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

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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.