Broad-Host-Range Plasmids for Red Fluorescent Protein Labeling of Gram-Negative Bacteria for Use in the Zebrafish Model System

Singer, John T.; Phennicie, Ryan; Sullivan, Matthew J.; Porter, Laura A.; Shaffer, Valerie J.; Kim, Carol H.

doi:10.1128/aem.01679-09

Cited by 47 publications

(39 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A red fluorescent protein-expressing derivative of Pseudomonas aeruginosa clinical isolate strain PA14 (PA14-dTom [80]) or the PA14-derived ΔlasR (16), transformed with the same plasmid, was grown on LB media (10 g/liter Bacto tryptone, 5 g/liter Bacto yeast extract, 10 g/liter sodium chloride, 1.2% agar; BD, San Jose, CA) supplemented with 750 g/ml ampicillin (EMD Millipore, Billerica, MA) overnight at 37°C. For infections, liquid cultures of P. aeruginosa were grown by rotating overnight in LB-Amp (750 g/ml) at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Candida albicans and Pseudomonas aeruginosa Interact To Enhance Virulence of Mucosal Infection in Transparent Zebrafish

et al. 2017

View full text Add to dashboard Cite

Polymicrobial infections often include both fungi and bacteria and can complicate patient treatment and resolution of infection. Cross-kingdom interactions among bacteria, fungi, and/or the immune system during infection can enhance or block virulence mechanisms and influence disease progression. The fungus Candida albicans and the bacterium Pseudomonas aeruginosa are coisolated in the context of polymicrobial infection at a variety of sites throughout the body, including mucosal tissues such as the lung. In vitro, C. albicans and P. aeruginosa have a bidirectional and largely antagonistic relationship. Their interactions in vivo remain poorly understood, specifically regarding host responses in mediating infection. In this study, we examine trikingdom interactions using a transparent juvenile zebrafish to model mucosal lung infection and show that C. albicans and P. aeruginosa are synergistically virulent. We find that high C. albicans burden, fungal epithelial invasion, swimbladder edema, and epithelial extrusion events serve as predictive factors for mortality in our infection model. Longitudinal analyses of fungal, bacterial, and immune dynamics during coinfection suggest that enhanced morbidity is associated with exacerbated C. albicans pathogenesis and elevated inflammation. The P. aeruginosa quorum-sensingdeficient ΔlasR mutant also enhances C. albicans pathogenicity in coinfection and induces extrusion of the swimbladder. Together, these observations suggest that C. albicans-P. aeruginosa cross talk in vivo can benefit both organisms to the detriment of the host.

show abstract

Section: Methodsmentioning

confidence: 99%

Candida albicans and Pseudomonas aeruginosa Interact To Enhance Virulence of Mucosal Infection in Transparent Zebrafish

et al. 2017

View full text Add to dashboard Cite

show abstract

“…P. aeruginosa(p67T1), which constitutively expresses the fluorescent protein dTomato, was incubated overnight in LB broth containing ampicillin at a concentration of 750 g/ml (30). Cultures were spun at 2,450 ϫ g for 5 min at 4°C.…”

Section: Methodsmentioning

confidence: 99%

Neutralization of Mitochondrial Superoxide by Superoxide Dismutase 2 Promotes Bacterial Clearance and Regulates Phagocyte Numbers in Zebrafish

et al. 2015

View full text Add to dashboard Cite

T he roles that mitochondria play in antiviral signaling, via mitochondrial antiviral-signaling protein and promotion of inflammation and apoptosis, are well established (1-3); however, their importance in innate immunity is only now becoming clear. While mitochondria can promote inflammation via NF-B signaling and NLRP3 inflammasome formation, inflammation can lead to mitochondrial dysfunction, which can compound the severity of exaggerated inflammatory conditions such as sepsis (4-10). As the site of the electron transport chain within cells, mitochondria are a major source of reactive oxygen species (ROS) (mainly superoxide anions). ROS play diverse roles in cellular and organismal health, especially in innate immunity and inflammation. While the use of ROS to clear infections is beneficial to the host, inappropriate ROS production or lack of ROS neutralization can damage host DNA, proteins, and cell membranes. ROS-induced cellular damage can contribute to the undesired side effects of infectious and inflammatory diseases. Mechanisms are in place in hosts, and even some pathogens, to chemically convert ROS into less toxic compounds; however, overproduction of ROS can overwhelm host antioxidants. Thus, a better understanding of the impact of mitochondria, ROS, and mechanisms for neutralization of ROS on innate immunity could lead to improved treatments for infectious diseases and inflammatory disorders.The cellular mechanisms to neutralize ROS include the glutathione system, catalases, and the superoxide dismutase (SOD) family of enzymes. As superoxide producers, mitochondria are equipped with nuclear-encoded, mitochondrially localized SODs (SOD2, MnSOD) that convert superoxide into hydrogen peroxide. The deleterious effects of mitochondrial superoxide are demonstrated by mutations in SOD2 being implicated in idiopathic cardiomyopathy, age-related macular degeneration, aberrant brain morphology, motor neuron disease, vascular complications of diabetes, and cancer, whereas overexpression of SOD2 increases the Drosophila life span (11,(69)(70)(71)(72). Despite the importance of SOD2 and the regulation of ROS for health, surprisingly little is known about the role of SOD2 in immunity. Numerous studies have implicated SOD2 in the immune response, but few have defined functional roles for SOD2 in immunity. SOD2 is upregulated in response to lipopolysaccharide (LPS), poly(I·C), beta-glucan, and numerous pathogens in multiple cell types and organisms (12-17). Functionally, SOD2 was found to be necessary for the phorbol myristate acetate-induced respiratory burst response and cell survival upon poly(I·C) exposure in vitro (16,17). In a mouse model with SOD2 deleted specifically from thymocytes, the animals did not mount an effective adaptive immune response to influenza virus infection because of disrupted T-cell

