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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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Luciferase expression in a mosquito cell line suggests the involvement of LL3 in the regulation of SRPN6 expression.Expression of LL3 and SRPN6 mRNAs in response to heat-killed E. cloacae was investigated in hemocyte-like Sua5B cells (A). LL3 and SRPN6 mRNA abundance was determined by qRT-PCR in cells that were non-induced (non) or after exposure to E. cloacae for 6 h (I). Transcript abundance was normalized to that of rpS7 in two independent biological samples and analyzed by the Student's t-test for significance. Asterisks denote significant changes upon bacterial induction (P<0.05). The constructs outlined in (B) were used to assess the ability of LL3 to modulate luciferase expression from a SRPN6 promoter. Red diamonds indicate the locations of LL3-binding sites in the wild type promoter (wt) and their presence/absence in each of the mutated promoter constructs. Individual mutants (R1, R2, or R3) correspond to those sites shown in Figure 5, and were combined to create double (R1,R2) or triple mutants (ALL). (C) Each construct was transfected into Sua5B cells and luciferase expression was measured under basal conditions (non-induced) or upon induction with heat-killed E. cloacae (induced). Expression was normalized to that of the non-induced wild type SRPN6 promoter in triplicate experiments. The values of two biological repeat experiments were pooled. Asterisks denote significant changes when compared to the wild type construct for each treatment (* = P<0.05, ** = P<0.01, or *** P<0.001) as determined by a Two-way ANOVA and Bonferroni post-test.
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ppat-1002965-g006: Luciferase expression in a mosquito cell line suggests the involvement of LL3 in the regulation of SRPN6 expression.Expression of LL3 and SRPN6 mRNAs in response to heat-killed E. cloacae was investigated in hemocyte-like Sua5B cells (A). LL3 and SRPN6 mRNA abundance was determined by qRT-PCR in cells that were non-induced (non) or after exposure to E. cloacae for 6 h (I). Transcript abundance was normalized to that of rpS7 in two independent biological samples and analyzed by the Student's t-test for significance. Asterisks denote significant changes upon bacterial induction (P<0.05). The constructs outlined in (B) were used to assess the ability of LL3 to modulate luciferase expression from a SRPN6 promoter. Red diamonds indicate the locations of LL3-binding sites in the wild type promoter (wt) and their presence/absence in each of the mutated promoter constructs. Individual mutants (R1, R2, or R3) correspond to those sites shown in Figure 5, and were combined to create double (R1,R2) or triple mutants (ALL). (C) Each construct was transfected into Sua5B cells and luciferase expression was measured under basal conditions (non-induced) or upon induction with heat-killed E. cloacae (induced). Expression was normalized to that of the non-induced wild type SRPN6 promoter in triplicate experiments. The values of two biological repeat experiments were pooled. Asterisks denote significant changes when compared to the wild type construct for each treatment (* = P<0.05, ** = P<0.01, or *** P<0.001) as determined by a Two-way ANOVA and Bonferroni post-test.

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)

Luciferase expression in a mosquito cell line suggests the involvement of LL3 in the regulation of SRPN6 expression.Expression of LL3 and SRPN6 mRNAs in response to heat-killed E. cloacae was investigated in hemocyte-like Sua5B cells (A). LL3 and SRPN6 mRNA abundance was determined by qRT-PCR in cells that were non-induced (non) or after exposure to E. cloacae for 6 h (I). Transcript abundance was normalized to that of rpS7 in two independent biological samples and analyzed by the Student's t-test for significance. Asterisks denote significant changes upon bacterial induction (P<0.05). The constructs outlined in (B) were used to assess the ability of LL3 to modulate luciferase expression from a SRPN6 promoter. Red diamonds indicate the locations of LL3-binding sites in the wild type promoter (wt) and their presence/absence in each of the mutated promoter constructs. Individual mutants (R1, R2, or R3) correspond to those sites shown in Figure 5, and were combined to create double (R1,R2) or triple mutants (ALL). (C) Each construct was transfected into Sua5B cells and luciferase expression was measured under basal conditions (non-induced) or upon induction with heat-killed E. cloacae (induced). Expression was normalized to that of the non-induced wild type SRPN6 promoter in triplicate experiments. The values of two biological repeat experiments were pooled. Asterisks denote significant changes when compared to the wild type construct for each treatment (* = P<0.05, ** = P<0.01, or *** P<0.001) as determined by a Two-way ANOVA and Bonferroni post-test.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475675&req=5

ppat-1002965-g006: Luciferase expression in a mosquito cell line suggests the involvement of LL3 in the regulation of SRPN6 expression.Expression of LL3 and SRPN6 mRNAs in response to heat-killed E. cloacae was investigated in hemocyte-like Sua5B cells (A). LL3 and SRPN6 mRNA abundance was determined by qRT-PCR in cells that were non-induced (non) or after exposure to E. cloacae for 6 h (I). Transcript abundance was normalized to that of rpS7 in two independent biological samples and analyzed by the Student's t-test for significance. Asterisks denote significant changes upon bacterial induction (P<0.05). The constructs outlined in (B) were used to assess the ability of LL3 to modulate luciferase expression from a SRPN6 promoter. Red diamonds indicate the locations of LL3-binding sites in the wild type promoter (wt) and their presence/absence in each of the mutated promoter constructs. Individual mutants (R1, R2, or R3) correspond to those sites shown in Figure 5, and were combined to create double (R1,R2) or triple mutants (ALL). (C) Each construct was transfected into Sua5B cells and luciferase expression was measured under basal conditions (non-induced) or upon induction with heat-killed E. cloacae (induced). Expression was normalized to that of the non-induced wild type SRPN6 promoter in triplicate experiments. The values of two biological repeat experiments were pooled. Asterisks denote significant changes when compared to the wild type construct for each treatment (* = P<0.05, ** = P<0.01, or *** P<0.001) as determined by a Two-way ANOVA and Bonferroni post-test.
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