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Anopheles Imd pathway factors and effectors in infection intensity-dependent anti-Plasmodium action.

Garver LS, Bahia AC, Das S, Souza-Neto JA, Shiao J, Dong Y, Dimopoulos G - PLoS Pathog. (2012)

Bottom Line: Silencing the expression of caspar, a negative regulator of the Imd pathway, or over-expressing rel2, an Imd pathway-controlled NFkappaB transcription factor, confers a resistant phenotype on A. gambiae mosquitoes that involves an array of immune effector genes.We further demonstrated that caspar silencing alone is sufficient to induce a robust anti-P. falciparum response even in the relative absence of resident gut microbiota.Finally, we established the relevance of the Imd pathway components and regulated effectors TEP1, APL1, and LRIM1 in parasite infection intensity-dependent defense, thereby shedding light on the relevance of laboratory versus natural infection intensity models.

View Article: PubMed Central - PubMed

Affiliation: W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, United States of America.

ABSTRACT
The Anopheles gambiae immune response against Plasmodium falciparum, an etiological agent of human malaria, has been identified as a source of potential anti-Plasmodium genes and mechanisms to be exploited in efforts to control the malaria transmission cycle. One such mechanism is the Imd pathway, a conserved immune signaling pathway that has potent anti-P. falciparum activity. Silencing the expression of caspar, a negative regulator of the Imd pathway, or over-expressing rel2, an Imd pathway-controlled NFkappaB transcription factor, confers a resistant phenotype on A. gambiae mosquitoes that involves an array of immune effector genes. However, unexplored features of this powerful mechanism that may be essential for the implementation of a malaria control strategy still remain. Using RNA interference to singly or dually silence caspar and other components of the Imd pathway, we have identified genes participating in the anti-Plasmodium signaling module regulated by Caspar, each of which represents a potential target to achieve over-activation of the pathway. We also determined that the Imd pathway is most potent against the parasite's ookinete stage, yet also has reasonable activity against early oocysts and lesser activity against late oocysts. We further demonstrated that caspar silencing alone is sufficient to induce a robust anti-P. falciparum response even in the relative absence of resident gut microbiota. Finally, we established the relevance of the Imd pathway components and regulated effectors TEP1, APL1, and LRIM1 in parasite infection intensity-dependent defense, thereby shedding light on the relevance of laboratory versus natural infection intensity models. Our results highlight the physiological considerations that are integral to a thoughtful implementation of Imd pathway manipulation in A. gambiae as part of an effort to limit the malaria transmission cycle, and they reveal a variety of previously unrecognized nuances in the Imd-directed immune response against P. falciparum.

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Some members of the Imd pathway have an effect on P. falciparum infection.(A–G) Dots represent individual oocyst counts following the indicated RNAi treatment; horizontal red bars represent the median number of oocysts per gut. P-values were derived from Mann-Whitney statistical tests and appear above each treatment and refer to that treatment as compared to the GFP dsRNA-treated control. Additional statistical analyses appear in Table S1) Filled portion of bars represent the % of all mosquitoes harboring at least one oocyst; open portion represents those in the group that were uninfected. All assays represent two to three independent biological replicate. Cpr, Caspar. (H) Prevalence of P. falciparum infection following the indicated RNAi treatment.
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ppat-1002737-g002: Some members of the Imd pathway have an effect on P. falciparum infection.(A–G) Dots represent individual oocyst counts following the indicated RNAi treatment; horizontal red bars represent the median number of oocysts per gut. P-values were derived from Mann-Whitney statistical tests and appear above each treatment and refer to that treatment as compared to the GFP dsRNA-treated control. Additional statistical analyses appear in Table S1) Filled portion of bars represent the % of all mosquitoes harboring at least one oocyst; open portion represents those in the group that were uninfected. All assays represent two to three independent biological replicate. Cpr, Caspar. (H) Prevalence of P. falciparum infection following the indicated RNAi treatment.

Mentions: Silencing of the Imd pathway factors imd, fadd, caspL1 (dredd), and rel2 resulted in median P. falciparum oocyst infection intensities that were at least two-fold greater than those of the control group treated with dsRNA against GFP (Figure 2A–2D, Table S1). Of these genes, only silencing of rel2 had a significant effect on infection prevalence (Figure 2H and Table S1). Co-silencing of the four pathway members (imd, fadd, caspL1, and rel2) with caspar completely reversed the typical resistance to infection observed when only caspar was silenced; i.e., median infection intensities were not significantly different from those of the single-silenced groups and did not exhibit the absence of infection typically observed following caspar silencing (Figure 3) and [4].


