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

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致病菌之一，开发新的治疗方法迫在眉睫 需要的。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 全球范围内的死因。它是囊性纤维化(CF)、心内膜炎、 伤口，或人工植入物的患者。铜绿假单胞菌致病性的多样性与 生物体出色的生理适应性，这部分归因于对 基因表达。传统上，铜绿假单胞菌被认为是一种臭名昭著的胞外病原体。 在生物表面或人造表面形成生物膜。然而，最近，随着铜绿假单胞菌的出现，这种模式发生了变化。 被证明内化到上皮细胞的细胞质中。虽然对其作用机制的研究 内化很少，但有证据表明3型分泌系统(T3SS)效应的重要性。它 很明显，为了控制目前无法治愈的假单胞菌感染，重要的是 不仅要了解生物被膜的形成机制，而且要了解生物膜的调节回路。 协调病原体在宿主细胞内的内化和复制。钙(钙)是主要的 真核细胞中的细胞内信使，调节大多数重要的细胞过程。它是众所周知的控制 T3SS效应子的表达。我们公布的数据表明，钙离子调节毒力的产生。 导致铜绿假单胞菌急性和慢性感染的因素。我们展示了 生物体能够维持细胞内钙稳态，并产生钙瞬变以响应 细胞外因子，并确定了钙信号转导和调控网络的几个关键组成部分。在这里，我们 假设钙信号在调节铜绿假单胞菌内化过程中起关键作用。为了测试这一点 假设，我们将(1)确定铜绿假单胞菌在培养前后差异表达的基因。 内化为上皮细胞；(2)确定钙信号的关键成分在P。 铜绿假单胞菌内化(3)监测钙离子亚细胞的时空变化 铜绿假单胞菌与上皮细胞相互作用过程中的浓度。拟议的研究是 旨在为本科生和研究生提供良好的培养环境。这个 独立目标利用一系列现代和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重要意义，因为它将是第一个研究 钙信号与铜绿假单胞菌内化的关系。第一次，我们将描述 病原菌内化后全基因组基因表达的变化及钙离子的作用 这一过程中的信令组件。确定铜绿假单胞菌内化的分子机制 制定有效的战略来控制病原体的毁灭性感染势在必行。

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.