP缺陷小鼠的PMN，并将进行小鼠感染实验以确定CRAMP是否在MtrCDE缺陷Gc或mtrR基因座突变体的体内表型中起作用。还将确定mtrE和mtrR基因座突变体对目的1中测试的因子的敏感性。将检查AP和PMN调节mtrCDE操纵子和lst表达的能力。我们还将构建突变菌株，其设计用于测试MtrC-MtrD- MtrE外排泵系统和Lst在逃避抑制性宿主因子中的相对贡献。最后，将测试靶向mtrC转录物的硫代磷酸酯修饰的反义寡脱氧核苷酸（PS-ODN）作为增加Gc对AP、抗生素和PMN杀伤的敏感性的潜在疗法。

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.