A Pseudomonas aeruginosa EF-hand protein, EfhP (PA4107), modulates stress responses and virulence at high calcium concentration

• Published in: PloS one Vol. 9; no. 2; p. e98985
• Main Authors: Sarkisova, Svetlana A, Lotlikar, Shalaka R, Guragain, Manita, Kubat, Ryan, Cloud, John, Franklin, Michael J, Patrauchan, Marianna A
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 11.06.2014; Public Library of Science (PLoS)
• Subjects: Alginic acid; Amino Acid Sequence; Bacteria; Bacterial infections; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biofilms; Biofilms - growth & development; Biology and Life Sciences; Biosynthesis; Calcium; Calcium (intracellular); Calcium - metabolism; Calcium content; Calcium homeostasis; Calcium-binding protein; Calcium-Binding Proteins - chemistry; Calcium-Binding Proteins - genetics; Calcium-Binding Proteins - metabolism; Cell division; Computational fluid dynamics; Cystic fibrosis; EF Hand Motifs; EF-hand; EF-hand protein; Endocarditis; Gene Deletion; Gene expression; Genomes; Hand protein; Health aspects; Homeostasis; Humans; Hydrogen peroxide; Infection; Iron; Lactuca - microbiology; Lung - microbiology; Medicine and Health Sciences; Molecular Sequence Data; Mutation; Nosocomial infection; Nosocomial infections; Oxidation resistance; Oxidative Stress; Patients; Physiological aspects; Physiology; Plant Diseases - microbiology; Protein Binding; Protein Structure, Tertiary; Proteins; Proteomes; Pseudomonas aeruginosa; Pseudomonas aeruginosa - chemistry; Pseudomonas aeruginosa - genetics; Pseudomonas aeruginosa - physiology; Pseudomonas Infections - metabolism; Pseudomonas Infections - microbiology; Pyocyanin; Pyocyanine - metabolism; Strain; Strains (organisms); Virulence; Virulence (Microbiology); Virulence factors; Virulence Factors - chemistry; Virulence Factors - genetics; Virulence Factors - metabolism; United States > US; Montana; Oklahoma
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0098985

Pseudomonas aeruginosa is a facultative human pathogen, and a major cause of nosocomial infections and severe chronic infections in endocarditis and in cystic fibrosis (CF) patients. Calcium (Ca2+) accumulates in pulmonary fluids of CF patients, and plays a role in the hyperinflammatory response to bacterial infection. Earlier we showed that P. aeruginosa responds to increased Ca2+ levels, primarily through the increased production of secreted virulence factors. Here we describe the role of putative Ca2+-binding protein, with an EF-hand domain, PA4107 (EfhP), in this response. Deletion mutations of efhP were generated in P. aeruginosa strain PAO1 and CF pulmonary isolate, strain FRD1. The lack of EfhP abolished the ability of P. aeruginosa PAO1 to maintain intracellular Ca2+ homeostasis. Quantitative high-resolution 2D-PAGE showed that the efhP deletion also affected the proteomes of both strains during growth with added Ca2+. The greatest proteome effects occurred when the pulmonary isolate was cultured in biofilms. Among the proteins that were significantly less abundant or absent in the mutant strains were proteins involved in iron acquisition, biosynthesis of pyocyanin, proteases, and stress response proteins. In support, the phenotypic responses of FRD1 ΔefhP showed that the mutant strain lost its ability to produce pyocyanin, developed less biofilm, and had decreased resistance to oxidative stress (H2O2) when cultured at high [Ca2+]. Furthermore, the mutant strain was unable to produce alginate when grown at high [Ca2+] and no iron. The effect of the ΔefhP mutations on virulence was determined in a lettuce model of infection. Growth of wild-type P. aeruginosa strains at high [Ca2+] causes an increased area of disease. In contrast, the lack of efhP prevented this Ca2+-induced increase in the diseased zone. The results indicate that EfhP is important for Ca2+ homeostasis and virulence of P. aeruginosa when it encounters host environments with high [Ca2+].