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Regulation of anti-Plasmodium immunity by a LITAF-like transcription factor in the malaria vector Anopheles gambiae.

Smith RC, Eappen AG, Radtke AJ, Jacobs-Lorena M - PLoS Pathog. (2012)

Bottom Line: Electrophoretic mobility shift assays identified specific LL3 DNA-binding motifs within the promoter of SRPN6, a gene that also mediates mosquito defense against Plasmodium.Further experiments indicated that these motifs play a direct role in LL3 regulation of SRPN6 expression.We conclude that LL3 is a transcription factor capable of modulating SRPN6 expression as part of the mosquito anti-Plasmodium immune response.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA.

ABSTRACT
The mosquito is the obligate vector for malaria transmission. To complete its development within the mosquito, the malaria parasite Plasmodium must overcome the protective action of the mosquito innate immune system. Here we report on the involvement of the Anopheles gambiae orthologue of a conserved component of the vertebrate immune system, LPS-induced TNFα transcription factor (LITAF), and its role in mosquito anti-Plasmodium immunity. An. gambiae LITAF-like 3 (LL3) expression is up-regulated in response to midgut invasion by both rodent and human malaria parasites. Silencing of LL3 expression greatly increases parasite survival, indicating that LL3 is part of an anti-Plasmodium defense mechanism. Electrophoretic mobility shift assays identified specific LL3 DNA-binding motifs within the promoter of SRPN6, a gene that also mediates mosquito defense against Plasmodium. Further experiments indicated that these motifs play a direct role in LL3 regulation of SRPN6 expression. We conclude that LL3 is a transcription factor capable of modulating SRPN6 expression as part of the mosquito anti-Plasmodium immune response.

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Immunofluorescence localization of LL3 in the mosquito midgut.To determine the localization of LL3 following P. berghei infection, midgut sheets were prepared 20 h PBM and visualized using a peptide-derived LL3 antibody. (A) Expression of LL3 (red) was detected in close proximity to invading ookinetes. Images are displayed as LL3 alone (left panel) or as a merged image (right panel) with ookinetes detected by an α-aldolase antibody (green) and DAPI staining (blue) to denote nuclei. (B) dsRNA-mediated silencing of LL3 (dsLL3) correlates with a dramatic reduction in the LL3 signal in comparison with dsGFP controls. Colors of the signals are as indicated on top of each panel. (C) Localization of LL3 following pervanadate treatment. LL3 protein staining was measured in control midgut sheets (−PV) or in midguts following treatment with pervanadate (+PV). Images are displayed as LL3 alone or as a merged image with DAPI staining as indicated on the top of each panel. All images are representative of multiple biological replicates. Scale bars denote 20 microns in all images.
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ppat-1002965-g002: Immunofluorescence localization of LL3 in the mosquito midgut.To determine the localization of LL3 following P. berghei infection, midgut sheets were prepared 20 h PBM and visualized using a peptide-derived LL3 antibody. (A) Expression of LL3 (red) was detected in close proximity to invading ookinetes. Images are displayed as LL3 alone (left panel) or as a merged image (right panel) with ookinetes detected by an α-aldolase antibody (green) and DAPI staining (blue) to denote nuclei. (B) dsRNA-mediated silencing of LL3 (dsLL3) correlates with a dramatic reduction in the LL3 signal in comparison with dsGFP controls. Colors of the signals are as indicated on top of each panel. (C) Localization of LL3 following pervanadate treatment. LL3 protein staining was measured in control midgut sheets (−PV) or in midguts following treatment with pervanadate (+PV). Images are displayed as LL3 alone or as a merged image with DAPI staining as indicated on the top of each panel. All images are representative of multiple biological replicates. Scale bars denote 20 microns in all images.

Mentions: LL3 induction following P. berghei infection was evaluated by immunofluorescence assays using a peptide-derived LL3 antibody. LL3 signal above background was detected only in midgut cells in close proximity to ookinetes (Figure 2A), suggesting that LL3 expression is induced by parasite invasion. This response was further confirmed by immunofluorescence assays following the dsRNA-mediated silencing of GFP (control) or LL3 to confirm the specificity of the LL3 signal (Figure 2B). These results are consistent with the weak fluorescence obtained when mosquitoes were fed with the invasion-deficient MAOP mutant parasites suggesting that LL3 protein expression correlates directly with LL3 transcript abundance (Figure S2). Moreover, the overall response of LL3 to parasite invasion resembles that previously described for SRPN6 [9].


