Scholarly Work - Center for Biofilm Engineering
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Item Calcium-induced virulence factors associated with the extracellular matrix of mucoid Pseudomonas aeruginosa biofilms(2005-06) Sarkisova, S. A.; Patrauchan, Marianna A.; Berglund, Deborah L.; Nivens, David E.; Franklin, Michael J.Pseudomonas aeruginosa colonizes the pulmonary tissue of patientswith cystic fibrosis (CF), leading to biofilm-associated infections.The pulmonary fluid of CF patients usually contains elevated concentrations of cations and may contain the P. aeruginosa redox-active pigment pyocyanin, which is known to disrupt calcium homeostasis of host cells. Since divalent cations are important bridging ions for bacterial polysaccharides and since they may play regulatory roles in bacterial gene expression, we investigated the effect of calcium ions on the extracellular matrix constituents of P. aeruginosa biofilms. For mucoid strain P. aeruginosa FRD1,calcium addition (1.0 and 10 mM as CaCl2) resulted in biofilmsthat were at least 10-fold thicker than biofilms without added calcium. Scanning confocal laser microscopy showed increased spacing between cells for the thick biofilms, and Fourier transform infrared spectroscopy revealed that the material between cells is primarily alginate. An algD transcriptional reporter demonstrated that calcium addition caused an eightfold increase in alg gene expression in FRD1 biofilms. Calcium addition also resulted in increased amounts of three extracellular proteases (AprA, LasB, and PrpL). Immunoblots of the biofilm extracellular material established that AprA was harbored within the biofilm extracellular matrix. An aprA deletion mutation and a mutation in gene for a putative P. aeruginosa calmodulin-like protein did not significantly affect calcium-induced biofilm structure. Two-dimensional gel electrophoresis showed increased amounts of phenazine biosynthetic proteins in FRD1 biofilms and in calcium-amended planktonic cultures. Spectrochemical analyses showed that the calcium addition causes a three- to fivefold increase in pyocyanin production. These results demonstrate that calcium addition affects the structure and extracellular matrix composition of mucoid P. aeruginosa biofilms, through increased expression and stability of bacterial extracellular products. The calcium-induced extracellular matrix of mucoid P. aeruginosa consists primarily of the virulence factor alginate and also harbors extracellular proteases and perhaps pyocyanin, a biomolecule that may further disrupt cellular calcium levels.Item Strain-specific proteome responses of Pseudomonas aeruginosa to biofilm-associated growth and to calcium(2007-11) Patrauchan, Marianna A.; Sarkisova, S. A.; Franklin, Michael J.Liposomal amikacin (Arikace™) is a liposome-encapsulated form of amikacin that is formulated to treat chronic P. aeruginosa infections in cystic fibrosis patients. These liposomes carry a zwitterionic surface charge and are composed of lipids found naturally within the lung. A key aspect of the activity of the formulation is the ability to penetrate to the sites of Pseudomonas biofilm-like growth in the lung. Experiments were designed to investigate the penetration of liposomes into P. aeruginosa biofilms and in vitro activity. Methods and Results: Model liposomes of the same size and lipid composition as liposomal amikacin (Arikace™) were prepared with membrane-associated or encapsulated fluorescent labels, a hydrophobic carbocyanine dye and calcein, respectively. A mucoid strain of Pseudomonas aeruginosa (PA3064) was used to establish biofilms in rectangular optical grade glass flow cells. Biofilms were observed after four days of growth by confocal laser scanning microscopy using a focal plane set to view within the biofilm cluster or outside as a control. Time dependent accumulation of fluorescent liposomes within the biofilms was measured by the spatial distribution of fluorescence intensity in regions within or outside of the biofilm. Images indicated significant penetration of liposomes into the interior of biofilms under these conditions. The rate of penetration was considerably slower than typical rates for small molecules, consistent with the size of the liposomes. Liposome concentrations were higher near the periphery than the interior. However, even the interior concentration was at least as high as the concentration of liposomes in the fluid outside of the biofilm, suggesting some binding or trapping of the liposomes within the biofilm. Penetration of liposomes was observed under flow or static conditions. In