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Updated: May 7, 2026
Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays
Published on: April 7, 2015
Calcium homeostasis in Pseudomonas aeruginosa requires multiple transporters and modulates swarming motility
Manita Guragain1, Dirk L Lenaburg, Frank S Moore
1Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK, United States.
Abstract:
Pseudomonas aeruginosa is an opportunistic human pathogen causing severe acute and chronic infections. Earlier we have shown that calcium (Ca(2+)) induces P. aeruginosa biofilm formation and production of virulence factors. To enable further studies of the regulatory role of Ca(2+), we characterized Ca(2+) homeostasis in P. aeruginosa PAO1 cells. By using Ca(2+)-binding photoprotein aequorin, we determined that the concentration of free intracellular Ca(2+) ([Ca(2+)]in) is 0.14±0.05μM. In response to external Ca(2+), the [Ca(2+)]in quickly increased at least 13-fold followed by a multi-phase decline by up to 73%. Growth at elevated Ca(2+) modulated this response. Treatment with inhibitors known to affect Ca(2+) channels, monovalent cations gradient, or P-type and F-type ATPases impaired [Ca(2+)]in response, suggesting the importance of the corresponding mechanisms in Ca(2+) homeostasis. To identify Ca(2+) transporters maintaining this homeostasis, bioinformatic and LC-MS/MS-based membrane proteomic analyses were used. [Ca(2+)]in homeostasis was monitored for seven Ca(2+)-affected and eleven bioinformatically predicted transporters by using transposon insertion mutants. Disruption of P-type ATPases PA2435, PA3920, and ion exchanger PA2092 significantly impaired Ca(2+) homeostasis. The lack of PA3920 and vanadate treatment abolished Ca(2+)-induced swarming, suggesting the role of the P-type ATPase in regulating P. aeruginosa response to Ca(2+).
Insights
Calcium (Ca2+) significantly impacts Pseudomonas aeruginosa infections. This study reveals key transporters, including P-type ATPases PA2435 and PA3920, crucial for maintaining calcium homeostasis and regulating bacterial virulence.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Pseudomonas aeruginosa is an opportunistic pathogen linked to severe infections.
- Calcium (Ca2+) is known to induce biofilm formation and virulence factor production in P. aeruginosa.
- Understanding Ca2+ regulation is vital for studying P. aeruginosa pathogenesis.
Purpose of the Study:
- To characterize Ca2+ homeostasis in P. aeruginosa PAO1 cells.
- To identify specific Ca2+ transporters involved in maintaining intracellular Ca2+ levels.
- To investigate the role of Ca2+ homeostasis in P. aeruginosa virulence and response to Ca2+.
Main Methods:
- Measurement of intracellular free Ca2+ ([Ca2+)]in) using the photoprotein aequorin.
- Analysis of Ca2+ response under varying external Ca2+ conditions and growth parameters.
- Utilizing bioinformatic and proteomic analyses to predict and identify Ca2+ transporters.
- Employing transposon insertion mutants to assess the function of candidate Ca2+ transporters.
- Investigating the effect of specific transporter disruptions and vanadate treatment on Ca2+-induced swarming.
Main Results:
- Basal intracellular free Ca2+ ([Ca2+)]in) in P. aeruginosa PAO1 was determined to be 0.14±0.05μM.
- External Ca2+ triggered a rapid, multi-phase change in [Ca2+)]in, modulated by growth conditions.
- Inhibitors targeting Ca2+ channels, gradients, and ATPases affected the [Ca2+)]in response.
- Disruption of P-type ATPases PA2435, PA3920, and ion exchanger PA2092 significantly impaired Ca2+ homeostasis.
- Loss of PA3920 abolished Ca2+-induced swarming, highlighting its role in Ca2+-mediated virulence.
Conclusions:
- P. aeruginosa possesses sophisticated mechanisms for maintaining Ca2+ homeostasis.
- Specific transporters, notably P-type ATPases PA2435 and PA3920, are critical for regulating intracellular Ca2+ levels.
- Impaired Ca2+ homeostasis affects P. aeruginosa virulence, including swarming motility.
- This study provides a foundation for understanding Ca2+ as a regulatory factor in P. aeruginosa pathogenesis.
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