Regulation of anti-Plasmodium immunity by a LITAF-like transcription factor in the malaria vector Anopheles gambiae.

Smith RC, Eappen AG, Radtke AJ, Jacobs-Lorena M - PLoS Pathog. (2012)

Immunofluorescence localization of LL3 in the mosquito midgut.To determine the localization of LL3 following P. berghei infection, midgut sheets were prepared 20 h PBM and visualized using a peptide-derived LL3 antibody. (A) Expression of LL3 (red) was detected in close proximity to invading ookinetes. Images are displayed as LL3 alone (left panel) or as a merged image (right panel) with ookinetes detected by an α-aldolase antibody (green) and DAPI staining (blue) to denote nuclei. (B) dsRNA-mediated silencing of LL3 (dsLL3) correlates with a dramatic reduction in the LL3 signal in comparison with dsGFP controls. Colors of the signals are as indicated on top of each panel. (C) Localization of LL3 following pervanadate treatment. LL3 protein staining was measured in control midgut sheets (−PV) or in midguts following treatment with pervanadate (+PV). Images are displayed as LL3 alone or as a merged image with DAPI staining as indicated on the top of each panel. All images are representative of multiple biological replicates. Scale bars denote 20 microns in all images.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3475675&req=5

ppat-1002965-g002: Immunofluorescence localization of LL3 in the mosquito midgut.To determine the localization of LL3 following P. berghei infection, midgut sheets were prepared 20 h PBM and visualized using a peptide-derived LL3 antibody. (A) Expression of LL3 (red) was detected in close proximity to invading ookinetes. Images are displayed as LL3 alone (left panel) or as a merged image (right panel) with ookinetes detected by an α-aldolase antibody (green) and DAPI staining (blue) to denote nuclei. (B) dsRNA-mediated silencing of LL3 (dsLL3) correlates with a dramatic reduction in the LL3 signal in comparison with dsGFP controls. Colors of the signals are as indicated on top of each panel. (C) Localization of LL3 following pervanadate treatment. LL3 protein staining was measured in control midgut sheets (−PV) or in midguts following treatment with pervanadate (+PV). Images are displayed as LL3 alone or as a merged image with DAPI staining as indicated on the top of each panel. All images are representative of multiple biological replicates. Scale bars denote 20 microns in all images.
Mentions: LL3 induction following P. berghei infection was evaluated by immunofluorescence assays using a peptide-derived LL3 antibody. LL3 signal above background was detected only in midgut cells in close proximity to ookinetes (Figure 2A), suggesting that LL3 expression is induced by parasite invasion. This response was further confirmed by immunofluorescence assays following the dsRNA-mediated silencing of GFP (control) or LL3 to confirm the specificity of the LL3 signal (Figure 2B). These results are consistent with the weak fluorescence obtained when mosquitoes were fed with the invasion-deficient MAOP mutant parasites suggesting that LL3 protein expression correlates directly with LL3 transcript abundance (Figure S2). Moreover, the overall response of LL3 to parasite invasion resembles that previously described for SRPN6 [9].

Bottom Line: Electrophoretic mobility shift assays identified specific LL3 DNA-binding motifs within the promoter of SRPN6, a gene that also mediates mosquito defense against Plasmodium.Further experiments indicated that these motifs play a direct role in LL3 regulation of SRPN6 expression.We conclude that LL3 is a transcription factor capable of modulating SRPN6 expression as part of the mosquito anti-Plasmodium immune response.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA.

ABSTRACT
The mosquito is the obligate vector for malaria transmission. To complete its development within the mosquito, the malaria parasite Plasmodium must overcome the protective action of the mosquito innate immune system. Here we report on the involvement of the Anopheles gambiae orthologue of a conserved component of the vertebrate immune system, LPS-induced TNFα transcription factor (LITAF), and its role in mosquito anti-Plasmodium immunity. An. gambiae LITAF-like 3 (LL3) expression is up-regulated in response to midgut invasion by both rodent and human malaria parasites. Silencing of LL3 expression greatly increases parasite survival, indicating that LL3 is part of an anti-Plasmodium defense mechanism. Electrophoretic mobility shift assays identified specific LL3 DNA-binding motifs within the promoter of SRPN6, a gene that also mediates mosquito defense against Plasmodium. Further experiments indicated that these motifs play a direct role in LL3 regulation of SRPN6 expression. We conclude that LL3 is a transcription factor capable of modulating SRPN6 expression as part of the mosquito anti-Plasmodium immune response.

Show MeSH
Related in: MedlinePlus