Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Open Access

Peer-reviewed

Research Article

The Endosymbiont Wolbachia pipientis Induces the Expression of Host Antioxidant Proteins in an Aedes albopictus Cell Line

  • Lesley J. Brennan,

    Affiliations Department of Biological Sciences, University of Alberta, Edmonton, Canada, Department of Biology and Environmental Science, Concordia University College of Alberta, Edmonton, Canada

    ⨯
  • B. Andrew Keddie,

    Affiliation Department of Biological Sciences, University of Alberta, Edmonton, Canada

    ⨯
  • Henk R. Braig,

    Affiliation School of Biological Sciences, University of Wales, Bangor, United Kingdom

    ⨯
  • Harriet L. Harris

    hharris@concordia.ab.ca

    Affiliations Department of Biological Sciences, University of Alberta, Edmonton, Canada, Department of Biology and Environmental Science, Concordia University College of Alberta, Edmonton, Canada

    ⨯

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.

Citation: Brennan LJ, Keddie BA, Braig HR, Harris HL (2008) The Endosymbiont Wolbachia pipientis Induces the Expression of Host Antioxidant Proteins in an Aedes albopictus Cell Line. PLoS ONE 3(5): e2083. https://doi.org/10.1371/journal.pone.0002083

Editor: Andy Alspaugh, Duke University, United States of America

Received: February 13, 2008; Accepted: March 14, 2008; Published: May 7, 2008

Copyright: © 2008 Brennan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work is supported by the Natural Sciences and Engineering Research Council of Canada.

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

Introduction

Wolbachia are maternally inherited obligate intracellular gram negative α-proteobacteria closely related to the Rickettsia. They were first described in the ovaries of Culex pipiens, and are common in insects and filarial nematodes. Wolbachia can induce diverse reproductive phenotypes in hosts, including feminization, male-killing, parthenogenesis, and cytoplasmic incompatibility [1]–[4] all of which contribute to the success of infected females at the expense of infected males.

How Wolbachia avoid destruction by the host innate immune response is unknown. In Drosophila, gram negative bacteria activate the IMD pathway inducing the synthesis of potent antimicrobial peptides (AMPs) such as attacin, cecropin, drosocin, and diptericin [5]. However, endosymbionts including Wolbachia [6] and Spiroplasma [7] fail to induce AMP synthesis in their insect hosts, nor do they suppress ectopic immune activation.

The generation of reactive oxygen species (ROS) is among the first lines of defense against invading microbes [8], [9]. ROS, including superoxide radicals, hydrogen peroxide, and hydroxyl radicals are formed as by-products of aerobic metabolism. In vertebrates, following phagocytosis of bacteria, superoxide is produced by an NADPH oxidase complex that assembles at the phagosomal membrane in a reaction called an oxidative burst [10]. From superoxide additional ROS are formed, all of which are active against bacteria [11]. In insects, superoxide generative reactions mimic the oxidative burst seen in vertebrates [12], [13]. In Anopheles gambiae high ROS levels generated after a blood meal confer resistance to Plasmodium infection [14] and bacterial challenge.

High concentrations of ROS create a state of oxidative stress, resulting in damage to lipids, nucleic acids, and proteins and reducing life span [15]. An unbalanced production of ROS has been implicated in human disease, including atherosclerosis, neurodegenerative and ophthalmologic diseases, and cancer [16]. Complex antioxidant defense systems have evolved to combat damaging ROS [17]. Detoxification of ROS is required for maintaining fecundity in mosquitoes [18], Drosophila [19], and mammals [20]. Herbivorous insects have developed defenses against prooxidant allelochemicals from host plants [21].

In order to elucidate mechanisms of host-microbe symbiosis, we have compared protein expression in an Aedes albopictus embryonic cell line (Aa23) naturally infected with Wolbachia and a parallel cell line cured of Wolbachia, using two dimensional polyacrylamide gel electrophoresis (2-D PAGE). Our results show that expression of host antioxidant proteins is induced in Aa23 cells infected with Wolbachia. Two bacterial antioxidant proteins were also identified. Futhermore, ROS production is significantly increased in infected cells compared to cured cells. These results illustrate, for the first time, an association between the insect antioxidant pathway and Wolbachia infection.

