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），假单胞菌 铜绿假单胞菌是关键的优先病原体之一，迫切需要开发新的治疗方法。 needed.这种病原体引起致命的急性和慢性呼吸道感染，是导致人类死亡的主要原因之一。 全世界的死亡原因。它是囊性纤维化（CF）、心内膜炎， 伤口或植入人工植入物的患者。铜绿假单胞菌致病性的多样性与 生物体的突出生理适应性，这部分是由于紧密协调的调节， 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的能力的细胞外病原体， 在生物或人造表面上形成生物膜。然而，最近的范例已经转移， 被证明内化到上皮细胞的细胞质中。虽然对这些机制的研究 尽管内化是罕见的，但有证据表明3型分泌系统（T3SS）效应子的重要性。它 很明显，为了控制目前无法治疗的假单胞菌感染， 不仅要了解生物膜形成的机制，还要了解生物膜形成的调控回路， 协调病原体在宿主细胞内的内化和复制。钙（Ca2+）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。众所周知，控制 T3SS效应子的表达。我们发表的数据表明，Ca 2+调节毒力的产生 导致铜绿假单胞菌急性和慢性感染发展的因素。我们发现， 生物体能够维持细胞内Ca2+稳态，并响应于 细胞外因子，并确定了Ca2+信号和调控网络的几个关键组成部分。这里我们 假设Ca2+信号在调节铜绿假单胞菌的内化中起关键作用。为了验证这一 假设，我们将（1）确定差异表达的基因在铜绿假单胞菌前后， （2）确定Ca~（2+）信号转导的关键成分在P. 铜绿假单胞菌内化（3）监测亚细胞内Ca2+的时空变化 与上皮细胞相互作用期间铜绿假单胞菌中的浓度。拟议的研究是 旨在为本科生和研究生提供良好的培训环境。的 独立的目标利用一系列现代和完善的实验方法，在分子 微生物学和组织培养。本研究具有创新性和重要意义，因为它将是第一个研究 Ca2+信号转导与铜绿假单胞菌内化的关系。我们将首次描述 病原体基因表达在内化后的全基因组变化，并确定Ca2+的作用 在这个过程中的信号成分。鉴定铜绿假单胞菌内化的分子机制是 因此，必须制定有效的战略来控制病原体的毁灭性感染。

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.