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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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Imd pathway components and effectors differ in their ability to affect P. falciparum infections of high, medium, and low infection intensities.Intensity of P. falciparum oocysts in A. gambiae silenced for given genes and subjected to low (A), medium (C) or high (E) infection exposures. Bars represent median numbers of oocysts per midgut, and dots represent individual midgut oocyst counts. Assays represent at least three independent biological replicates and were subject to Mann-Whitney statistical tests. P-values appear above each treatment and refer to that treatment as compared to the GFP dsRNA-treated control. Non-significant p-values were not included in the figure. Additional statistical analyses appear in Table S4. D-F: Prevalence of infection in A. gambiae subjected to low (B), medium (D) and high (F) loads of P. falciparum. Filled portion of bars represent the % of all mosquitoes harboring at least one oocyst; open portion represents those in the group that were uninfected. wAPL1 (whole APL1) dsRNA – dsRNA for a conserved region of APL1 genes, which results in the silencing of all three APL1 proteins (APL1A, APL1B and APL1C).
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ppat-1002737-g005: Imd pathway components and effectors differ in their ability to affect P. falciparum infections of high, medium, and low infection intensities.Intensity of P. falciparum oocysts in A. gambiae silenced for given genes and subjected to low (A), medium (C) or high (E) infection exposures. Bars represent median numbers of oocysts per midgut, and dots represent individual midgut oocyst counts. Assays represent at least three independent biological replicates and were subject to Mann-Whitney statistical tests. P-values appear above each treatment and refer to that treatment as compared to the GFP dsRNA-treated control. Non-significant p-values were not included in the figure. Additional statistical analyses appear in Table S4. D-F: Prevalence of infection in A. gambiae subjected to low (B), medium (D) and high (F) loads of P. falciparum. Filled portion of bars represent the % of all mosquitoes harboring at least one oocyst; open portion represents those in the group that were uninfected. wAPL1 (whole APL1) dsRNA – dsRNA for a conserved region of APL1 genes, which results in the silencing of all three APL1 proteins (APL1A, APL1B and APL1C).

Mentions: Earlier studies have featured a model system composed of a highly virulent parasite strain and highly susceptible mosquito strain. While we have previously shown that the Imd pathway is effective against P. falciparum in other Anopheles strains [4], recent data have revealed the importance of infection intensity in the generation of immune responses directed against Plasmodium[17]. To determine whether Imd-derived responses are more or less effective against different levels of infection, we subjected mosquitoes to a standard administration of dsRNA against various Imd pathway members and downstream effectors; then, at 3 to 4 days after the dsRNA injection, we fed them on P. falciparum-infected blood with a more dilute or more concentrated gametocyte culture than that used in the standard protocol. Oocyst counts indicated that, indeed, low-, medium-, and high-intensity infections had been achieved, with roughly log-fold differences (median = 1, 7, and 86, respectively) from the levels in GFP dsRNA- treated control mosquitoes (Figure 5, Tables S4A–C) and that the infection phenotypes observed following silencing were not consistent for the three levels of infection intensity (Figure 5). For example, silencing all forms of apl1 significantly increased the infection levels (when compared to the GFP dsRNA-treated group) only at low (a 2-fold increase) and medium (a 2.6 -fold increase) infection intensities. Specific paralog silencing suggested that APL1C was most effective in influencing low-level infections (a 2-fold increase), while APL1B was the most influential paralog at medium infection intensities (a 2.3-fold increase). In our experiments, APL1A depletion had no effect on P. falciparum infection under any condition.


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)

Imd pathway components and effectors differ in their ability to affect P. falciparum infections of high, medium, and low infection intensities.Intensity of P. falciparum oocysts in A. gambiae silenced for given genes and subjected to low (A), medium (C) or high (E) infection exposures. Bars represent median numbers of oocysts per midgut, and dots represent individual midgut oocyst counts. Assays represent at least three independent biological replicates and were subject to Mann-Whitney statistical tests. P-values appear above each treatment and refer to that treatment as compared to the GFP dsRNA-treated control. Non-significant p-values were not included in the figure. Additional statistical analyses appear in Table S4. D-F: Prevalence of infection in A. gambiae subjected to low (B), medium (D) and high (F) loads of P. falciparum. Filled portion of bars represent the % of all mosquitoes harboring at least one oocyst; open portion represents those in the group that were uninfected. wAPL1 (whole APL1) dsRNA – dsRNA for a conserved region of APL1 genes, which results in the silencing of all three APL1 proteins (APL1A, APL1B and APL1C).
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Related In: Results  -  Collection

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ppat-1002737-g005: Imd pathway components and effectors differ in their ability to affect P. falciparum infections of high, medium, and low infection intensities.Intensity of P. falciparum oocysts in A. gambiae silenced for given genes and subjected to low (A), medium (C) or high (E) infection exposures. Bars represent median numbers of oocysts per midgut, and dots represent individual midgut oocyst counts. Assays represent at least three independent biological replicates and were subject to Mann-Whitney statistical tests. P-values appear above each treatment and refer to that treatment as compared to the GFP dsRNA-treated control. Non-significant p-values were not included in the figure. Additional statistical analyses appear in Table S4. D-F: Prevalence of infection in A. gambiae subjected to low (B), medium (D) and high (F) loads of P. falciparum. Filled portion of bars represent the % of all mosquitoes harboring at least one oocyst; open portion represents those in the group that were uninfected. wAPL1 (whole APL1) dsRNA – dsRNA for a conserved region of APL1 genes, which results in the silencing of all three APL1 proteins (APL1A, APL1B and APL1C).
Mentions: Earlier studies have featured a model system composed of a highly virulent parasite strain and highly susceptible mosquito strain. While we have previously shown that the Imd pathway is effective against P. falciparum in other Anopheles strains [4], recent data have revealed the importance of infection intensity in the generation of immune responses directed against Plasmodium[17]. To determine whether Imd-derived responses are more or less effective against different levels of infection, we subjected mosquitoes to a standard administration of dsRNA against various Imd pathway members and downstream effectors; then, at 3 to 4 days after the dsRNA injection, we fed them on P. falciparum-infected blood with a more dilute or more concentrated gametocyte culture than that used in the standard protocol. Oocyst counts indicated that, indeed, low-, medium-, and high-intensity infections had been achieved, with roughly log-fold differences (median = 1, 7, and 86, respectively) from the levels in GFP dsRNA- treated control mosquitoes (Figure 5, Tables S4A–C) and that the infection phenotypes observed following silencing were not consistent for the three levels of infection intensity (Figure 5). For example, silencing all forms of apl1 significantly increased the infection levels (when compared to the GFP dsRNA-treated group) only at low (a 2-fold increase) and medium (a 2.6 -fold increase) infection intensities. Specific paralog silencing suggested that APL1C was most effective in influencing low-level infections (a 2-fold increase), while APL1B was the most influential paralog at medium infection intensities (a 2.3-fold increase). In our experiments, APL1A depletion had no effect on P. falciparum infection under any condition.

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