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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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Plasmodium oocyst intensity increases in LL3 knockdown mosquitoes.GFP (control) or LL3 dsRNA was injected into adult female mosquitoes and two days later, surviving mosquitoes were given an infectious blood meal containing P. berghei (A) or P. falciparum (B) parasites. Oocyst numbers were determined respectively at 10 or 7 days following the blood meal. Data from six (P. berghei) or three (P. falciparum) independent experiments were pooled and analyzed by the Mann-Whitney test. The horizontal bars denote the median value for each experimental group. N: number of midguts assayed; Prevalence: percent of midguts that had at least one oocyst. (C) Efficiency of LL3 knockdown and its effect on SRPN6 mRNA abundance were assessed in midguts of P. berghei-infected mosquitoes 24 h PBM. The relative gene expression represents transcript abundance normalized to the GFP control across four separate experiments. Data were compared with the Student's t-test to determine differences in LL3 and SRPN6 expression between GFP control and LL3 knockdown samples. Asterisks denote a P value of <0.05.
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ppat-1002965-g003: Plasmodium oocyst intensity increases in LL3 knockdown mosquitoes.GFP (control) or LL3 dsRNA was injected into adult female mosquitoes and two days later, surviving mosquitoes were given an infectious blood meal containing P. berghei (A) or P. falciparum (B) parasites. Oocyst numbers were determined respectively at 10 or 7 days following the blood meal. Data from six (P. berghei) or three (P. falciparum) independent experiments were pooled and analyzed by the Mann-Whitney test. The horizontal bars denote the median value for each experimental group. N: number of midguts assayed; Prevalence: percent of midguts that had at least one oocyst. (C) Efficiency of LL3 knockdown and its effect on SRPN6 mRNA abundance were assessed in midguts of P. berghei-infected mosquitoes 24 h PBM. The relative gene expression represents transcript abundance normalized to the GFP control across four separate experiments. Data were compared with the Student's t-test to determine differences in LL3 and SRPN6 expression between GFP control and LL3 knockdown samples. Asterisks denote a P value of <0.05.

Mentions: We used RNAi-mediated gene silencing to determine whether LL3 plays a role in the mosquito response to Plasmodium infection. Silencing of LL3 led to a substantial increase in P. berghei oocyst numbers and infection prevalence when compared to dsGFP controls (Figure 3A). Infection by P. falciparum parasites was similarly affected as the LL3 knockdown mosquitoes displayed double the oocyst load when compared to controls (Figure 3B). A small, but non-significant increase in the P. falciparum infection prevalence was detected, despite the high intensity of the controls. Previous reports have suggested that the immune responses of the mosquito to P. berghei and P. falciparum are quite divergent [15]. Several molecules that have been implicated in anti-Plasmodium defenses only function against a specific parasite species [8]. However, the dsRNA-mediated silencing of LL3 results in a significant increase in the number of developing oocysts for both rodent and human parasite species, suggesting that LL3 is a universal component of the mosquito anti-Plasmodium response. The presumed role of LL3 as a transcription factor suggests that it regulates the immune response at the transcriptional level.


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)

Plasmodium oocyst intensity increases in LL3 knockdown mosquitoes.GFP (control) or LL3 dsRNA was injected into adult female mosquitoes and two days later, surviving mosquitoes were given an infectious blood meal containing P. berghei (A) or P. falciparum (B) parasites. Oocyst numbers were determined respectively at 10 or 7 days following the blood meal. Data from six (P. berghei) or three (P. falciparum) independent experiments were pooled and analyzed by the Mann-Whitney test. The horizontal bars denote the median value for each experimental group. N: number of midguts assayed; Prevalence: percent of midguts that had at least one oocyst. (C) Efficiency of LL3 knockdown and its effect on SRPN6 mRNA abundance were assessed in midguts of P. berghei-infected mosquitoes 24 h PBM. The relative gene expression represents transcript abundance normalized to the GFP control across four separate experiments. Data were compared with the Student's t-test to determine differences in LL3 and SRPN6 expression between GFP control and LL3 knockdown samples. Asterisks denote a P value of <0.05.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002965-g003: Plasmodium oocyst intensity increases in LL3 knockdown mosquitoes.GFP (control) or LL3 dsRNA was injected into adult female mosquitoes and two days later, surviving mosquitoes were given an infectious blood meal containing P. berghei (A) or P. falciparum (B) parasites. Oocyst numbers were determined respectively at 10 or 7 days following the blood meal. Data from six (P. berghei) or three (P. falciparum) independent experiments were pooled and analyzed by the Mann-Whitney test. The horizontal bars denote the median value for each experimental group. N: number of midguts assayed; Prevalence: percent of midguts that had at least one oocyst. (C) Efficiency of LL3 knockdown and its effect on SRPN6 mRNA abundance were assessed in midguts of P. berghei-infected mosquitoes 24 h PBM. The relative gene expression represents transcript abundance normalized to the GFP control across four separate experiments. Data were compared with the Student's t-test to determine differences in LL3 and SRPN6 expression between GFP control and LL3 knockdown samples. Asterisks denote a P value of <0.05.
Mentions: We used RNAi-mediated gene silencing to determine whether LL3 plays a role in the mosquito response to Plasmodium infection. Silencing of LL3 led to a substantial increase in P. berghei oocyst numbers and infection prevalence when compared to dsGFP controls (Figure 3A). Infection by P. falciparum parasites was similarly affected as the LL3 knockdown mosquitoes displayed double the oocyst load when compared to controls (Figure 3B). A small, but non-significant increase in the P. falciparum infection prevalence was detected, despite the high intensity of the controls. Previous reports have suggested that the immune responses of the mosquito to P. berghei and P. falciparum are quite divergent [15]. Several molecules that have been implicated in anti-Plasmodium defenses only function against a specific parasite species [8]. However, the dsRNA-mediated silencing of LL3 results in a significant increase in the number of developing oocysts for both rodent and human parasite species, suggesting that LL3 is a universal component of the mosquito anti-Plasmodium response. The presumed role of LL3 as a transcription factor suggests that it regulates the immune response at the transcriptional level.

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