Characterization of peptidoglycan from the human pathogen Chlamydia trachomatis

人类病原体沙眼衣原体肽聚糖的表征

• 批准号: 9055546
• 负责人: GEORGE WARREN LIECHTI
• 金额: $ 3.9万
• 依托单位: HENRY M. JACKSON FDN FOR THE ADV MIL/MED
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-26 至 2016-09-05
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9055546
• 关键词: Affect; Amidohydrolases; Amino Acids; Anabolism; Antibiotics; Azides; Bacteria; Binding; Biochemical; Blindness; Cell Shape; Cell Wall; Cell division; Cell physiology; Cells; Chemical Structure; Chemicals; Chemistry; Chlamydia; Chlamydia trachomatis; Chlamydiales; Cytosol; Data; Development; Dipeptides; Disaccharides; Enzymes; Escherichia coli; Exhibits; Fluorescence Microscopy; Focal Infection; Genes; Genome; Goals; Gram-Negative Bacteria; Health; Hour; Human; Hydrostatic Pressure; Immune response; Immune system; Individual; Infection; Knowledge; Label; Life Cycle Stages; Literature; Mass Spectrum Analysis; Measures; Microbe; Modeling; Molecular; Monobactams; Muramidase; Organism; Pathway interactions; Pattern recognition receptor; Peptide Hydrolases; Peptidoglycan; Physiologic pulse; Plant Resins; Play; Polymers; Predisposition; Procedures; Process; Proteins; Recycling; Replication-Associated Process; Research; Research Personnel; Role; Scaffolding Protein; Sexually Transmitted Diseases; Signal Transduction; Structural Models; Structure; Techniques; Testing; Type III Secretion System Pathway; Work; amidase; antimicrobial; base; beta-Lactams; crosslink; enzyme biosynthesis; extracellular; inhibitor/antagonist; meetings; microbial; mutant; novel; pathogen; peptidoglycan monomer; public health relevance; transpeptidation; uptake

 DESCRIPTION (provided by applicant): Chlamydia trachomatis is the leading cause of infectious blindness and bacterial sexually transmitted infection globally. For the last 20 years, the Gram-negative bacteria comprising the order Chlamydiales were thought to lack peptidoglycan (PG), a bacterial polymer critical for maintaining microbial cell shape and hydrostatic pressure and essential for all bacteria with few exceptions. Despite encoding genes for the complete PG biosynthesis pathway and exhibiting susceptibility to -lactam antibiotics, all attempts to detect PG in any chlamydial species have proven unsuccessful, giving rise to the 'chlamydial anomaly'. Research exploring chlamydial PG to date has consisted of complementation studies in E. coli mutants and biochemical analysis of chlamydial proteins expressed in E. coli. While structural models for the assembly of chlamydial PG have been proposed based on these studies, there has been no way to validate their accuracy as there has been no direct evidence that PG exists in this bacterium, until now. By utilizing chemically altered di D-amino acid probes, we have succeeded in labeling the PG of replicating, intracellular Chlamydia trachomatis, allowing us to track cell wall biosynthesis throughout its biphasic life cycle. Probe uptake and incorporation are apparent as early as 8 hours post infection and present throughout development. [Unlike all other bacteria studied to date, Chlamydia does not appear to maintain a PG sacculus (uniform covering over entire microbe) but rather a single PG-ring present at the bacterial division plane, strongly hinting at a role for chlamydial PG in replication. Despite Chlamydia lacking classical PG degradation and recycling pathways, this PG ring appears to be dynamic, with rapid turnover rates even when cell division is artificially arrested with antibiotics.] The long term goal of this project is to fully and completely characterize how Chlamydia creates and re-models its PG and elucidate the consequences of these processes on the mammalian host cell. To meet this goal, the specific aims of this application are 1) to track the biosynthesis and turnover of PG within intracellular and extracellular Chlamydia via use of a novel fluorescent dipeptide labeling strategy, 2) to purify this labeled PG and determine its chemical composition and structure via mass spectrometry, and 3) to examine the eventual degradation and release of chlamydial PG into the host cell cytosol and characterize those enzymes that play a role in this process. Thus, the central hypothesis to be tested in this proposal is that C. trachomatis synthesizes PG and that its ability to synthesize and turnover PG plays an important role in cell division, extracellular survival, and the stimulation of the intracellular innate immune response. The data generated from this research will significantly enhance our understanding of the process of replication in this organism, as well as how the host immune system reacts to the microbe's cellular processes. Additionally, the actual targets of β-lactam antibiotics on this human pathogen and their effects on PG biosynthesis will finally be elucidated, thereby generating knowledge that has the potential to enhance health and reduce illness for the millions of infected individuals worldwide.

 描述（由申请方提供）：沙眼衣原体是全球感染性失明和细菌性传播感染的主要原因。在过去的20年里，包括衣原体目的革兰氏阴性细菌被认为缺乏肽聚糖（PG），肽聚糖是一种对维持微生物细胞形状和静水压力至关重要的细菌聚合物，并且对于几乎所有细菌都是必需的。尽管编码完整PG生物合成途径的基因并表现出对β-内酰胺抗生素的敏感性，但在任何衣原体物种中检测PG的所有尝试都被证明是不成功的，导致了“衣原体异常”。迄今为止，探索衣原体PG的研究包括在大肠杆菌中的互补研究。大肠杆菌突变体和衣原体蛋白在大肠杆菌中表达的生化分析。杆菌虽然基于这些研究提出了衣原体PG组装的结构模型，但直到现在还没有方法验证其准确性，因为到目前为止还没有直接证据表明PG存在于这种细菌中。通过利用化学改变的二D-氨基酸探针，我们已经成功地标记PG的复制，细胞内沙眼衣原体，使我们能够跟踪细胞壁的生物合成在其双相生命周期。探针摄取和掺入早在感染后8小时就很明显，并在整个发育过程中存在。[与迄今为止研究的所有其他细菌不同，衣原体似乎没有保持PG球囊（均匀覆盖整个微生物），而是在细菌分裂平面上存在单个PG环，强烈暗示了其在细菌分裂中的作用。 衣原体PG的复制。尽管衣原体缺乏经典的PG降解和再循环途径，但这种PG环似乎是动态的，即使在用抗生素人工阻止细胞分裂时也具有快速的周转率。 该项目的长期目标是充分和完整地表征衣原体如何创建和重新建模其PG，并阐明这些过程对哺乳动物宿主细胞的影响。为了实现这一目标，本申请的具体目的是1）通过使用新的荧光二肽标记策略来追踪胞内和胞外衣原体内PG的生物合成和周转，2）纯化这种标记的PG并通过质谱法确定其化学组成和结构，和3）检查衣原体PG最终降解和释放到宿主细胞胞质溶胶中，并表征在该过程中起作用的那些酶。因此，在这个提议中要检验的中心假设是C。沙眼衣原体合成PG，并且其合成和周转PG的能力在细胞分裂、细胞外存活和刺激细胞内先天免疫应答中起重要作用。从这项研究中产生的数据将大大提高我们对这种生物体复制过程的理解，以及宿主免疫系统如何对微生物的细胞过程作出反应。此外，最终将阐明β-内酰胺抗生素对这种人类病原体的实际靶点及其对PG生物合成的影响，从而产生有可能增强全球数百万感染者健康和减少疾病的知识。