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Adenylyl cyclase alpha and cAMP signaling mediate Plasmodium sporozoite apical regulated exocytosis and hepatocyte infection.

Ono T, Cabrita-Santos L, Leitao R, Bettiol E, Purcell LA, Diaz-Pulido O, Andrews LB, Tadakuma T, Bhanot P, Mota MM, Rodriguez A - PLoS Pathog. (2008)

Bottom Line: We have generated P. berghei parasites deficient in adenylyl cyclase alpha (ACalpha), a gene containing regions with high homology to adenylyl cyclases.PbACalpha-deficient sporozoites do not exocytose in response to migration through host cells and present more than 50% impaired hepatocyte infectivity in vivo.These effects are specific to ACalpha, as re-introduction of ACalpha in deficient parasites resulted in complete recovery of exocytosis and infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Parasitology, New York University School of Medicine, New York, New York, United States of America.

ABSTRACT
Malaria starts with the infection of the liver of the host by Plasmodium sporozoites, the parasite form transmitted by infected mosquitoes. Sporozoites migrate through several hepatocytes by breaching their plasma membranes before finally infecting one with the formation of an internalization vacuole. Migration through host cells induces apical regulated exocytosis in sporozoites. Here we show that apical regulated exocytosis is induced by increases in cAMP in sporozoites of rodent (P. yoelii and P. berghei) and human (P. falciparum) Plasmodium species. We have generated P. berghei parasites deficient in adenylyl cyclase alpha (ACalpha), a gene containing regions with high homology to adenylyl cyclases. PbACalpha-deficient sporozoites do not exocytose in response to migration through host cells and present more than 50% impaired hepatocyte infectivity in vivo. These effects are specific to ACalpha, as re-introduction of ACalpha in deficient parasites resulted in complete recovery of exocytosis and infection. Our findings indicate that ACalpha and increases in cAMP levels are required for sporozoite apical regulated exocytosis, which is involved in sporozoite infection of hepatocytes.

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Extracellular K+ is required for sporozoite apical regulated exocytosis.(A) P. yoelii sporozoites were pre-incubated for 15 min in regular medium or K+-free medium before addition or not of uracil derivatives (UD) for 45 min. (B) Sporozoites were incubated with regular medium or K+-free medium for 45 min, followed by incubation in regular medium in the presence or absence of UD for another 45 min. (C,D) Sporozoites were pre-incubated with the K+-channel inhibitors charybdotoxin (C) or margatoxin (D) for 15 min before addition of UD for 45 min. (E,F) sporozoites were pre-incubated for 15 min in regular medium or K+-free medium before addition or not of forskolin (E) or 8Br-cAMP (F). (G) Sporozoites were incubated with UD, ionomycin or 8Br-cAMP for 45 min. (H) Sporozoites were pre-incubated for 15 min in regular medium or Ca++-free medium before addition or not of UD for 45 min. (I) Sporozoites were pre-incubated with the membrane permeant calcium chelator BAPTA-AM for 15 min before addition of UD for 45 min. Results are expressed as mean of triplicates±SD. ** p<0.01 when compared to control by ANOVA. (J) Possible model consistent with the results. UD activate directly or indirectly the K+ channel domain of ACα (1) and trigger the activation of AC activity (2). The increase in cAMP activates PKA (3), which leads to the activation of exocytosis.
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ppat-1000008-g004: Extracellular K+ is required for sporozoite apical regulated exocytosis.(A) P. yoelii sporozoites were pre-incubated for 15 min in regular medium or K+-free medium before addition or not of uracil derivatives (UD) for 45 min. (B) Sporozoites were incubated with regular medium or K+-free medium for 45 min, followed by incubation in regular medium in the presence or absence of UD for another 45 min. (C,D) Sporozoites were pre-incubated with the K+-channel inhibitors charybdotoxin (C) or margatoxin (D) for 15 min before addition of UD for 45 min. (E,F) sporozoites were pre-incubated for 15 min in regular medium or K+-free medium before addition or not of forskolin (E) or 8Br-cAMP (F). (G) Sporozoites were incubated with UD, ionomycin or 8Br-cAMP for 45 min. (H) Sporozoites were pre-incubated for 15 min in regular medium or Ca++-free medium before addition or not of UD for 45 min. (I) Sporozoites were pre-incubated with the membrane permeant calcium chelator BAPTA-AM for 15 min before addition of UD for 45 min. Results are expressed as mean of triplicates±SD. ** p<0.01 when compared to control by ANOVA. (J) Possible model consistent with the results. UD activate directly or indirectly the K+ channel domain of ACα (1) and trigger the activation of AC activity (2). The increase in cAMP activates PKA (3), which leads to the activation of exocytosis.

