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The Anopheles gambiae oxidation resistance 1 (OXR1) gene regulates expression of enzymes that detoxify reactive oxygen species - PubMed

  • ️Fri Jan 01 2010

The Anopheles gambiae oxidation resistance 1 (OXR1) gene regulates expression of enzymes that detoxify reactive oxygen species

Giovanna Jaramillo-Gutierrez et al. PLoS One. 2010.

Abstract

Background: OXR1 is an ancient gene, present in all eukaryotes examined so far that confers protection from oxidative stress by an unknown mechanism. The most highly conserved region of the gene is the carboxyl-terminal TLDc domain, which has been shown to be sufficient to prevent oxidative damage.

Methodology/principal findings: OXR1 has a complex genomic structure in the mosquito A. gambiae, and we confirm that multiple splice forms are expressed in adult females. Our studies revealed that OXR1 regulates the basal levels of catalase (CAT) and glutathione peroxidase (Gpx) expression, two enzymes involved in detoxification of hydrogen peroxide, giving new insight into the mechanism of action of OXR1. Gene silencing experiments indicate that the Jun Kinase (JNK) gene acts upstream of OXR1 and also regulates expression of CAT and GPx. Both OXR1 and JNK genes are required for adult female mosquitoes to survive chronic oxidative stress. OXR1 silencing decreases P. berghei oocyst formation. Unexpectedly, JNK silencing has the opposite effect and enhances Plasmodium infection in the mosquito, suggesting that JNK may also mediate some, yet to be defined, antiparasitic response.

Conclusion: The JNK pathway regulates OXR1 expression and OXR1, in turn, regulates expression of enzymes that detoxify reactive oxygen species (ROS) in Anopheles gambiae. OXR1 silencing decreases Plasmodium infection in the mosquito, while JNK silencing has the opposite effect and enhances infection.

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Conflict of interest statement

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

Figures

Figure 1
Figure 1. Genomic organization, phylogeny, and expression of the An. gambiae OXR1 gene.

(A) Genomic organization of AgOXR1 gene and splice forms transcript B, C, and I. (B) Phylogenetic tree based on the sequence alignment of the deduced amino acid sequence of members of the OXR1 family from different species. (Hs  =  Homo sapiens, Mm =  Mus musculus, Dm  =  Drosophila melanogaster, Ae  =  Aedes aegypti, Ag  =  Anopheles gambiae, At  =  Arabidopsis thaliana and Sc  =  Saccharomyces cerevisiae). The sequence alignment used to build the dendrogram is shown in Figure S1. (C) Changes in Catalase, Glutathione peroxidase, JNK and OXR1 (TLDc domain region) mRNA levels 6 h after H2O2 injection (T) compared with water-injected control mosquitoes (C) in A. gambiae. Values were normalized using An. gambiae ribosomal protein S7 mRNA levels as an internal reference. Mean ± SEM. (D) Changes in mRNAs levels of specific OXR-1 isoforms (OXR1-C, OXR1-I and OXR1-E18) and of all OXR1 transcripts (OXR1-all, in which primers from the shared TLDc domain were used) were determined 6 h after injection of H2O2 (T  =  treated group) or water control (C  =  control group) in An. gambiae (G3 strain). Values were normalized using An. gambiae ribosomal protein S7 mRNA levels as an internal reference. Mean ± SEM. * indicates significant differences (p<0.05) by ANOVA.

Figure 2
Figure 2. Effect of OXR1 silencing on JNK and ROS detoxification enzymes mRNA levels and effect of JNK silencing on OXR1 and ROS detoxification enzymes mRNA levels.

(A) Changes in OXR1, Catalase, Glutathione peroxidase, JNK mRNA levels in dsOXR1 injected mosquitoes compared to dsLacZ control injected mosquitoes. (B) Changes in JNK, Catalase, Glutathione peroxidase, OXR1 mRNA levels in dsJNK-injected mosquitoes compared to dsLacZ control injected mosquitoes All transcript measurements were performed using qRT-PCR. Data are shown as Mean + SE. * indicates significant differences (p<0.05) by ANOVA.(C) Diagram of the proposed organization of the OXR1 signaling cascade in A. gambiae adult females.

Figure 3
Figure 3. Effect of OXR1 midgut silencing on A. gambiae G3 adult females survival when challenged by H2O2 chronic oral administration.

(A) Relative amounts of midgut OXR1 mRNA after injection with dsOXR1 compared with dsLacZ injected control mosquitoes. (B) Relative amounts of midgut JNK mRNA after injection with dsJNK compared with dsLacZ injected control mosquitoes. (C) % Survival of control dsLacZ and dsOXR1-injected mosquitoes after 1% H2O2 chronic feeding over 6 days. (D) % Survival of control dsLacZ and dsJNK-injected mosquitoes after 1% H2O2 chronic feeding over 6 days. Significance was determined by Logrank test. * * * significance(p<0.001).

Figure 4
Figure 4. AgOXR1 expression in response to P. berghei infection and effect of AgOXR1 silencing on parasite survival in Plasmodium-infected An. gambiae (G3 Strain) females.

(A) Changes in OXR1 mRNA levels in response to P. berghei infection (0, 24, 28 h hr PI) in midgut samples. (B) Changes in OXR1 mRNA levels in response to P. berghei infection (0, 24, 28 h hr PI) in carcass samples (whole body without the midgut). (C) Effect of dsOXR1 injection on OXR1 mRNA levels in G3 mosquitoes relative to a control group injected with LacZ dsRNA (dsLacZ). All transcript determinations were performed using qRT-PCR. (D) Effect of OXR1 silencing on H2O2 hemolymph levels of individual female mosquitoes (n = 10) that were either sugar-fed and kept at 27°C (left panel) or blood-fed on a healthy mouse and kept at 20°C (right panel), the temperature at which Plasmodium-infected mosquitoes are kept. Data are shown as Mean + SE. The asterisk indicates significant differences (p<0.05) by ANOVA. (E–F) Effect of dsRNA-mediated knockdown of An. gambiae OXR1 in P. berghei midgut infection after 48 h post-infection in dsOXR1 or dsLacZ injected mosquitoes. (D) Representative fields of immunofluorescence stainings of early P. berghei oocysts 48 h post-infection. Nuclei are in blue and P. berghei oocysts are in red. Scale bar 10 µm. (E) The dots represent the number of parasites present on individual midguts and the median number of oocysts is indicated by the horizontal line. The distributions are compared using the Kolmogorov-Smirnov test (KS test), n  =  number of mosquitoes, * significantly different with respect to dsLacZ injected controls (Kolgomorof-Smirnoff test, p<0.05).

Figure 5
Figure 5. Effect of AgOXR1 silencing on parasite survival in Plasmodium-infected A. gambiae (G3 Strain) females.

(A) Relative amounts of JNK mRNA in G3 mosquitoes after injection with JNK dsRNA (dsJNK) compared with controls injected with LacZ dsRNA (dsLacZ). (B) Effect of dsRNA-mediated knockdown of An. gambiae JNK in P. berghei midgut infection after 6 days post-infection in dsJNK or dsLacZ injected mosquitoes. The dots represent the number of parasites present on individual midguts and the median number of oocysts is indicated by the horizontal line. The distributions are compared using the Kolmogorov-Smirnov test (KS test), n  =  number of mosquitoes, * significantly different with respect to dsLacZ injected controls (Kolgomorof-Smirnoff test, p<0.05).

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