Results

PCR analysis of cell lines

DNA from Wolbachia-infected Aa23 cells amplified with primers for Wolbachia surface protein (wsp) generated a band at 590 bp (Figure 1A, top). Rifampicin treatment resulted in the total loss of the Wolbachia signal over the course of 7 passages. All DNA extracts produced a band at approximately 400 bp using 28S arthropod primers, confirming DNA template quality (Figure 1A, bottom). The two cell lines are morphologically indistinguishable (Figure 1B).

thumbnail
Download:
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.

https://doi.org/10.1371/journal.pone.0002083.g001

Protein induction

A consistent 2 dimensional profile (Figure 2A) was obtained from protein extracts representing 3 biological replicates (using independently cured Aa23T cell lines). Six proteins (Protein ID #1–6) shown in Figure 2B from Wolbachia-infected Aa23 cells failed to appear on the gel from Wolbachia – free Aa23 cells. These proteins are antioxidant proteins (Table 1). Proteins 1 (glutathione peroxidase; GPx); 3, 4, 5 (CuZn superoxide dismutase; CuZnSOD) and 6 (peroxiredoxin; Prx) are host proteins. In addition to these, spot 5 contains Wolbachia chaperone protein GroES, and spot 3 contains Wolbachia bacterioferritin (Bfr). A third Wolbachia protein, iron superoxide dismutase (FeSOD) was identified in spot 2.

thumbnail
Download:
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.

https://doi.org/10.1371/journal.pone.0002083.g002

thumbnail
Download:
Table 1. Identification of proteins visualized by 2D PAGE.

https://doi.org/10.1371/journal.pone.0002083.t001

Several antioxidant enzymes exist as multiple isoforms, exhibiting small variations in isolectric point and molecular mass, which are likely the result of post-translational modifications [22], [23]. CuZn SOD is known to undergo phosphorylation [24] and glycosylation [25] in addition to copper and zinc binding [26]. Similarly, phosphorylation of peroxiredoxin [27], and glutathione peroxidase [28] is common. Such modifications would explain the appearance of CuZn superoxide dismutase at three locations (spots 3,4, and 5) within close proximity of one another (Figure 2A).

The presence of the Wolbachia chaperonin GroES is not surprising. Expression of this protein in conjunction with its cochaperonin GroEL is common in endosymbiotic bacteria, and is believed to play an essential role in successfully maintaining an intracellular lifestyle by managing deleterious mutations[29].

Flow cytometric analysis

The increase in host CuZnSOD, Prx, and GPx levels in response to a Wolbachia symbiosis suggests an increase in ROS within this system. To investigate this further we labeled infected and uninfected cells with the fluorescent ROS indicator carboxy-H2DCFDA, and evaluated ROS formation by flow cytometry and microscopy. Only 1.54% of uninfected Aa23 cells exhibited ROS formation when examined by flow cytometry (Figure 3A, top). This number rose to 5.47% following induction with TBHP (Figure 3A, middle). In contrast, 9.90% of cells infected with Wolbachia fluoresced, demonstrating a substantial increase in ROS formation (Figure 3A, bottom). Microscopic analysis shows that ROS generation is associated with Wolbachia in the cytoplasm of Aa23 cells (Figure 3B).

thumbnail
Download:
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.

https://doi.org/10.1371/journal.pone.0002083.g003

Discussion

Host antioxidants

Superoxide dismutases (SOD) are conserved metalloenzymes which catalyze the dismutation of superoxide radicals into hydrogen peroxide and oxygen and are essential in combating oxidative stress [30]. Insects have 3 families of SOD enzymes – a mitochondrial MnSOD and two CuZnSODs, one of which is primarily cytoplasmic and one which is extracellular [31]. The mosquito CuZnSOD identified here (gi|94468490) is homologous to the cytoplasmic Drosophila CuZnSOD (gi|17136496) (http://www.ncbi.nlm.nih.gov/blast). Insects deficient in cytoplasmic CuZnSOD suffer from a number of detrimental effects, including a reduction in lifespan and fertility, and an increase in spontaneous DNA damage [32], while insects overexpressing CuZnSOD in combination with catalase show a significant extension in lifespan, emphasizing the physiological importance of these enzymes [33]. Cytoplasmic SOD in the scallop Chlamys farreri has been shown to be inducible upon challenge with Listeria anguillarium and Micrococcus luteus [34].

Peroxiredoxins (Prx) are ubiquitous antioxidant enzymes which reduce peroxides in a thiol dependent manner [35]. Two subgroups have been identified, the 1-Cys and 2-Cys Prxs, based on the number of conserved cysteine residues present [36]. Most 2-Cys Prxs utilize reduced thioredoxin (Trx) as an electron donor and are therefore referred to as thioredoxin peroxidases (TPx) [37]. Prx mutant yeast strains demonstrate a growth rate significantly less than wild-type strains under aerobic conditions, suggesting this group of proteins is essential for cell growth and division [38]. The mosquito Prx induced by Wolbachia (gi|55233150) in this study shares 58% sequence identity with human Prx-5 (gi|83405871) (http://www.ncbi.nlm.nih.gov/BLAST) and further investigation is required to characterize its antioxidant function.

