Calcium signaling controls Pseudomonas aeruginosa invasion and adaptation to the host intracellular environment

钙信号控制铜绿假单胞菌入侵和适应宿主细胞内环境

• 批准号: 10611028
• 负责人: Marianna Patrauchan
• 金额: $ 3.85万
• 依托单位: OKLAHOMA STATE UNIVERSITY STILLWATER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10611028
• 关键词: Acute; Apoptosis; Artificial Implants; Bacteria; Behavior; Calcium; Calcium Signaling; Cause of Death; Cell Communication; Cell Differentiation process; Cell division; Cell physiology; Cells; Centers for Disease Control and Prevention (U.S.); Chemotaxis; Chronic; Clinical; Complement; Confocal Microscopy; Cytoplasm; Data; Development; Endocarditis; Ensure; Environment; Epithelial Cells; Eukaryota; Eukaryotic Cell; Event; Extracellular Space; Future; Gene Expression; Gene Expression Regulation; Genes; Homeostasis; Human; Infection; Knowledge; Learning; Life; Measures; Medical; Membrane; Microbial Biofilms; Microbiology; Modernization; Molecular; Monitor; Organism; Pathogenicity; Patients; Physiological; Physiological Processes; Play; Pneumonia; Process; Production; Prokaryotic Cells; Pseudomonas Infections; Pseudomonas aeruginosa; Publishing; Regulation; Reproduction spores; Research; Resistance; Respiratory Tract Infections; Role; Sepsis; Series; Signal Transduction; Subcellular Spaces; Surface; Surgical Wound Infection; Testing; Therapeutic; Time; Training; Transcriptional Regulation; Type III Secretion System Pathway; Urinary tract infection; Virulence; Virulence Factors; Work; World Health Organization; acute infection; base; chronic infection; cystic fibrosis patients; design; differential expression; extracellular; genome-wide; graduate student; healthcare-associated infections; human pathogen; innovation; mutant; pathogen; pathogenic bacteria; periplasm; priority pathogen; response; tissue culture; transcriptome sequencing; undergraduate student; wound

SUMMARY______________________________________________________________________________ According to the World Health Organization (WHO) and the Center of Disease Control (CDC), Pseudomonas aeruginosa is one of the critical priority pathogens, for which development of new treatments is urgently needed. This pathogen causes lethal acute and chronic respiratory infections that represent one of the leading causes of death worldwide. It is responsible for fatal infections in patients with cystic fibrosis (CF), endocarditis, wounds, or patients with artificial implants. The versatility of P. aeruginosa pathogenicity is associated with an outstanding physiological adaptability of the organism, which is due in part to a tightly coordinated regulation of gene expression. Traditionally, P. aeruginosa was considered an extracellular pathogen with notorious ability to form biofilms on living or artificial surfaces. However, recently the paradigm has shifted as P. aeruginosa was shown to internalize into the cytoplasm of epithelial cells. Although studies on the mechanisms of internalization are scarce, there is evidence of the importance of type 3 secretion system (T3SS) effectors. It became obvious that in order to gain control over currently untreatable Pseudomonas infections, it is important to not only understand the mechanisms of biofilm formation, but to also learn the regulatory circuits coordinating the pathogen’s internalization and replication within host cells. Calcium (Ca2+) is a primary intracellular messenger in eukaryotic cells, regulating most vital cellular processes. It is well known to control the expression of T3SS effectors. Our published data indicate that Ca2+ regulates production of virulence factors that contribute to the development of acute and chronic infections in P. aeruginosa. We showed that the organism is able to maintain cellular Ca2+ homeostasis, and to produce Ca2+ transients in response to extracellular factors, and identified several key components of Ca2+ signaling and regulatory network. Here, we hypothesize that Ca2+-signaling plays a key role in regulating internalization of P. aeruginosa. To test this hypothesis, we will (1) identify the genes differentially expressed in P. aeruginosa pre- and post- internalization into epithelial cells; (2) determine the role of the key components of Ca2+ signaling in P. aeruginosa internalization (3) monitor the temporal and spatial changes in Ca2+ subcellular concentrations in P. aeruginosa during interactions with epithelial cells. The proposed research is designed to provide an excellent training environment for undergraduate and graduate students. The independent aims utilize an array of modern and well-established experimental approaches in molecular microbiology and tissue culture. The research is innovative and significant because it will be first to study the relationship between Ca2+-signaling and P. aeruginosa internalization. For the first time, we will characterize the genome-wide changes in the pathogen’s gene expression upon internalization and identify the role of Ca2+ signaling components in this process. Identifying the molecular mechanisms of P. aeruginosa internalization is imperative for the development of efficient strategies to control the pathogen’s devastating infections.

概括______________________________________________________________________________ 根据世界卫生组织 (WHO) 和疾病控制中心 (CDC) 的数据，假单胞菌 铜绿假单胞菌是重要的优先病原体之一，迫切需要开发新的治疗方法 需要。这种病原体会引起致命的急性和慢性呼吸道感染，这是主要的呼吸道感染之一 全世界的死亡原因。它是导致囊性纤维化 (CF)、心内膜炎、 伤口或有人工植入物的患者。铜绿假单胞菌致病性的多功能性与 生物体具有出色的生理适应性，部分原因在于紧密协调的调节 基因表达。传统上，铜绿假单胞菌被认为是一种具有臭名昭著能力的细胞外病原体 在活体或人造表面上形成生物膜。然而，最近范式发生了变化，铜绿假单胞菌 显示内化到上皮细胞的细胞质中。尽管对其机制的研究 内化很少，有证据表明 3 型分泌系统 (T3SS) 效应器的重要性。它 很明显，为了控制目前无法治疗的假单胞菌感染，重要的是 不仅要了解生物膜形成的机制，还要了解调节回路 协调病原体在宿主细胞内的内化和复制。钙 (Ca2+) 是主要元素 真核细胞中的细胞内信使，调节最重要的细胞过程。众所周知，控制 T3SS效应子的表达。我们发表的数据表明 Ca2+ 调节毒力的产生 导致铜绿假单胞菌急性和慢性感染发生的因素。我们证明了 生物体能够维持细胞 Ca2+ 稳态，并产生 Ca2+ 瞬态响应 细胞外因子，并确定了 Ca2+ 信号传导和调节网络的几个关键组成部分。在这里，我们 假设 Ca2+ 信号传导在调节铜绿假单胞菌的内化中起关键作用。为了测试这个 假设，我们将（1）识别铜绿假单胞菌前后差异表达的基因 内化至上皮细胞； (2)确定Ca2+信号传导关键成分在P中的作用。 铜绿假单胞菌内化（3）监测Ca2+亚细胞的时空变化 与上皮细胞相互作用期间铜绿假单胞菌的浓度。拟议的研究是 旨在为本科生和研究生提供优良的培养环境。这 独立的目标利用一系列现代且成熟的分子实验方法 微生物学和组织培养。这项研究具有创新性和意义，因为它将首先研究 Ca2+ 信号传导与铜绿假单胞菌内化之间的关系。我们将首次描述 内化后病原体基因表达的全基因组变化并确定 Ca2+ 的作用 在此过程中的信号组件。确定铜绿假单胞菌内化的分子机制是 制定有效策略来控制病原体的破坏性感染势在必行。