Limits...
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.

Show MeSH

Related in: MedlinePlus

Caspar-mediated killing of P. falciparum is not dependent on midgut bacteria.(A) Blue bars represent bacteria colony-forming unit (CFUs) from midguts of mosquitoes undergoing the indicated treatments. Pluses and minuses indicate the presence or absence of antibiotic in GFP or Cpr dsRNA treated group. Each bar represents the average of at least 15 mosquitoes tested, with each mosquito's CFU count determined by averaging counts from three serial dilutions. Bars represent the standard deviation for all mosquitoes in a given treatment group. Cpr, Caspar. (B) Dots represent individual oocyst counts following the indicated RNAi treatment; horizontal red bars represent the median number of oocysts per gut. Pluses and minuses indicate the presence or absence of antibiotic in the GFP or Cpr dsRNA-treated group. Assays represent three independent biological replicates and were subject to Mann-Whitney statistical tests. P-values appear below each treatment and refer to that treatment as compared to the GFP dsRNA-treated control. Additional statistical analyses appear in Table S3. Filled portion of bars represent the % of all mosquitoes harboring at least one oocyst; open portion represents those in the group that were uninfected. Cpr, Caspar.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3369948&req=5

ppat-1002737-g004: Caspar-mediated killing of P. falciparum is not dependent on midgut bacteria.(A) Blue bars represent bacteria colony-forming unit (CFUs) from midguts of mosquitoes undergoing the indicated treatments. Pluses and minuses indicate the presence or absence of antibiotic in GFP or Cpr dsRNA treated group. Each bar represents the average of at least 15 mosquitoes tested, with each mosquito's CFU count determined by averaging counts from three serial dilutions. Bars represent the standard deviation for all mosquitoes in a given treatment group. Cpr, Caspar. (B) Dots represent individual oocyst counts following the indicated RNAi treatment; horizontal red bars represent the median number of oocysts per gut. Pluses and minuses indicate the presence or absence of antibiotic in the GFP or Cpr dsRNA-treated group. Assays represent three independent biological replicates and were subject to Mann-Whitney statistical tests. P-values appear below each treatment and refer to that treatment as compared to the GFP dsRNA-treated control. Additional statistical analyses appear in Table S3. Filled portion of bars represent the % of all mosquitoes harboring at least one oocyst; open portion represents those in the group that were uninfected. Cpr, Caspar.

Mentions: Several studies have illuminated the importance of tripartite interactions between the mosquito's immune system, the parasite, and the mosquito's intestinal flora during Plasmodium infection [7], [9], [15], [16]. For some anti-parasite gene regulation and killing mechanisms, the presence and appropriate composition of the bacterial populations in the gut are required [9], [15], [16]. In Drosophila, the Imd pathway is a primary regulator of the response against intestinal bacteria [18]–[20], and we have observed that REL2 over-expression exclusively in the mosquito midgut offers resistance to parasites [10], leading us to question whether bacteria are necessary for the activation of the Imd pathway during caspar silencing, or whether the Imd pathway activation provided by the bacteria under normal conditions is simply supplanted by the artificial Imd pathway activation mediated by caspar silencing. To answer this question, we administered an antibiotic cocktail to mosquitoes before treating them with dsRNA against caspar or GFP and subjecting them to P. falciparum infection. We have previously shown that antibiotics can be used to eliminate the majority of the bacterial population from the mosquito midgut [9], [21]. As had previously been shown, we found that GFP dsRNA-treated mosquitoes were more susceptible to Plasmodium infection if they had been pre-treated with antibiotics; aseptic mosquitoes harbored almost twice as many oocysts as their GFP dsRNA-treated septic counterparts (Figure 4A, Table S3). However, the lack of bacteria in antibiotic-treated mosquitoes had no effect on the ability of caspar silencing to severely limit parasite development; both septic and aseptic mosquitoes treated with dsRNA against caspar displayed median oocyst levels of 0 and had much lower prevalence of infection than did either GFP dsRNA-treated group, though not significantly different from one another (Figure 4B, Table S3).