Glutathione peroxidase (GPx) (gi|108871563) expression is also enhanced in Wolbachia infected cells. GPx catalyzes the reduction of hydrogen peroxide and organic hydroperoxides using reduced glutathione (GSH) as an electron donor [39]. The flavoenzyme glutathione reductase (GR) recycles the oxidized form of glutathione, glutathione disulfide (GSSH) back to its reduced form, maintaining high concentrations of GSH [40]. Drosophila melanogaster lacks a functional GR which seems to support early findings that insects lack GPx activity [41], [42]. However, Trx is capable of reducing GSSH, suggesting that a Trx system compensates for the absence of GR in Drosophila [42]. The Drosophila genome contains two GPx homologs [43], one of which utilizes reduced Trx as an electron donor; as a result, it has been described as a GPx homolog with TPx (thioredoxin peroxidase) activity (GTPx-1) [44]. GTPx-1 maintains a key role in the oxidative stress response [44]. The second GPx homolog of Drosophila (termed-GPx-like) has not yet been biochemically characterized [44].

Endosymbiont antioxidants

Intracellular free iron reacts with H2O2 to generate highly reactive hydroxyl radicals. Superoxide radicals can destroy enzymes containing Fe-S clusters, and release additional free iron into the intercellular environment [45]. Ferritins are a broad superfamily of iron-storage proteins common to both aerobic and anaerobic organisms and are essential components of antioxidant pathways [46]. Iron is sequestered by ferritins and strictly regulated under normal circumstances and during times of oxidative stress. Although ferritins generally lack haem groups, some bacterial forms are haem-containing, and are referred to as bacterioferritins (Bfr) [47]. Like their eukaryotic homologs, Bfr are integral factors in bacterial iron storage and in combating iron-mediated oxidative stress [48]. Bfr mutants of P. aeruginosa demonstrate an increased sensitivity to peroxides, emphasizing the importance of Bfr in the redox stress response [49]. Our data suggest that Wolbachia Bfr (gi|42521044) may protect the endosymbiont from ROS generated in its intracellular compartment.

Bacterial superoxide dismutase (Fe-SOD) contains iron at its catalytic center and plays an important role in the pathogenesis of numerous bacteria, including Shigella flexneri [50], Pseudomonas aeruginosa [51] and Bordetella pertussis [52]. In E. coli, Fe-SOD is constitutively expressed, while two other forms, a manganese-containing form (MnSOD) and a copper and zinc-containing form (CuZnSOD) are induced in response to oxygen [53], [54] . In contrast to E. coli, the genome of the Wolbachia endosymbiont of Drosophila melanogaster (wMel) has only one SOD gene, which corresponds to the FeSOD identified here (gi|42520581). This enzyme likely plays a fundamental role in the management of oxidative stress within the vacuoles harboring Wolbachia.

Antioxidant pathways and the endosymbiont

For the first time, we report a significant increase in ROS which may be a host-mediated immune response to Wolbachia or, alternatively, may be generated by the aerobic metabolism of Wolbachia themselves [55]. In either case, increased host antioxidant expression is an adaptation to symbiosis and our evidence suggests that the neutralization of potentially deadly ROS by elevated antioxidant levels is important for maintaining this unique relationship.

The E. coli genome has numerous antioxidant proteins, including three SOD's – a CuZnSOD [56], an MnSOD, and an FeSOD [57], two catalases [58], four peroxidases – GPx [59], thiol peroxidase [60], bacterioferritin comigratory protein [61], alkyl hydroperoxide reductase [62], and Bfr [63] (http://wishart.biology.ualberta.ca/BacMap/) [64]. The genome of wMel contains four homologs of these ten E. coli proteins - FeSOD, Bfr, bacterioferritin comigratory protein, and alkyl hydroperoxide reductase (http://www.ncbi.nlm.nih.gov/blast). Wolbachia, like most obligate endosymbionts, has a reduced genome [55], the result of an ongoing adaptation to an intracellular lifestyle. The induction of host antioxidant proteins by Wolbachia may be an adaptive mechanism for coping with ROS despite having lost genes coding for bacterial antioxidants.

