Calcium-Regulated Protein CarP Responds to Multiple Host Signals and Mediates Regulation of Pseudomonas aeruginosa Virulence by Calcium

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen causing life-threatening infections. Previously, we showed that elevated calcium (Ca2+) levels increase the production of virulence factors in P. aeruginosa. In the effort to characterize the Ca2+ regulatory network, we identified a Ca2+–regulated β-propeller protein, CarP, and showed that expression of the encoding gene is controlled by the Ca2+-regulated two-component system CarSR. Here, by using a Galleria melonella model, we showed that CarP plays a role in regulating P. aeruginosa virulence. By using RNA-Seq, RT-PCR, RT-qPCR, and promoter fusions, we determined that carP is transcribed into at least two transcripts and regulated by several bacterial and host factors. The transcription of carP is elevated in response to Ca2+ in P. aeruginosa cystic fibrosis isolates and PAO1 laboratory strain. Elevated Fe2+ also induces carP. Simultaneous addition of Ca2+ and Fe2+ increased the carP promoter activity synergistically, which requires the presence of CarR. In silico analysis of the intergenic sequence upstream of carP predicted recognition sites of RhlR/LasR, OxyR, and LexA, suggesting regulation by quorum sensing (QS) and oxidative stress. In agreement, the carP promoter was activated in response to stationary-phase PAO1 supernatant and required the presence of elevated Ca2+ and CarR, but remained silent in the triple mutant lacking rhlI, lasI, and pqsA synthases. We also showed that carP transcription is regulated by oxidative stress and that CarP contributes to P. aeruginosa Ca2+-dependent H2O2 tolerance. The multifactorial regulation of carP suggests that CarP plays an important role in P. aeruginosa adaptations to host environments. IMPORTANCE P. aeruginosa is a human pathogen causing life-threatening infections. It is particularly notorious for its ability to adapt to diverse environments within the host. Understanding the signals and the signaling pathways enabling P. aeruginosa adaptation is imperative for developing effective therapies to treat infections caused by this organism. One host signal of particular importance is calcium. Previously, we identified a component of P. aeruginosa calcium-signaling network, CarP, whose expression is induced by elevated levels of calcium. Here we show that carP plays an important role in P. aeruginosa virulence and is up-regulated in P. aeruginosa strains isolated from sputa of patients with cystic fibrosis. We also identified several bacterial and host factors that regulate the transcription of carP. Such multifactorial regulation highlights the interconnectedness between regulatory circuits and together with the pleotropic effect of CarP on virulence suggests the importance of this protein in P. aeruginosa adaptations to the host.

EF-Hand Calcium Sensor, EfhP, Controls Transcriptional Regulation of Iron Uptake by Calcium inPseudomonas aeruginosa

“…Previous studies have identified Ca 2+ regulation of numerous Pa virulence factors ( 33, 34, 79 ) contributing to increased mortality in in vivo infection models ( 35, 49, 80 ). The EF hand Ca 2+ sensor, EfhP, was shown to control Pa Ca 2+ responses including the induction of pyoverdine ( 25 ) and pyocyanin production ( 49 ) and elevated virulence in lettuce leaf and G. mellonella infection models ( 49, 50 ).…”

“…To count the bacterial cell number, the samples were 10x diluted and spotted onto LB agar plates, and CFU were calculated. For quantitative RT-PCR (RT-qPCR), total RNA was extracted as described above, and cDNA was synthesized using a Transcriptor first-strand cDNA synthesis kit (Roche) and RT-qPCR was performed using SYBR green master mix (Roche, Indianapolis, IN) and a Light Cycler 480 Instrument II (Roche) as described previously ( 111 ). The sequences of PCR primers used for RT-qPCR are provided in Table 1.…”

“…To quantify efhP transcription via RT-qPCR, the strains were grown in LB medium until mid-log phase (OD 600 of 0.30 ± 0.05). Total RNA was isolated, and cDNA was synthesized as described in (35). Absence of genomic DNA (gDNA) in the RNA samples was verified by PCR amplification of 16S rRNA genes prior to cDNA synthesis by using DreamTaq green PCR master mix, alongside a gDNA positive control.…”

“…Cases of calcification of the lungs, vascular and extravascular regions have also been reported (30)(31)(32). Such elevated level of Ca 2+ may act as a signaling cue and lead to modulation of the physiology and virulence of the invading bacteria, including Pa (25,(33)(34)(35)(36)(37). Therefore, a better understanding of Ca 2+ -dependent responses in Pa will inform about the host-pathogen relationship and help in the development of antimicrobial strategies.…”

EF-hand protein, EfhP, specifically binds Ca2+ and mediates Ca2+ regulation of virulence in a human pathogen Pseudomonas aeruginosa

“…Our earlier studies showed that P. aeruginosa responds to elevated levels of extracellular Ca 2+ through increased production of biofilm, secreted virulence factors such as pyocyanin, pyoverdine, and proteases 16 , enhanced virulence in plant 17 and worm 18 models, and increased resistance to antibiotics 19 . Two major Ca 2+ regulatory pathways have been reported, including the Ca 2+ -induced two-component regulatory system CarSR 20 and the membrane Ca 2+ sensor LadS kinase which activates the Gac/Rsm system, controlling the switch to sessile mode of growth 21 .…”