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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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Related in: MedlinePlus

rLL3 binds to specific regions of the SRPN6 promoter.Experiments identified that recombinant LL3 protein interacts with two ∼40 bp regions within the putative SRPN6 promoter (Figs. S5 and S6). Gel shift assays including competition with specific and non-specific (NS) competitors are illustrated for the two regions, with the respective sequences provided above or below. The nucleotides identified by mutational analysis (Figure S6) as being critical for LL3-DNA interactions are in bold italics and labeled as R1 through R3.
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ppat-1002965-g005: rLL3 binds to specific regions of the SRPN6 promoter.Experiments identified that recombinant LL3 protein interacts with two ∼40 bp regions within the putative SRPN6 promoter (Figs. S5 and S6). Gel shift assays including competition with specific and non-specific (NS) competitors are illustrated for the two regions, with the respective sequences provided above or below. The nucleotides identified by mutational analysis (Figure S6) as being critical for LL3-DNA interactions are in bold italics and labeled as R1 through R3.

Mentions: For both methods, the most frequently recovered consensus sequence was a GGG[A/T]G motif (Figures 4B, 4C and S4), providing validation of our approach and suggesting that this is a high affinity DNA-binding site for LL3. This motif also shares a striking resemblance to the CTCCC motif (reverse complement of the LL3 motif) described for LITAF on the TNF-α promoter [3]. An additional, highly degenerate motif was also identified within the affinity-based enrichment (Figure 4B). The two motifs were chosen from those identified by MEME analysis (Figure S4) based on their presence in the SRPN6 promoter and likely role in SRPN6 regulation (Figures 5 and 6).


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)

rLL3 binds to specific regions of the SRPN6 promoter.Experiments identified that recombinant LL3 protein interacts with two ∼40 bp regions within the putative SRPN6 promoter (Figs. S5 and S6). Gel shift assays including competition with specific and non-specific (NS) competitors are illustrated for the two regions, with the respective sequences provided above or below. The nucleotides identified by mutational analysis (Figure S6) as being critical for LL3-DNA interactions are in bold italics and labeled as R1 through R3.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002965-g005: rLL3 binds to specific regions of the SRPN6 promoter.Experiments identified that recombinant LL3 protein interacts with two ∼40 bp regions within the putative SRPN6 promoter (Figs. S5 and S6). Gel shift assays including competition with specific and non-specific (NS) competitors are illustrated for the two regions, with the respective sequences provided above or below. The nucleotides identified by mutational analysis (Figure S6) as being critical for LL3-DNA interactions are in bold italics and labeled as R1 through R3.
Mentions: For both methods, the most frequently recovered consensus sequence was a GGG[A/T]G motif (Figures 4B, 4C and S4), providing validation of our approach and suggesting that this is a high affinity DNA-binding site for LL3. This motif also shares a striking resemblance to the CTCCC motif (reverse complement of the LL3 motif) described for LITAF on the TNF-α promoter [3]. An additional, highly degenerate motif was also identified within the affinity-based enrichment (Figure 4B). The two motifs were chosen from those identified by MEME analysis (Figure S4) based on their presence in the SRPN6 promoter and likely role in SRPN6 regulation (Figures 5 and 6).

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