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)

Caspar-mediated killing of P. falciparum is not dependent on midgut bacteria.(A) Blue bars represent bacteria colony-forming unit (CFUs) from midguts of mosquitoes undergoing the indicated treatments. Pluses and minuses indicate the presence or absence of antibiotic in GFP or Cpr dsRNA treated group. Each bar represents the average of at least 15 mosquitoes tested, with each mosquito's CFU count determined by averaging counts from three serial dilutions. Bars represent the standard deviation for all mosquitoes in a given treatment group. Cpr, Caspar. (B) Dots represent individual oocyst counts following the indicated RNAi treatment; horizontal red bars represent the median number of oocysts per gut. Pluses and minuses indicate the presence or absence of antibiotic in the GFP or Cpr dsRNA-treated group. Assays represent three independent biological replicates and were subject to Mann-Whitney statistical tests. P-values appear below each treatment and refer to that treatment as compared to the GFP dsRNA-treated control. Additional statistical analyses appear in Table S3. Filled portion of bars represent the % of all mosquitoes harboring at least one oocyst; open portion represents those in the group that were uninfected. Cpr, Caspar.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002737-g004: Caspar-mediated killing of P. falciparum is not dependent on midgut bacteria.(A) Blue bars represent bacteria colony-forming unit (CFUs) from midguts of mosquitoes undergoing the indicated treatments. Pluses and minuses indicate the presence or absence of antibiotic in GFP or Cpr dsRNA treated group. Each bar represents the average of at least 15 mosquitoes tested, with each mosquito's CFU count determined by averaging counts from three serial dilutions. Bars represent the standard deviation for all mosquitoes in a given treatment group. Cpr, Caspar. (B) Dots represent individual oocyst counts following the indicated RNAi treatment; horizontal red bars represent the median number of oocysts per gut. Pluses and minuses indicate the presence or absence of antibiotic in the GFP or Cpr dsRNA-treated group. Assays represent three independent biological replicates and were subject to Mann-Whitney statistical tests. P-values appear below each treatment and refer to that treatment as compared to the GFP dsRNA-treated control. Additional statistical analyses appear in Table S3. Filled portion of bars represent the % of all mosquitoes harboring at least one oocyst; open portion represents those in the group that were uninfected. Cpr, Caspar.
Mentions: Several studies have illuminated the importance of tripartite interactions between the mosquito's immune system, the parasite, and the mosquito's intestinal flora during Plasmodium infection [7], [9], [15], [16]. For some anti-parasite gene regulation and killing mechanisms, the presence and appropriate composition of the bacterial populations in the gut are required [9], [15], [16]. In Drosophila, the Imd pathway is a primary regulator of the response against intestinal bacteria [18]–[20], and we have observed that REL2 over-expression exclusively in the mosquito midgut offers resistance to parasites [10], leading us to question whether bacteria are necessary for the activation of the Imd pathway during caspar silencing, or whether the Imd pathway activation provided by the bacteria under normal conditions is simply supplanted by the artificial Imd pathway activation mediated by caspar silencing. To answer this question, we administered an antibiotic cocktail to mosquitoes before treating them with dsRNA against caspar or GFP and subjecting them to P. falciparum infection. We have previously shown that antibiotics can be used to eliminate the majority of the bacterial population from the mosquito midgut [9], [21]. As had previously been shown, we found that GFP dsRNA-treated mosquitoes were more susceptible to Plasmodium infection if they had been pre-treated with antibiotics; aseptic mosquitoes harbored almost twice as many oocysts as their GFP dsRNA-treated septic counterparts (Figure 4A, Table S3). However, the lack of bacteria in antibiotic-treated mosquitoes had no effect on the ability of caspar silencing to severely limit parasite development; both septic and aseptic mosquitoes treated with dsRNA against caspar displayed median oocyst levels of 0 and had much lower prevalence of infection than did either GFP dsRNA-treated group, though not significantly different from one another (Figure 4B, Table S3).

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