The endosymbiont Wolbachia pipientis induces the expression of host antioxidant proteins in an Aedes albopictus cell line - PubMed (original) (raw)

The endosymbiont Wolbachia pipientis induces the expression of host antioxidant proteins in an Aedes albopictus cell line

Lesley J Brennan et al. PLoS One. 2008.

Abstract

Wolbachia are obligate intracellular bacteria which commonly infect arthropods. They are maternally inherited and capable of altering host development, sex determination, and reproduction. Reproductive manipulations include feminization, male-killing, parthenogenesis, and cytoplasmic incompatibility. The mechanism by which Wolbachia avoid destruction by the host immune response is unknown. Generation of antimicrobial peptides (AMPs) and reactive oxygen species (ROS) by the host are among the first lines of traditional antimicrobial defense. Previous work shows no link between a Wolbachia infection and the induction of AMPs. Here we compare the expression of protein in a cell line naturally infected with Wolbachia and an identical cell line cured of the infection through the use of antibiotics. Protein extracts of each cell line were analyzed by two dimensional gel electrophoresis and LC/MS/MS. Our results show the upregulation of host antioxidant proteins, which are active against ROS generated by aerobic cell metabolism and during an immune response. Furthermore, flow cytometric and microscopic analysis demonstrates that ROS production is significantly greater in Wolbachia-infected mosquito cells and is associated with endosymbiont-containing vacuoles located in the host cell cytoplasm. This is the first empirical data supporting an association between Wolbachia and the insect antioxidant system.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Wolbachia stably infects Aa23 cells and can be cured by antibiotic treatment.

(A) PCR analysis using Wolbachia wsp primers (top) and arthropod 28S primers (bottom) of Aa23 cells treated with 10 ug/ml rifampicin for seven passages. Lane L: molecular ladder. Lane 1: stably infected Aa23 cells. Lanes 2 through 8: cells treated with rifampicin for 1, 2, 3, 4, 5, 6, and 7 passages. Lane 9: negative control. (B) Aa23 cells stably infected with Wolbachia (I) and Aa23 cells cured of Wolbachia using rifampicin (II). Bar, 100 µm.

Figure 2

Figure 2. 2-D Page of _Wolbachia_-infected and uninfected Aa23 cells.

(A) Identification of proteins unique to _Wolbachia-_infected Aa23 cells. Approximately 750 ug of protein extract from an Aa23 cell line stably infected with Wolbachia (I) and a parallel cell line cured of a Wolbachia infection (II) were analyzed. Proteins expressed only in the presence of a Wolbachia infection (ID #1–6) are identified. (B) Gel sections showing proteins selected for LC/MS/MS analysis.

Figure 3

Figure 3. Analysis of ROS formation in Wolbachia -infected and uninfected Aa23 cells.

(A) Flow cytometric analysis of Wolbachia –infected and uninfected Aa23 cells using the fluorescent ROS marker carboxy-H2DCFDA. Histograms representative of three replicates are shown. The negative control (shaded) consists of unlabeled cells. Test samples (black lines) include: uninfected Aa23 cells (top panel), uninfected Aa23 cells induced to produce ROS using TBHP (middle panel), and infected Aa23 cells (bottom panel). Carboxy-H2DCFDA positive cells are represented on each histogram. (B) Microscopic analysis of _Wolbachia_-infected (I) and uninfected (II) Aa23 cells. Hoechst stain was used to label DNA (left panel). Carboxy-H2DCFDA was used to label ROS (right panel). Bar, 10 µm.

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