R. rickettsii, the agent of Rocky Mountain Spotted Fever, stimulates the production of ROS in mammalian endothelial cells and inflicts host cell damage via lipid peroxidation of membranes [65]. Expression of host antioxidants is modified in a manner that is consistent with the generation of intracellular peroxides [66]–[68]. Such evidence lends support to the premise that Wolbachia manipulates host antioxidant systems in a manner that is beneficial to its survival.

Microarray analyses using Drosophila have found little induction of host antioxidant transcription in response to pathogenic bacteria (E. coli and M. luteus) or fungi (B. bassiana). While one study [69] discovered a single transcript induced in response to septic injury (a peroxidase), a second study identified none [70]. Recently, DNA microarray analysis of Drosophila S2 cells infected with Wolbachia demonstrated an increase in transcripts belonging to the Toll and Imd immune signaling pathways, and the antimicrobial peptides attacin and diptericin [71]. In this experiment the Wolbachia were introduced artificially using the shell vial technique and the infection was subsequently lost after 18 passages. In contrast, our results show that a stable symbiotic interaction with Wolbachia in mosquito cells involves ROS generation and induction of antioxidant enzymes. Although the role of ROS and antioxidants in maintaining symbiosis in vivo has not yet been determined, it will be interesting to see if these pathways differentiate between insects that can establish a long term association with symbionts and those that cannot.

Materials and Methods

Cell culture

The Aa23 cell line was provided by Steven Dobson and cultured as previously described [72]. The growth medium consisted of equal volumes of Mitsuhashi-Maramorosch (MM) and Schneider's Insect medium (Sigma) supplemented with 20% heat-inactivated fetal bovine serum (Sigma). Three uninfected cell lines were independently generated from the original cell line by adding 10 µg/ml rifampicin to the culture medium for 7 passages [73].

PCR analysis

PCR analysis using Wolbachia wsp primers [74] confirmed the presence or absence of Wolbachia. DNA was isolated using the Sigma GenElute Mammalian Genomic DNA MiniPrep kit and stored until use at −20°C. PCR conditions have been previously described [75]. The presence of Wolbachia was confirmed by the existence of a 590 bp product. Universal 28S ribosomal DNA primers D3A and D3B [76] produced a band at 400 bp, confirming DNA template quality.

Protein purification

Protein extracts were prepared as described by Adrain et al. [77]. From each cell line, 5×108 cells were packed into a 2-ml Dounce homogenizer with an equal volume of ice-cold cell extract buffer (CEB: 20 mM HEPES-KOH, pH 7.5, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 250 mM sucrose, 10 mM KCl, 100 µM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 2 µg/ml aprotinin). Cells were incubated for 20 minutes on ice then homogenized. The homogenate was centrifuged for 15 minutes at 15,000× g. The pellet was resuspended in ice-cold CEB with 6% IGEPAL CA-630 (Sigma), and again subjected to the extraction procedure previously described. The supernatant was collected, and a Bradford Assay was used to determine the protein concentration (µg/µl). Protein (∼750 µg) was precipitated in acetone overnight, and cleared of contaminants using the Bio-Rad 2-D Clean-Up Kit.

2 dimensional PAGE

Protein pellets were suspended in 322 µl of Amersham DeStreak Rehydration Solution containing 0.5% Amersham IPG buffer (pH 3–10 NL) and 10 mM DTT, then loaded onto 17 cm (pH 5–8) Bio-Rad ReadyStrip IPG strips and rehydrated passively overnight at room temperature. Isoelectric focusing was performed on an Amersham IPGphor Isoelectric focusing unit. Following equilibration for 15 minutes in each of two equilibration buffers (50 mM Tris-HCl, pH 8.8, 6M urea, 30% glycerol, 2% SDS, 0.002% bromophenol blue), one containing 1% DTT, the other containing 2.5% IAA, the strips were run on 12% homogenous SDS-PAGE gels with a 4% stacking gel at 280V for 4 hours. Gels were stained with Deep Purple (Amersham) and imaged on a Fugifilm FLA-500 scanner at 473 nm, then stained in Coomassie Blue (Sigma).

Protein identification

Protein spots were excised manually and analyzed by LC/MS/MS at the Southern Alberta Mass Spectrometry (SAMS) Centre for Proteomics at the University of Calgary. Matching protein sequences were identified by the MASCOT search engine (www.matrixscience.com). From the significant hits generated by MASCOT, those corresponding to the primary sequence databases of Ae. Aegypti and wMel were considered most probable.