a “washout” experiment, where medium is passed through the biofilms previously treated with liposomes, a significant portion of the liposomes remained associated with the biofilms for an extended period of time. The penetration of liposomes was reflected in the observation of killing of bacteria in colonies in the interior of agar beads. Exposure of these cultures to liposomal amikacin resulted in a large reduction of viable bacteria throughout the beads as monitored by a fluorescent DNA content assay. Similar colony forming unit reductions in animal models (to be shown in other poster presentations) suggest that these principles also operate in vivo. Conclusions: Liposomes similar to liposomal amikacin (Arikace™) readily penetrate into biofilms of Pseudomonas aeruginosa and may even have enhanced binding to biofilms. This binding along with localized release can explain the substantial efficacy observed in animal models.Item The Pseudomonas aeruginosa PAO1 Two-Component Regulator CarSR Regulates Calcium Homeostasis and Calcium-Induced Virulence Factor Production through Its Regulatory Targets CarO and CarP(2016-01) Guragain, Manita; Kinga, Michelle M.; Williamson, Kerry S.; Akiyama, Tatsuya; Khanam, Sharmily; Perez-Osorio, Ailyn C.; Patrauchan, Marianna A.; Franklin, Michael J.Pseudomonas aeruginosa is an opportunistic human pathogen that causes severe, life threatening infections in patients with cystic fibrosis (CF), endocarditis, wounds, or with artificial implants. During CF pulmonary infections, P. aeruginosa often encounters environments where the levels of calcium (Ca2+) are elevated. Previously, we showed that P. aeruginosa responds to externally added Ca2+ through enhanced biofilm formation, increased production of several secreted virulence factors, and by developing a transient increase in the intracellular Ca2+ followed by its removal to the basal sub-micromolar level. However, the molecular mechanisms responsible for regulating Ca2+-induced virulence factor production and Ca2+ homeostasis are not known. Here, we characterized the genome-wide transcriptional response of P. aeruginosa to elevated [Ca2+] in both planktonic cultures and in biofilms. Among the genes induced by CaCl2 in strain PAO1 was an operon containing the two-component regulator PA2656-PA2657 (here called carS and carR), while the closely related two-component regulators, phoPQ and pmrAB, were repressed by CaCl2 addition. To identify the regulatory targets of CarSR, we constructed a deletion mutant of carR, and performed transcriptome analysis of the mutant strain at low and high [Ca2+]. Among the genes regulated by CarSR in response to CaCl2 are the predicted periplasmic OB-fold protein, PA0320 (here called carO) and the inner membrane-anchored five-bladed β-propeller protein, PA0327 (here called carP). Mutations in both carO and carP affected Ca2+ homeostasis, reducing the ability of P. aeruginosa to export excess Ca2+. In addition, a mutation in carP had a pleotropic effect in a Ca2+-dependent manner, altering swarming motility, pyocyanin production, and tobramycin sensitivity. Overall, the results indicate that the two-component system CarSR is responsible for sensing high levels of external Ca2+, and responding through its regulatory targets that modulate Ca2+ homeostasis, surface-associated motility, and production of the virulence factor, pyocyanin. IMPORTANCE During infectious disease, Pseudomonas aeruginosa encounters environments with high calcium (Ca2+) concentration, yet the cells maintain intracellular Ca2+ at levels that are orders of magnitude less than the external environment. In addition, Ca2+ signals P. aeruginosa to induce production of several virulence factors. Compared to eukaryotes, little is known about how bacteria maintain Ca2+ homeostasis, or how Ca2+ acts as a signal. In this study, we identified a two-component regulatory system in P. aeruginosa PAO1, termed CarRS, that is induced at elevated Ca2+. CarRS modulates Ca2+ signaling and Ca2+ homeostasis through its regulatory targets, CarO and CarP. The results demonstrate that P. aeruginosa uses a two-component regulatory system to sense external Ca2+, and relays that information for Ca2+-dependent cellular processes.Item A Pseudomonas aeruginosa EF-Hand Protein, EfhP (PA4107), Modulates Stress Responses and Virulence at High Calcium Concentration(2014-06) Sarkisova, S. A.; Guragain, Manita; Lotikar, S. R.; Kubat, R.; Cloud, J.; Franklin, Michael J.; Patrauchan, Marianna A.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 hyperinflamatory 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+].