Drosophila are protected from Pseudomonas aeruginosa lethality by transgenic expression of paraoxonase-1
David A. Stoltz, … , Matthew R. Parsek, Joseph Zabner
David A. Stoltz, … , Matthew R. Parsek, Joseph Zabner
Published August 14, 2008
Citation Information: J Clin Invest. 2008;118(9):3123-3131. https://doi.org/10.1172/JCI35147.
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Research Article Infectious disease

Drosophila are protected from Pseudomonas aeruginosa lethality by transgenic expression of paraoxonase-1

Abstract

Pseudomonas aeruginosa uses quorum sensing, an interbacterial communication system, to regulate gene expression. The signaling molecule N-3-oxododecanoyl homoserine lactone (3OC12-HSL) is thought to play a central role in quorum sensing. Since 3OC12-HSL can be degraded by paraoxonase (PON) family members, we hypothesized that PONs regulate P. aeruginosa virulence in vivo. We chose Drosophila melanogaster as our model organism because it has been shown to be a tractable model for investigating host-pathogen interactions and lacks PONs. By using quorum-sensing–deficient P. aeruginosa, synthetic acyl-HSLs, and transgenic expression of human PON1, we investigated the role of 3OC12-HSL and PON1 on P. aeruginosa virulence. We found that P. aeruginosa virulence in flies was dependent upon 3OC12-HSL. PON1 transgenic flies expressed enzymatically active PON1 and thereby exhibited arylesterase activity and resistance to organophosphate toxicity. Moreover, PON1 flies were protected from P. aeruginosa lethality, and protection was dependent on the lactonase activity of PON1. Our findings show that PON1 can interfere with quorum sensing in vivo and provide insight into what we believe is a novel role for PON1 in the innate immune response to quorum-sensing–dependent pathogens. These results raise intriguing possibilities about human-pathogen interactions, including potential roles for PON1 as a modifier gene and for PON1 protein as a regulator of normal bacterial florae, a link between infection/inflammation and cardiovascular disease, and a potential therapeutic modality.

Authors

David A. Stoltz, Egon A. Ozer, Peter J. Taft, Marilyn Barry, Lei Liu, Peter J. Kiss, Thomas O. Moninger, Matthew R. Parsek, Joseph Zabner

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Figure 3

PON1 expression and activity in PON1 transgenic Drosophila.

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PON1 expression and activity in PON1 transgenic Drosophila. (A) West...
(A) Western blot analysis for PON1 in da-GAL4/+ (control) and UAS-PON1/da-GAL4 flies. PON1 flies were constructed using the GAL4-UAS binary system under control of the da promoter driving ubiquitous PON1 expression. Fly heads were obtained from 1- to 3-day-old flies, homogenized, and used for immunoblotting with PON1 antibody. Lysates from CHO cells infected with an adenovirus-expressing human PON1 served as the positive control. All PON1 fly lines obtained were tested for PON1 protein. β-tubulin was used as a loading control. (B) Arylesterase activity was tested in fly homogenates from da-GAL4/+ and UAS-PON1/da-GAL4 transgenic fly lines by measuring phenylacetate degradation. mOD, optical density × 10–3. (C) Correlation of arylesterase activity and PON1 protein expression in control and PON1 transgenic flies. Densitometry analysis of PON1 and β-tubulin immunoreactive bands was performed, and data are expressed in relative units. (D) Lactonase activity in control and PON1 transgenic flies. Whole-fly lysates were combined with the synthetic lactone TBBL (0.25 mM), and lactonase activity (thiol moiety release) was monitored with DTNB (0.5 mM) at an absorbance of 412 nm. Data represent the mean ± SEM with n = 3–4 per group. *P < 0.01, lactonase activity between da-GAL4/+ and UAS-PON1/da-GAL4 flies; Student’s t test.