ROS flow cytometric analysis

The presence of ROS within Wolbachia-infected and uninfected Aa23 cells was evaluated using the fluorogenic marker carboxy-H2DCFDA (Molecular Probes). Prior to labeling, cells were suspended in PBS containing 1.26 mM CaCl2, 0.81 mM MgSO4 and 5 mM EDTA (Buffer A). For measurement of ROS, cells were incubated in Buffer A containing 25 µM of carboxy-H2DCFDA for 30 min at 27°C, washed twice in Buffer A, and analyzed immediately. Positive controls were generated by incubating cells in 150 µM tert-butyl hydroperoxide (TBHP) in cell culture medium for 90 min followed by labeling. A total of 10 000 events were aquired using the CellQuest software on a FACScan flow cytometer (Beckton Dickinson).

Microscopic ROS assay

Infected and uninfected Aa23 cells were permitted to adhere to glass slides, labeled as described above using carboxy-H2DCFDA, and counterstained with Hoechst 333342. Slides were imaged on a Zeiss Axiomat 40 fluorescent microscope with a Canon PowerShot camera.

Acknowledgments

We thank Anthony Cornish and Troy Locke at the University of Alberta Molecular Biology Services Unit for technical assistance, and Jamie Mader and Dorothy Rutkowski for assistance with the flow cytometric analysis.

Author Contributions

Conceived and designed the experiments: HB LB HH BK. Performed the experiments: LB. Analyzed the data: LB HH. Contributed reagents/materials/analysis tools: HB HH BK. Wrote the paper: LB HH.

