Interaction between c-di-GMP and cAMP signaling in Pseudomonas aeruginosa biofilms

University Of Washington, Seattle WA

Investigators

Abstract

PROJECT SUMMARY/ABSTRACT Bacterial biofilms are microbial communities with aggregates of bacterial cells enclosed in an extracellular matrix. Biofilms present a significant healthcare challenge because of their prevalence in infectious diseases and resistance against treatment. Second messengers, which are critical intracellular signaling molecules that are produced in response to environmental stimuli, play a key role in biofilms. The regulation of these signaling pathways is a fundamental question in microbiology and biofilm research. In particular, the second messenger molecules cyclic diguanylate monophosphate (c-di-GMP) and cyclic adenosine monophosphate (cAMP) play critical roles in biofilm formation in many bacterial species. The opportunistic pathogen Pseudomonas aeruginosa is a model species for studying biofilms. My postdoctoral work has developed a tricolor reporter system to simultaneously study c-di-GMP and cAMP signaling at the single cell level in P. aeruginosa. Using this system, I found that c-di-GMP and cAMP signaling are activated under distinct surface conditions during the initial stages of biofilm formation, when bacteria attach and respond to a surface. This proposal aims to elucidate the molecular coordination between c-di-GMP and cAMP signaling and their roles in later stages of biofilm formation. Aim 1 will characterize how cAMP influences the enzymatic activity of c-di-GMP cyclase and phosphodiesterase, identify the enzyme targeted by cAMP, and investigate the role of an unknown protein PA3413. Aim 2 will examine how c-di-GMP impacts the three key proteins controlling cAMP signaling and employ an unbiased screen to identify additional genetic factors involved. Aim 3 will elucidate the coordination of c-di- GMP and cAMP signaling during biofilm maturation and dispersion, particularly under host-related environmental conditions. These aims will reveal the regulatory mechanisms between these two critical second messengers, deepen our understanding of phenotypic heterogeneity within biofilm communities, and are expected to facilitate future research into the broader regulatory networks of second messengers. This proposal also includes a career development plan for me to successfully transition to an independent faculty position. I will leverage the guidance of highly experienced mentor and co-mentors during the K99 mentored phase, gaining training in 1) second messenger signaling, 2) biofilm cultivation and dispersion, and 3) live cell confocal microscopy. I will also enhance my skills in mentoring, lab management, and scientific communication. In transition to the independent R00 phase, I will apply these skills and use the data generated from the proposed studies to position myself for a successful R01 grant prior to the end of this award.