Anopheles Imd pathway factors and effectors in infection intensity-dependent anti-Plasmodium action.

Garver LS, Bahia AC, Das S, Souza-Neto JA, Shiao J, Dong Y, Dimopoulos G - PLoS Pathog. (2012)

Some members of the Imd pathway have an effect on P. falciparum infection.(A–G) Dots represent individual oocyst counts following the indicated RNAi treatment; horizontal red bars represent the median number of oocysts per gut. P-values were derived from Mann-Whitney statistical tests and appear above each treatment and refer to that treatment as compared to the GFP dsRNA-treated control. Additional statistical analyses appear in Table S1) Filled portion of bars represent the % of all mosquitoes harboring at least one oocyst; open portion represents those in the group that were uninfected. All assays represent two to three independent biological replicate. Cpr, Caspar. (H) Prevalence of P. falciparum infection following the indicated RNAi treatment.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369948&req=5

ppat-1002737-g002: Some members of the Imd pathway have an effect on P. falciparum infection.(A–G) Dots represent individual oocyst counts following the indicated RNAi treatment; horizontal red bars represent the median number of oocysts per gut. P-values were derived from Mann-Whitney statistical tests and appear above each treatment and refer to that treatment as compared to the GFP dsRNA-treated control. Additional statistical analyses appear in Table S1) Filled portion of bars represent the % of all mosquitoes harboring at least one oocyst; open portion represents those in the group that were uninfected. All assays represent two to three independent biological replicate. Cpr, Caspar. (H) Prevalence of P. falciparum infection following the indicated RNAi treatment.
Mentions: Silencing of the Imd pathway factors imd, fadd, caspL1 (dredd), and rel2 resulted in median P. falciparum oocyst infection intensities that were at least two-fold greater than those of the control group treated with dsRNA against GFP (Figure 2A–2D, Table S1). Of these genes, only silencing of rel2 had a significant effect on infection prevalence (Figure 2H and Table S1). Co-silencing of the four pathway members (imd, fadd, caspL1, and rel2) with caspar completely reversed the typical resistance to infection observed when only caspar was silenced; i.e., median infection intensities were not significantly different from those of the single-silenced groups and did not exhibit the absence of infection typically observed following caspar silencing (Figure 3) and [4].

Bottom Line: Silencing the expression of caspar, a negative regulator of the Imd pathway, or over-expressing rel2, an Imd pathway-controlled NFkappaB transcription factor, confers a resistant phenotype on A. gambiae mosquitoes that involves an array of immune effector genes.We further demonstrated that caspar silencing alone is sufficient to induce a robust anti-P. falciparum response even in the relative absence of resident gut microbiota.Finally, we established the relevance of the Imd pathway components and regulated effectors TEP1, APL1, and LRIM1 in parasite infection intensity-dependent defense, thereby shedding light on the relevance of laboratory versus natural infection intensity models.

View Article: PubMed Central - PubMed

Affiliation: W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, United States of America.

ABSTRACT
The Anopheles gambiae immune response against Plasmodium falciparum, an etiological agent of human malaria, has been identified as a source of potential anti-Plasmodium genes and mechanisms to be exploited in efforts to control the malaria transmission cycle. One such mechanism is the Imd pathway, a conserved immune signaling pathway that has potent anti-P. falciparum activity. Silencing the expression of caspar, a negative regulator of the Imd pathway, or over-expressing rel2, an Imd pathway-controlled NFkappaB transcription factor, confers a resistant phenotype on A. gambiae mosquitoes that involves an array of immune effector genes. However, unexplored features of this powerful mechanism that may be essential for the implementation of a malaria control strategy still remain. Using RNA interference to singly or dually silence caspar and other components of the Imd pathway, we have identified genes participating in the anti-Plasmodium signaling module regulated by Caspar, each of which represents a potential target to achieve over-activation of the pathway. We also determined that the Imd pathway is most potent against the parasite's ookinete stage, yet also has reasonable activity against early oocysts and lesser activity against late oocysts. We further demonstrated that caspar silencing alone is sufficient to induce a robust anti-P. falciparum response even in the relative absence of resident gut microbiota. Finally, we established the relevance of the Imd pathway components and regulated effectors TEP1, APL1, and LRIM1 in parasite infection intensity-dependent defense, thereby shedding light on the relevance of laboratory versus natural infection intensity models. Our results highlight the physiological considerations that are integral to a thoughtful implementation of Imd pathway manipulation in A. gambiae as part of an effort to limit the malaria transmission cycle, and they reveal a variety of previously unrecognized nuances in the Imd-directed immune response against P. falciparum.

Show MeSH
Related in: MedlinePlus