References

  1. 1. Harris HL, Braig HR (2003) Sperm chromatin remodelling and Wolbachia-induced cytoplasmic incompatibility in Drosophila. Biochem Cell Biol 81: 229–240.
  2. 2. Hurst GDD, Jiggins FM, Graf von der Schulenburg JH, Bertrand D, West SA, et al. (1999) Male-killing Wolbachia in two species of insect. Proc R Soc Lond B Biol Sci 266: 735–740.
  3. 3. Rousset F, Bouchon D, Pintureau B, Juchault P, Solignac M (1992) Wolbachia endosymbionts responsible for various alterations of sexuality in arthropods. Proc R Soc Lond B Biol Sci 250: 91–98.
  4. 4. Stouthamer R, Breeuwer JAJ, Luck RF, Werren JH (1993) Molecular identification of microorganisms associated with parthenogenesis. Nature 361: 66–68.
  5. 5. Lemaitre B, Hoffmann J (2007) The host defense of Drosophila melanogaster. Annu Rev Immunol 25: 697–743.
  6. 6. Bourtzis K, Pettigrew MM, O'Neill SL (2000) Wolbachia neither induces nor suppresses transcripts encoding antimicrobial peptides. Insect Mol Biol 9: 635–639.
  7. 7. Hurst GDD, Anbutsu H, Kutsukake M, Fukatsu T (2003) Hidden from the host: Spiroplasma bacteria infecting Drosophila do not cause an immune response, but are suppressed by ectopic immune activation. Insect Mol Biol 12: 93–97.
  8. 8. Ha E-M, Oh C-T, Ryu J-H, Bae Y-S, Kang S-W, et al. (2005) An antioxidant system required for host protection against gut infection in Drosophila. Dev Cell 8: 125–132.
  9. 9. Hoffmann JA (2003) The immune responses of Drosophila. Nature 426: 33–38.
  10. 10. Hampton MB, Kettle AJ, Winterbourn CC (1998) Inside the neutrophil phagosome: oxidants, myeloperoxidase, and bacterial killing. Blood 92: 3007–3017.
  11. 11. Roos D, van Bruggen R, Meischl C (2003) Oxidative killing of microbes by neutrophils. Microbes Infect 5: 1307–1315.
  12. 12. Ha E-M, Oh C-T, Bae Y-S, Lee W-J (2005) A direct role for dual oxidase in Drosophila gut immunity. Science 310: 847–850.
  13. 13. Whitten MMA, Ratcliffe NA (1999) In vitro superoxide activity in the haemolymph of the west indian leaf cockroach, Blaberus discoidalis. J Insect Physiol 45: 667–675.
  14. 14. Kumar S, Christophides GK, Cantera R, Charles B, Han YS, et al. (2003) The role of reactive oxygen species on Plasmodium melanotic encapsulation in Anopheles gambiae. Proc Natl Acad Sci U S A 100: 14139–14144.
  15. 15. Fridell Y-WC, Sanchez-Blanco A, Silvia BA, Helfand SL (2005) Targeted expression of the human uncoupling protein 2 (hUCP2) to adult neurons extends life span in the fly. Cell Metab 1: 145–152.
  16. 16. Dalle-Donne I, Rossi R, Colombo R, Giustarini D, Milzani A (2006) Biomarkers of oxidative damage in human disease. Clin Chem 52: 601–623.
  17. 17. Sies H (1993) Strategies of antioxidant defense. Eur J Biochem 215: 213–219.
  18. 18. DeJong RJ, Miller LM, Molina-Cruz A, Gupta L, Kumar S, et al. (2007) Reactive oxygen species detoxification by catalase is a major determinant of fecundity in the mosquito Anopheles gambiae. Proc Natl Acad Sci U S A 104: 2121–2126.
  19. 19. Parkes TL, Kirby K, Phillips JP, Hilliker AJ (1998) Transgenic analysis of the cSOD-null phenotypic syndrome in Drosophila. Genome 41: 642–651.
  20. 20. Ho Y-S, Magnenat J-L, Gargano M, Cao J (1998) The nature of antioxidant defense mechanisms: a lesson from transgenic studies. Environ Health Perspect 106: 1219–1228.
  21. 21. Mittapalli O, Neal JJ, Shukle RH (2007) Antioxidant defense response in a galling insect. Proc Natl Acad Sci U S A 104: 1889–1894.
  22. 22. Sarto C, Frutiger S, Cappellano F, Sanchez J-C, Doro G, et al. (1999) Modified expression of plasma glutathione peroxidase and manganese superoxide dismutase in human renal cell carcinoma. Electrophoresis 20: 3458–3466.
  23. 23. Manduzio H, Monsinjon T, Rocher B, Leboulenger F, Galap C (2003) Characterization of an inducible isoform of the Cu/Zn superoxide dismutase in the blue mussel Mytilus edulis. Aquat Toxicol 64: 73–83.
  24. 24. Csar XF, Wilson NJ, Strike P, Sparrow L, McMahon KA, et al. (2001) Copper/zinc superoxide dismutase is phosphorylated and modulated specifically by granulocyte-colony stimulating factor in myeloid cells. Proteomics 1: 435–443.
  25. 25. Arai K, Maguchi S, Fujii S, Ishibashi H, Oikawa K, et al. (1987) Glycation and inactivation of human Cu-Zn-superoxide dismutase. J Biol Chem 262: 16969–16972.
  26. 26. Furukawa Y, O'Halloran TV (2006) Posttranslational modifications in Cu,Zn-superoxide dismutase and mutations associated with amylotrophic lateral sclerosis. Antioxid Redox Signal 8: 847–867.
