Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Nov;78(11):4542-50.
doi: 10.1128/IAI.00302-10. Epub 2010 Aug 23.

Specific resistance to Pseudomonas aeruginosa infection in zebrafish is mediated by the cystic fibrosis transmembrane conductance regulator

Ryan T Phennicie  1 , Matthew J Sullivan, John T Singer, Jeffrey A Yoder, Carol H Kim
Affiliations

Specific resistance to Pseudomonas aeruginosa infection in zebrafish is mediated by the cystic fibrosis transmembrane conductance regulator

Ryan T Phennicie et al. Infect Immun. 2010 Nov.

Abstract

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 ΔLasR 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.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Zebrafish are infected by microinjected P. aeruginosa. PA14 (50 CFU) was injected into the duct of Cuvier of 48 hpf fish. (a) Kaplan-Meier survival curve showing approximately 50% mortality in PA14-infected embryos by 48 hpi in wild-type AB zebrafish. This mortality is significantly higher than in mock-injected controls by log-rank test and by the Wilcoxon test (P < 0.0001). (b) Red fluorescent bacteria (white arrows) phagocytosed by green fluorescent neutrophils in the Tg(mpx:GFP)i114 transgenic zebrafish embryo. Scale bars represent 10 um. (c) PCR amplification of 200-bp fragment of cftr from zebrafish tissues. K, kidney; S, spleen; I, intestine; V, liver; L, lymphocytes; M, myeloid cells; C, no-template control.
FIG. 2.
FIG. 2.
Dampened respiratory burst response in Cftr morphant embryos. At 48 and 56 hpf respiratory burst is significantly dampened in morphant embryos versus that in controls, indicating a decrease in the production of ROS in morphants. Results are representative of three independent experiments. Error bars represent standard errors of the means. Statistical significance was determined by two-factor ANOVA.
FIG. 3.
FIG. 3.
Neutrophil migration is impaired in Cftr morphants. (a) Neutrophil migration to the hindbrain ventricle (*) of Tg(mpx:GFP)i114 zebrafish embryos at 48 hpf, and the site of infection was monitored for 8 h postinfection. Total numbers of neutrophils within the boundary of the hindbrain ventricle (white lines) were counted at 180 mpi. (b) Significantly fewer neutrophils were observed for the Cftr morphants than for the control-infected embryos when challenged with 50 CFU of PA14(p67T1) (n = 42), but no difference in neutrophil migration was observed when morphants and controls were infected with 50 CFU of E. coli XL-10 (n = 35) or injected with 5 nl of sterile PBS (n = 30). Error bars represent standard errors of the means, and statistical significance was determined by Student's t test.
FIG. 4.
FIG. 4.
Bacterial clearance is impaired at 8 h postinfection in Cftr morphants. (a) Cftr morphant embryos infected with 50 CFU of PA14 at 48 hpf exhibit a 3.47-fold-greater bacterial burden than control-infected embryos at 8 hpi. (b) No difference in bacterial burden was observed when the Cftr knockdown was rescued. (c and d) Results were visually confirmed using fish infected with PA14(p67T1). Fluorescence of Cftr morphants (d) was observably greater than that of control fish (c). Results are representative of three independent experiments. Error bars represent standard errors of the means, and statistical significance was determined by Student's t test.
FIG. 5.
FIG. 5.
Increased bacterial burden in Cftr morphants is specific for P. aeruginosa. Infection of Cftr morphants with PA14 shows a statistically significant 3.47-fold change in bacterial burden over that of the control. P. aeruginosa clinical isolate SCM573 shows a similar 2.9-fold higher bacterial burden in morphants compared to controls. Infection of morphants with Edwardsiella tarda, Escherichia coli, Burkholderia cenocepacia, Staphylococcus aureus, and Haemophilus influenzae shows no significant change in bacterial load over that of the controls. Results are representative of three independent experiments. Error bars represent standard errors of the means. Hib, Haemophilus influenzae type b.
FIG. 6.
FIG. 6.
Quorum-sensing transcriptional regulator LasR contributes to the reduced bacterial clearance of P. aeruginosa in Cftr morphant zebrafish embryos. Morphant (48 h postfertilization) and control embryos were challenged with 50 CFU of PA14 mutants defective in either T3SS or the LasR transcriptional regulator and incubated for 8 h. Morphant embryos infected with the T3SS mutant exhibit a bacterial burden 4.5-fold-greater than that of the controls. No difference in bacterial burden was observed upon infection with PA14 defective in LasR. Results are representative of three independent experiments. Error bars represent standard errors of the means, and statistical significance was determined by Student's t test.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bates, J. M., J. Akerlund, E. Mittge, and K. Guillemin. 2007. Intestinal alkaline phosphatase detoxifies lipopolysaccharide and prevents inflammation in zebrafish in response to the gut microbiota. Cell Host Microbe 2:371-382. - PMC - PubMed
    1. Brannon, M. K., J. M. Davis, J. R. Mathias, C. J. Hall, J. C. Emerson, P. S. Crosier, A. Huttenlocher, L. Ramakrishnan, and S. M. Moskowitz. 2009. Pseudomonas aeruginosa type III secretion system interacts with phagocytes to modulate systemic infection of zebrafish embryos. Cell. Microbiol. 11:755-768. - PMC - PubMed
    1. Chen, J. M., C. Cutler, C. Jacques, G. Boeuf, E. Denamur, G. Lecointre, B. Mercier, G. Cramb, and C. Ferec. 2001. A combined analysis of the cystic fibrosis transmembrane conductance regulator: implications for structure and disease models. Mol. Biol. Evol. 18:1771-1788. - PubMed
    1. Chroneos, Z. C., S. E. Wert, J. L. Livingston, D. J. Hassett, and J. A. Whitsett. 2000. Role of cystic fibrosis transmembrane conductance regulator in pulmonary clearance of Pseudomonas aeruginosa in vivo. J. Immunol. 165:3941-3950. - PubMed
    1. Clatworthy, A. E., J. S. Lee, M. Leibman, Z. Kostun, A. J. Davidson, and D. T. Hung. 2009. Pseudomonas aeruginosa infection of zebrafish involves both host and pathogen determinants. Infect. Immun. 77:1293-1303. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources