Gonococcal Infection and Gene Expression in Female Mice

描述（由申请人提供）：淋病奈瑟菌（Gc）诱导强烈的炎症反应，其特征是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，但不是通过依赖氧的防御。纯化的组织蛋白酶G （CG）和在PMNs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。在目前的资助期内，我们发现Gc唾液转移酶（Lst）通过阻断人体自噬细胞的摄取，在逃避小鼠PMNs的杀伤中起着重要作用，唾液化增加了PMNs内Gc的存活。重要的是，在实验性雌性小鼠生殖道感染期间，最后一个突变体显着减弱。我们还证明了Gc MtrC-MtrD-MtrE主动外排泵系统，在对大环内酯类抗生素、青霉素和宿主先天防御的抗菌因子的抗性中起作用，对小鼠感染至关重要，并且由于mtrR抑制位点突变而过度表达mtrCDE操纵子的突变体在体内比野生型Gc更适合。与MtrC-MtrD-MtrE外排系统有助于逃避宿主防御一致，我们发现mtrcde缺陷突变体对抗菌蛋白CRAMP更敏感，这是人类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，细菌表面的唾液化和APs的主动外排。在这里，我们将明确确定杀死Gc的PMN因子，并确定Lst如何保护内化的Gc免受PMN的杀死（目的1）。为此，纯化的CG、LL37、CRAMP和BPI的活性将与唾液化和非唾液化的Gc进行对比。将进行结合研究，以确定唾液化保护Gc的机制。我们将检测来自人类和CG或camp缺失小鼠的PMNs对Gc的杀伤活性，并将野生型和等基因突变体感染在正常小鼠和CG或camp缺失小鼠中进行比较。bpi缺陷小鼠将用于类似的研究。在Aim 2中，我们将确定mtrCDE突变体在小鼠模型中衰减的基础，并测量mtrCDE和Lst在mtrCDE表达或Lst活性不同的菌株中逃避APs和pmn的相对贡献。我们将检测人类pmn和来自正常小鼠和CRAMP缺陷小鼠的pmn，并进行小鼠感染实验，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发挥作用。还将确定mtrE和mtrR位点突变体对目标1中测试的因素的敏感性。我们将研究ap和pmn对mtrCDE操纵子和lst表达的调节能力。我们还将构建突变株，旨在测试MtrC-MtrD- MtrE外排泵系统和Lst在逃避抑制宿主因子中的相对贡献。最后，针对mtrC转录本的磷硫酸修饰的反义寡脱氧核苷酸（PS-ODNs）将作为一种潜在的治疗方法进行测试，以增加Gc对ap、抗生素和PMN杀伤的敏感性。

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.