  27. 27. Chang T-S, Jeong W, Choi SY, Yu S, Kang S-W, et al. (2002) Regulation of peroxiredoxin I activity by Cdc2-mediated phosphorylation. J Biol Chem 277: 25370–25376.
  28. 28. Cao C, Leng Y, Kufe D (2003) Catalase activity is regulated by c-Abl and Arg in the oxidative stress response. J Biol Chem 278: 29667–29675.
  29. 29. Fares MA, Moyes A, Barrio E (2004) GroEL and the maintenance of bacterial endosymbiosis. Trends Genet 20: 413–416.
  30. 30. Fridovich I (1995) Superoxide radical and superoxide dismutases. Annu Rev Biochem 64: 97–112.
  31. 31. Parker JD, Parker KM, Keller L (2004) Molecular phylogenetic evidence for an extracellular Cu Zn superoxide dismutase gene in insects. Insect Mol Biol 13: 587–594.
  32. 32. Woodruff RC, Phillips JP, Hilliker AJ (2004) Increased spontaneous DNA damage in Cu/Zn superoxide dismutase (SOD1) deficient Drosophila. Genome 47: 1029–1035.
  33. 33. Orr WC, Sohal RS (1994) Extension of life-span by overexpression of superoxide dismutase and catalase in Drosophila melanogaster. Science 263: 1128–1130.
  34. 34. Ni D, Song L, Gao Q, Wu L, Yu Y, et al. (2007) The cDNA cloning and mRNA expression of cytoplasmic Cu, Zn superoxide dismutase (SOD) gene in scallop Chlamys farreri. Fish Shellfish Immunol 23: 1032–1042.
  35. 35. Wood ZA, Schroder E, Harris JR, Poole LB (2003) Structure, mechanism and regulation of peroxiredoxins. Trends Biochem Sci 28: 32–40.
  36. 36. McGonigle S, Dalton JP, James ER (1998) Peroxiredoxins: a new antioxidant family. Parasitol Today 14: 139–145.
  37. 37. Radyuk SN, Klichko VI, Spinola B, Sohal RS, Orr WC (2001) The peroxiredoxin gene family in Drosophila melanogaster. Free Radic Biol Med 31: 1090–1100.
  38. 38. Chae HZ, Kim I-H, Kim K, Rhee SG (1993) Cloning, sequencing, and mutation of thiol-specific antioxidant gene of Saccharomyces cerevisiae. J Biol Chem 268: 16815–16821.
  39. 39. Ursini F, Maiorino M, Brigelius-Flohe R, Aumann KD, Roveri A, et al. (1995) Diversity of glutathione peroxidases. Methods Enzymol 252: 38–48.
  40. 40. Kehrer JP, Lund LG (1994) Cellular reducing equivalents and oxidative stress. Free Radic Biol Med 17: 65–75.
  41. 41. Smith J, Shrift A (1979) Phylogenetic distribution of glutathione peroxidase. Comp Biochem Physiol B Biochem Mol Biol 63: 39–44.
  42. 42. Kanzok SM, Fechner A, Bauer H, Ulschmid JK, Muller H-M, et al. (2001) Substitution of the thioredoxin system for glutathione reductase in Drosophila melanogaster. Science 291: 643–646.
  43. 43. Adams MD, Celniker SE, Holt RA, Evans CA, Gocayne JD, et al. (2000) The genome sequence of Drosophila melanogaster. Science 287: 2185–2195.
  44. 44. Missirlis F, Rahlfs S, Dimopoulos N, Bauer H, Becker K, et al. (2003) A putative glutathione peroxidase of Drosophila encodes a thioredoxin peroxidase that provides resistance against oxidative stress but fails to complement a lack of catalase activity. Biol Chem 384: 463–472.
  45. 45. Imlay JA (2003) Pathways of oxidative damage. Annu Rev Microbiol 57: 395–418.
  46. 46. Carrondo MA (2003) Ferritins, iron uptake and storage from the bacterioferritin viewpoint. EMBO J 22: 1959–1968.
  47. 47. Andrews SC, Robinson AK, Rodriguez-Quinones F (2003) Bacterial iron homeostasis. FEMS Microbiol Rev 27: 215–237.
  48. 48. Chen C-Y, Morse SA (1999) Neisseria gonorrhoeae bacterioferritin: structural heterogeneity, involvement in iron storage and protection against oxidative stress. Microbiology 145: 2967–2975.
  49. 49. Ma J-F, Ochsner UA, Klotz MG, Nanayakkara VK, Howell ML, et al. (1999) Bacterioferritin A modulates catalase A (KatA) activity and resistance to hydrogen peroxide in Pseudomonas aeruginosa. J Bacteriol 181: 3730–3742.
  50. 50. Franzon VL, Arondel J, Sansonetti PJ (1990) Contribution of superoxide dismutase and catalase activities to Shigella flexneri pathogenesis. Infect Immun 58: 529–535.
  51. 51. Iiyama K, Chieda Y, Lee JM, Kusakabe T, Yasunaga-Aoki C, et al. (2007) Effect of superoxide dismutase gene inactivation on virulence of Pseudomonas aeruginosa PAO1 toward the silkworm, Bombyx mori. Appl Environ Microbiol 73: 1569–1575.
  52. 52. Khelef N, DeShazer D, Friedman RL, Guiso N (1996) In vivo and in vitro analysis of Bordatella pertussis catalase and Fe-superoxide dismutase mutants. FEMS Microbiol Lett 142: 231–235.
  53. 53. Benov LT, Fridovich I (1994) Escherichia coli expresses a copper- and zinc-containing superoxide dismutase. J Biol Chem 269: 25310–25314.
  54. 54. Hassan HM, Fridovich I (1977) Regulation of the synthesis of superoxide dismutase in Escherichia coli. J Biol Chem 252: 7667–7672.
