Mechanisms regulating peptidoglycan fragment production

调节肽聚糖片段产生的机制

基本信息

批准号：
9896150
负责人：
Joseph P Dillard
金额：
$ 24.28万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-02-18 至 2022-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9896150
关键词：
Affect Agonist Antisense RNA Bacteria Binding Binding Sites Biochemical Genetics Cause of Death Cell Death Cell Wall Cells Cervical Cervix Uteri Cicatrix Colony-forming units Data Defect Disease Ectopic Pregnancy Endotoxins Enzymes Family Female Genes Genetic Transcription Growth Helix-Turn-Helix Motifs Heptoses Host Defense Human In Vitro Infection Infertility Inflammation Inflammatory Inflammatory Response Knock-out Lytic Male urethral structure Mammalian Oviducts Measures Messenger RNA Metabolism Methods Molecular Monitor Muramidase Mutation Neisseria gonorrhoeae Pain Pattern recognition receptor Pelvic Inflammatory Disease Peptidoglycan Phenotype Physiologic pulse Point Mutation Process Production Promoter Regions Proteins Proteomics Publishing RNA Recycling Regulation Regulon Reporter Rest Risk Symptoms Testing Tissues Transcript Transcriptional Activation Transcriptional Regulation Tube VDAC1 gene Vas deferens structure chronic pelvic pain dimer human tissue mutant pathogen pathogenic bacteria peptidoglycan monomer receptor reproductive tract response small molecule libraries survival outcome transcriptome transcriptome sequencing transcriptomics

项目摘要

In symptomatic Neisseria gonorrhoeae infections, the inflammatory response to bacterial products results in tissue damage and pain in the human host. Peptidoglycan fragments released by the bacteria during growth represent a major pro-inflammatory factor in these infections. In particular, the peptidoglycan monomers are known to cause the death and sloughing of ciliated cells in human Fallopian tubes, and one of the peptidoglycan monomers is an agonist for the intracellular pattern-recognition receptor NOD1. Peptidoglycan dimers, also released by the bacteria, are processed by host lysozyme to create peptidoglycan fragments that are agonists for another peptidoglycan sensing receptor, NOD2. Peptidoglycan monomers and dimers are created by enzymes called lytic transglycosylases that function to degrade strands of peptidoglycan in the cell wall so that additional strands can be inserted to facilitate growth. The lytic transglycosylase LtgA produces nearly half of the peptidoglycan monomers released by gonococci and a vast majority of the peptidoglycan fragments that are recycled. LtgD produces the rest of the peptidoglycan monomers released by N. gonorrhoeae. Published transcriptomic studies indicate that ltgA is subject to transcriptional regulation, and this regulation may therefore affect gonococcal cell wall metabolism and release of pro-inflammatory peptidoglycan fragments. To understand how N. gonorrhoeae might control peptidoglycan fragment metabolism or release, we previously performed a proteomics analysis of five different mutants that have distinct defects in peptidoglycan fragment recycling. This analysis identified a putative transcriptional regulator, NGO1982, as significantly increased in three of the mutants. Deletion of ngo1982 resulted in 8-9 fold increased transcript levels for ltgA, suggesting that NGO1982 is a repressor of ltgA. Since published studies had identified MtrR as an activator of ltgA and the antisense RNA NgncR_246 as an additional regulator of ltgA expression, ltgA appears to be regulated by three distinct factors. To understand the consequences of this regulation for infection, we will: 1) Determine how growth in human tissues affect ltgA and ltgD regulation, and how this regulation affects cell wall-related phenotypes in infection, and 2) Use biochemical and genetic methods to determine the molecular mechanisms used by these regulators. Preliminary results demonstrate that ltgA is strongly regulated, and that regulatory factors can greatly increase or decrease peptidoglycan fragment release. In cervical infection, LtgA and LtgD levels decreased even as colony forming units increased. Significantly reducing or greatly increasing peptidoglycan fragment release may allow gonococci to cause asymptomatic infection in some tissues and highly inflammatory infections other tissues.

在有症状的淋病奈瑟菌感染中，对细菌产物的炎症反应会导致人类宿主的组织损伤和疼痛。细菌在生长过程中释放的肽聚糖片段代表这些感染中的主要促炎因子。特别地，肽聚糖单体是已知会导致人类输卵管中纤毛细胞的死亡和脱落，并且是其中之一肽聚糖单体是细胞内模式识别受体 NOD1 的激动剂。肽聚糖二聚体也是由细菌释放的，由宿主溶菌酶处理以产生肽聚糖片段是另一种肽聚糖传感受体 NOD2 的激动剂。肽聚糖单体和二聚体是由称为裂解性转糖基酶的酶产生的，其功能是降解细胞壁中的肽聚糖链，以便插入额外的链以促进生长。裂解性转糖基酶 LtgA 产生近一半的肽聚糖单体淋球菌和绝大多数回收的肽聚糖片段。 LtgD 产生剩余的淋病奈瑟菌释放的肽聚糖单体。已发表的转录组学研究表明 ltgA 受到转录调控，因此这种调控可能影响淋球菌细胞壁促炎肽聚糖片段的代谢和释放。为了了解淋病奈瑟菌如何控制肽聚糖片段代谢或释放，我们之前对五种不同的突变体进行了蛋白质组学分析，这些突变体在肽聚糖片段回收。该分析确定了一个假定的转录调节因子 NGO1982，其中三个突变体显着增加。删除ngo1982导致转录本增加8-9倍 ltgA 水平，表明 NGO1982 是 ltgA 的抑制因子。由于已发表的研究已确定 MtrR 作为 ltgA 的激活剂，反义 RNA NgncR_246 作为 ltgA 表达的附加调节剂，ltgA 似乎受到三个不同因素的调节。为了了解这一规定对感染的影响，我们将： 1) 确定感染的增长方式人体组织影响 ltgA 和 ltgD 调节，以及这种调节如何影响细胞壁相关表型感染，以及 2) 使用生化和遗传学方法来确定感染所使用的分子机制这些监管机构。初步结果表明 ltgA 受到强烈调控，并且调控因素可以大大增加或减少肽聚糖片段的释放。在宫颈感染中，LtgA 和 LtgD 水平即使集落形成单位增加，也会减少。显着减少或大幅增加肽聚糖片段的释放可能使淋球菌在某些组织中引起无症状感染其他组织高度炎症感染。