show abstract

“…Wild-type nonmucoid P. aeruginosa strains PA14, PA14 ⌬LasR, and PA14 ⌬T3SS were generously provided by George A. O'Toole and Deborah A. Hogan (Dartmouth Medical School, Hanover, NH). Strain PA14(p67T1) was constructed recently (51) and carries a constitutive plasmid-encoded red fluorescent protein (RFP) variant of d-Tomato that was engineered by Shaner et al (49). A P. aeruginosa CF lung isolate designated SMC573 was provided by Joe Schwartzman (Dartmouth-Hitchcock Medical Center, Lebanon, NH).…”

Section: Methodsmentioning

confidence: 99%

“…4c and d). We have previously shown that RFP-expressing and wild-type strains of P. aeruginosa are equally virulent and that the production of RFP has no effect on bacterial burden (51). Increased bacterial burden is unique to P. aeruginosa.…”

Section: Assessment Of P Aeruginosa Infection By Microinjection Pmentioning

confidence: 99%

Specific Resistance to Pseudomonas aeruginosa Infection in Zebrafish Is Mediated by the Cystic Fibrosis Transmembrane Conductance Regulator

et al. 2010

Self Cite

View full text Add to dashboard Cite

Cystic fibrosis (CF) is a genetic disease caused by recessive mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and is associated with prevalent and chronic Pseudomonas aeruginosa lung infections. Despite numerous studies that have sought to elucidate the role of CFTR in the innate immune response, the links between CFTR, innate immunity, and P. aeruginosa infection remain unclear. The present work highlights the zebrafish as a powerful model organism for human infectious disease, particularly infection by P. aeruginosa. Zebrafish embryos with reduced expression of the cftr gene (Cftr morphants) exhibited reduced respiratory burst response and directed neutrophil migration, supporting a connection between cftr and the innate immune response. Cftr morphants were infected with P. aeruginosa or other bacterial species that are commonly associated with infections in CF patients, including Burkholderia cenocepacia, Haemophilus influenzae, and Staphylococcus aureus. Intriguingly, the bacterial burden of P. aeruginosa was found to be significantly higher in zebrafish Cftr morphants than in controls, but this phenomenon was not observed with the other bacterial species. Bacterial burden in Cftr morphants infected with a P. aeruginosa ⌬LasR mutant, a quorum sensing-deficient strain, was comparable to that in control fish, indicating that the regulation of virulence factors through LasR is required for enhancement of infection in the absence of Cftr. The zebrafish system provides a multitude of advantages for studying the pathogenesis of P. aeruginosa and for understanding the role that innate immune cells, such as neutrophils, play in the host response to acute bacterial infections commonly associated with cystic fibrosis.Cystic fibrosis (CF) is a multiorgan genetic disorder characterized by chronic pulmonary infections as well as gastrointestinal and reproductive abnormalities. In 1989, it was discovered that CF results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (25), which encodes a chloride ion channel present in most epithelial cells. Today, 80 to 95% of CF patients succumb to respiratory failure resulting from chronic lung infections that begin in early childhood and that are caused primarily by Pseudomonas aeruginosa (7, 32). Chronic infection with the bacterium leads to hyperinflammation characterized by the excessive recruitment of neutrophils to the airways and the subsequent release of oxidants and enzymes from activated neutrophils, which damages the lung tissue over time (32).Pseudomonas aeruginosa is a flagellated Gram-negative opportunistic pathogen of plants and animals and is found naturally in soil and water. As a nosocomial pathogen, it is the third most commonly isolated organism, leading primarily to urinary tract or wound infections in immunocompromised patients (17). Due to its production of alginate, the low permeability of its outer membrane and the presence of multidrug efflux pumps, P. aeruginosa exhibits a high degree of ...

show abstract

Broad-Host-Range Plasmids for Red Fluorescent Protein Labeling of Gram-Negative Bacteria for Use in the Zebrafish Model System

Cited by 47 publications

References 29 publications

Candida albicans and Pseudomonas aeruginosa Interact To Enhance Virulence of Mucosal Infection in Transparent Zebrafish

Candida albicans and Pseudomonas aeruginosa Interact To Enhance Virulence of Mucosal Infection in Transparent Zebrafish

Neutralization of Mitochondrial Superoxide by Superoxide Dismutase 2 Promotes Bacterial Clearance and Regulates Phagocyte Numbers in Zebrafish

Specific Resistance to Pseudomonas aeruginosa Infection in Zebrafish Is Mediated by the Cystic Fibrosis Transmembrane Conductance Regulator

Contact Info

Product

Resources

About