  55. 55. Wu M, Sun LV, Vamathevan J, Riegler M, Deboy R, et al. (2004) Phylogenomics of the reproductive parasite Wolbachia pipientis wMel: a streamlined genome overrun by mobile genetic elements. PLoS Biology 2: 0327–0341.
  56. 56. Gort AS, Ferber DM, Imlay JA (1999) The regulation and role of the periplasmic copper, zinc superoxide dismutase of Escherichia coli. Mol Microbiol 32: 179–191.
  57. 57. Geslin C, Llanos J, Prieur D, Jeanthon C (2001) The manganese and iron superoxide dismutases protect Escherichia coli from heavy metal toxicity. Res Microbiol 152: 901–905.
  58. 58. Loewen PC, Switala J, Triggs-Raine BL (1985) Catalases HPI and HPII in Escherichia coli are induced independently. Arch Biochem Biophys 243: 144–149.
  59. 59. Cabiscol E, Tamarit J, Ros J (2000) Oxidative stress in bacteria and protein damage by reactive oxygen species. Int Microbiol 2: 3–8.
  60. 60. Cha M-K, Kim H-K, Kim H-K (1996) Mutation and mutagenesis of thiol peroxidase of Escherichia coli and a new type of thiol peroxidase family. J Bacteriol 178: 5610–5614.
  61. 61. Jeong W, Cha M-K, Kim I-H (2000) Thioredoxin-dependent hydroperoxide peroxidase activity of bacterioferritin comigratory protein (BCP) as a new member of the thiol-specific antioxidant protein (TSA)/alkyl hydroperoxide peroxidase C (ahpC) family. J Biol Chem 275: 2924–2930.
  62. 62. Seaver LC, Imlay JA (2001) Alkyl hydroperoxide reductase is the primary scavenger of endogenous hydrogen peroxide in Escherichia coli. J Bacteriol 183: 7173–7181.
  63. 63. Andrews SC, Harrison PM, Guest JR (1989) Cloning, sequencing, and mapping of the bacterioferritin gene (bfr) of Escherichia coli K-12. J Bacteriol 171: 3940–3947.
  64. 64. Stothard P, Van Domselaar G, Shrivastava S, Guo A, O'Neill B, et al. (2005) BacMap: an interactive picture atlas of annotated bacterial genomes. Nucleic Acids Res 33: D317–D320.
  65. 65. Silverman DJ, Santucci LA (1988) Potential for free radical-induced lipid peroxidation as a cause of endothelial cell injury in Rocky Mountain spotted fever. Infect Immun 56: 3110–3115.
  66. 66. Santucci LA, Gutierrez PL, Silverman DJ (1992) Rickettsia rickettsii induces superoxide radical and superoxide dismutase in human endothelial cells. Infect Immun 60: 5113–5118.
  67. 67. Devamanoharan PS, Santucci LA, Hong JE, Tian X, Silverman DJ (1994) Infection of human endothelial cells by Rickettsia rickettsii causes a significant reduction in the levels of key enzymes involved in protection against oxidative injury. Infect Immun 62: 2619–2621.
  68. 68. Eremeeva ME, Silverman DJ (1998) Rickettsia rickettsii infection of the EA.hy 926 endothelial cell line: morphological response to infection and evidence for oxidative injury. Microbiology 144: 2037–2048.
  69. 69. De Gregorio E, Spellman PT, Rubin GM, Lemaitre B (2001) Genome-wide analysis of the Drosophila immune response by using oligonucleotide microarrays. Proc Natl Acad Sci U S A 98: 12590–12595.
  70. 70. Irving P, Troxler L, Heuer TS, Belvin M, Kopczynski C, et al. (2001) A genome-wide analysis of immune responses in Drosophila. Proc Natl Acad Sci U S A 98: 15119–15124.
  71. 71. Xi Z, Gavotte L, Xie Y, Dobson SL (2008) Genome-wide analysis of the interaction between the endosymbiotic bacterium Wolbachia and its Drosophila host. BMC Genomics 9:
  72. 72. O'Neill SL, Pettigrew MM, Sinkins SP, Braig HR, Andreadis TG, et al. (1997) In vitro cultivation of Wolbachia pipientis in an Aedes albopictus cell line. Insect Mol Biol 6: 33–39.
  73. 73. Hermans PG, Hart CA, Trees AJ (2001) In vitro activity of antimicrobial agents against the endosymbiont Wolbachia pipientis. J Antimicrob Chemother 47: 659–663.
  74. 74. Braig HR, Zhou W, Dobson SL, O'Neill SL (1998) Cloning and characterization of a gene encoding the major surface protein of the bacterial endosymbiont Wolbachia pipientis. J Bacteriol 180: 2373–2378.
  75. 75. Zhou W, Rousset F, O'Neill S (1998) Phylogeny and PCR-based classification of Wolbachia strains using wsp gene sequences. Proc R Soc Lond B Biol Sci 265: 509–515.
  76. 76. Singh OP, Chandra D, Nanda N, Raghavendra K, Sunil S, et al. (2004) Differentiation of members of the Anopheles fluviatilis species complex by an allele-specific polymerase chain reaction based on 28S ribosomal DNA sequences. Am J Trop Med Hyg 70: 27–32.
  77. 77. Adrain C, Creagh EM, Cullen SP, Martin SJ (2004) Caspase-dependent inactivation of proteasome function during programmed cell death in Drosophila and man. J Biol Chem 279: 36923–36930.