Calcium homeostasis in Pseudomonas aeruginosa requires multiple transporters and modulates swarming motility

Abstract

Pseudomonas aeruginosa is an opportunistic human pathogen causing severe acute and chronic infections. Earlier we have shown that calcium (Ca2+) induces P. aeruginosa biofilm formation and production of virulence factors. To enable further studies of the regulatory role of Ca2+, we characterized Ca2+ homeostasis in P. aeruginosa PAO1 cells. By using Ca2+-binding photoprotein aequorin, we determined that the concentration of free intracellular Ca2+ ([Ca2+]in) is 0.14±0.05 μM. In response to external Ca2+, the [Ca2+]in quickly increased at least 13 fold followed by a multi-phase decline by up to 73%. Growth at elevated Ca2+ modulated this response. Treatment with inhibitors known to affect Ca2+ channels, monovalent cations gradient, or P-type and F-type ATPases impaired [Ca2+]in response, suggesting the importance of the corresponding mechanisms in Ca2+ homeostasis. To identify Ca2+ transporters maintaining this homeostasis, bioinformatic and LC-MS/MS-based membrane proteomic analyses were used. [Ca2+]in homeostasis was monitored for seven Ca2+-affected and eleven bioinformatically predicted transporters by using transposon insertion mutants. Disruption of P-type ATPases PA2435, PA3920, and ion exchanger PA2092 significantly impaired Ca2+ homeostasis. The lack of PA3920 and vanadate treatment abolished Ca2+- induced swarming, suggesting the role of the P-type ATPase in regulating P. aeruginosa response to Ca2+.

The Pseudomonas aeruginosa PAO1 Two-Component Regulator CarSR Regulates Calcium Homeostasis and Calcium-Induced Virulence Factor Production through Its Regulatory Targets CarO and CarP

“…[Ca 2ϩ ] in was calculated by using the formula pCa ϭ 0.612 (Ϫlog 10 k) ϩ 3.745, where k is a rate constant for luminescence decay (per second) (54). The results were normalized against the total amount of available aequorin as described previously (6). The discharge was per-formed by permeabilizing cells with 2% Nonidet 40 (NP-40) in the presence of 12.5 mM CaCl 2 .…”

“…4F, inset) 2؉ -induced swarming motility and pyocyanin production. In previous work, we noted that Ca 2ϩ addition results in phenotypic changes to P. aeruginosa (6). In particular, growth at elevated Ca 2ϩ results in changes in swarming motility and pyocyanin production in the wild-type strain.…”

“…In a previous study, we demonstrated that P. aeruginosa maintains a submicromolar intracellular concentration of Ca 2ϩ ([Ca 2ϩ ] in ) (6). However, when the cells are exposed to high levels of extracellular Ca 2ϩ , characteristic of the environments described above, the cells undergo a transient increase of [Ca 2ϩ ] in .…”

“…Interestingly, in addition to the maintenance of Ca 2ϩ homeostasis, P. aeruginosa recognizes the external concentration of Ca 2ϩ as a physiological signal and responds through changes in the abundances of intracellular proteins and secreted virulence factors, alginate, pyocyanin, and secreted proteases (19,20). This Ca 2ϩ -triggered change in P. aeruginosa physiology leads to enhanced plant infectivity (21), biofilm formation, and swarming motility (6,19,20). Furthermore, Ca 2ϩ alters the abundance of P. aeruginosa proteins involved in iron acquisition, quinolone signaling, nitrogen metabolism, and stress responses (19,20).…”

“…In prokaryotes, Ca 2ϩ is an essential nutrient, since it is a necessary cofactor for many enzymes. However, Ca 2ϩ can be toxic to cells at high concentrations; therefore, bacteria maintain a low-submicromolar intracellular concentration of Ca 2ϩ (6). P. aeruginosa may encounter environments where external Ca 2ϩ levels are in the millimolar range, varying from 10 mM in soil (7) to 40 mM in hypersaline lakes (8).…”