Dissertation
c-di-GMP fine tunes the Pil-Chp system to control type IV pilus-mediated interspecies chemotaxis
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Summer 2024
DOI: 10.25820/etd.007732
Abstract
To survive in complex environments, it is critical for bacteria to have the ability to sense and respond to interspecies and environmental signals. We have observed that the pathogen Pseudomonas aeruginosa detects secreted peptides from bacterial competitors and navigates through interspecies signal gradients using pilus-based motility. Yet, it was unknown whether P. aeruginosa utilizes a designated chemosensory system for this behavior. Here, we performed a systematic genetic analysis of a putative pilus chemosensory system, followed by high-speed live-imaging and single-cell tracking, to reveal behaviors of mutants that retain motility but are blind to interspecies signals. The enzymes predicted to methylate (PilK) and demethylate (ChpB) the putative pilus chemoreceptor, PilJ, are necessary for cells to control the direction of migration. While these findings implicate PilJ as a bona fide chemoreceptor, such function had yet to be experimentally defined, as full-length PilJ is essential for motility. Thus, we constructed systematic genetic modifications of PilJ and found that without the predicted ligand binding domains or predicted methylation sites, cells lose the ability to detect competitor gradients, despite retaining pilus-mediated motility. Chemotaxis trajectory analysis revealed that increased probability and size of P. aeruginosa pilus-mediated steps towards S. aureus peptides, versus steps away, determines motility bias in wild type cells. However, PilJ mutants blind to interspecies signals take less frequent steps towards S. aureus or steps of equal size towards and away. In addition to confirming a chemotactic role for Pil-Chp, we also found that P. aeruginosa increases global levels of the second messenger, cyclic adenosine monophosphate (cAMP) in response to interspecies chemoeffectors, but cellular levels of cyclic diguanosine monophosphate (c-di-GMP), another second messenger, remain constant. However, we identified a subset of four of the 40 enzymes that regulate c-di-GMP levels (SiaD, MorA, BifA, and PA2870) as well as two previously understudied c-di-GMP effector proteins (PA0012 and PA2989) are each critical for response to S. aureus peptides, which suggests a local c-di-GMP signaling network modulates type IV pili-mediated chemotaxis. Further investigation revealed that the two effector proteins directly interact with Pil-Chp components to promote biased motility up a gradient. Specifically, ChpB, the response regulator, PilH, and the retraction ATPases, PilT and PilU, are each targeted by PA0012 or PA2989, in a c-di-GMP-dependent manner. Collectively, this work uncovers the chemosensory nature of PilJ and the role of a c-di-GMP network consisting of specific enzymes and effectors that can fine-tune Pil-Chp signaling to promote chemotaxis. This knowledge provides insight into how cell movements are biased during pilus-based chemotaxis and clues about how c-di-GMP effectors specifically target and alter function of proteins in Pil-Chp to increase biased movement on a surface. The chemotactic interactions necessary for bacterial survival in polymicrobial communities that are identified here further reveal potential pathways where therapeutic intervention might disrupt bacterial communication.
Details
- Title: Subtitle
- c-di-GMP fine tunes the Pil-Chp system to control type IV pilus-mediated interspecies chemotaxis
- Creators
- Kaitlin D. Yarrington
- Contributors
- Dominique Limoli (Advisor)Noah Butler (Committee Member)Anthony Fischer (Committee Member)Michael Gebhardt (Committee Member)David Weiss (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Microbiology
- Date degree season
- Summer 2024
- DOI
- 10.25820/etd.007732
- Publisher
- University of Iowa
- Number of pages
- xiv, 166 pages
- Copyright
- Copyright 2024 Kaitlin D. Yarrington
- Language
- English
- Date submitted
- 06/12/2024
- Description illustrations
- illustrations (some color)
- Description bibliographic
- Includes bibliographical references (pages 156-164) and videography (pages 165-166).
- Public Abstract (ETD)
Bacteria often live in communities composed of multiple bacterial species. To survive, they must detect what other species are in their environment so that they can cooperate or compete. We previously found that one bacterial species, Pseudomonas aeruginosa, can sense secreted peptides from another bacterial species, Staphylococcus aureus, and move towards it. However, it was unclear how P. aeruginosa could detect these signals and direct its movements towards S. aureus. P. aeruginosa and S. aureus frequently co-infect chronic wounds and the airways of people with the genetic disease, cystic fibrosis. When together, these species increase production of factors that can be harmful to human hosts, suggesting that their interactions negatively impact patients.
Here, to understand how P. aeruginosa cells can find and surround S. aureus cells, we explored the role of a specific pathway in P. aeruginosa called Pil-Chp. This pathway consists of specialized proteins, which can either sense signals or coordinate directional movement in response to these signals, a behavior known as chemotaxis. We found that the predicted Pil-Chp receptor is responsible for sensing S. aureus signals and that without the receptor, P. aeruginosa cells are unable to move towards S. aureus. Furthermore, we discovered that a small molecule called c-di-GMP indirectly promotes directional movements through enhancing interactions between different proteins within the Pil-Chp pathway. Collectively, the knowledge we gained about this pathway provides clues to how bacterial species can find each other and may be helpful in future treatment development for patients that are infected with multiple species.
- Academic Unit
- Microbiology and Immunology
- Record Identifier
- 9984698251202771
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