Mentions: To determine whether extracellular K+ is required for sporozoite exocytosis, we stimulated exocytosis in P. yoelii sporozoites in regular medium (containing K+) or in K+-free medium. We found that exocytosis stimulated with uracil derivatives was inhibited in K+-free medium (Fig. 4A). To confirm that sporozoites were not impaired by the incubation in K+-free medium, we transferred sporozoites to regular medium after the K+-free medium incubation. We found that exocytosis in these sporozoites was similar to exocytosis in sporozoites that were never incubated in K+-free medium (Fig. 4B).


Adenylyl cyclase alpha and cAMP signaling mediate Plasmodium sporozoite apical regulated exocytosis and hepatocyte infection.

Ono T, Cabrita-Santos L, Leitao R, Bettiol E, Purcell LA, Diaz-Pulido O, Andrews LB, Tadakuma T, Bhanot P, Mota MM, Rodriguez A - PLoS Pathog. (2008)

Extracellular K+ is required for sporozoite apical regulated exocytosis.(A) P. yoelii sporozoites were pre-incubated for 15 min in regular medium or K+-free medium before addition or not of uracil derivatives (UD) for 45 min. (B) Sporozoites were incubated with regular medium or K+-free medium for 45 min, followed by incubation in regular medium in the presence or absence of UD for another 45 min. (C,D) Sporozoites were pre-incubated with the K+-channel inhibitors charybdotoxin (C) or margatoxin (D) for 15 min before addition of UD for 45 min. (E,F) sporozoites were pre-incubated for 15 min in regular medium or K+-free medium before addition or not of forskolin (E) or 8Br-cAMP (F). (G) Sporozoites were incubated with UD, ionomycin or 8Br-cAMP for 45 min. (H) Sporozoites were pre-incubated for 15 min in regular medium or Ca++-free medium before addition or not of UD for 45 min. (I) Sporozoites were pre-incubated with the membrane permeant calcium chelator BAPTA-AM for 15 min before addition of UD for 45 min. Results are expressed as mean of triplicates±SD. ** p<0.01 when compared to control by ANOVA. (J) Possible model consistent with the results. UD activate directly or indirectly the K+ channel domain of ACα (1) and trigger the activation of AC activity (2). The increase in cAMP activates PKA (3), which leads to the activation of exocytosis.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2279260&req=5

ppat-1000008-g004: Extracellular K+ is required for sporozoite apical regulated exocytosis.(A) P. yoelii sporozoites were pre-incubated for 15 min in regular medium or K+-free medium before addition or not of uracil derivatives (UD) for 45 min. (B) Sporozoites were incubated with regular medium or K+-free medium for 45 min, followed by incubation in regular medium in the presence or absence of UD for another 45 min. (C,D) Sporozoites were pre-incubated with the K+-channel inhibitors charybdotoxin (C) or margatoxin (D) for 15 min before addition of UD for 45 min. (E,F) sporozoites were pre-incubated for 15 min in regular medium or K+-free medium before addition or not of forskolin (E) or 8Br-cAMP (F). (G) Sporozoites were incubated with UD, ionomycin or 8Br-cAMP for 45 min. (H) Sporozoites were pre-incubated for 15 min in regular medium or Ca++-free medium before addition or not of UD for 45 min. (I) Sporozoites were pre-incubated with the membrane permeant calcium chelator BAPTA-AM for 15 min before addition of UD for 45 min. Results are expressed as mean of triplicates±SD. ** p<0.01 when compared to control by ANOVA. (J) Possible model consistent with the results. UD activate directly or indirectly the K+ channel domain of ACα (1) and trigger the activation of AC activity (2). The increase in cAMP activates PKA (3), which leads to the activation of exocytosis.
Mentions: To determine whether extracellular K+ is required for sporozoite exocytosis, we stimulated exocytosis in P. yoelii sporozoites in regular medium (containing K+) or in K+-free medium. We found that exocytosis stimulated with uracil derivatives was inhibited in K+-free medium (Fig. 4A). To confirm that sporozoites were not impaired by the incubation in K+-free medium, we transferred sporozoites to regular medium after the K+-free medium incubation. We found that exocytosis in these sporozoites was similar to exocytosis in sporozoites that were never incubated in K+-free medium (Fig. 4B).

Bottom Line: We have generated P. berghei parasites deficient in adenylyl cyclase alpha (ACalpha), a gene containing regions with high homology to adenylyl cyclases.PbACalpha-deficient sporozoites do not exocytose in response to migration through host cells and present more than 50% impaired hepatocyte infectivity in vivo.These effects are specific to ACalpha, as re-introduction of ACalpha in deficient parasites resulted in complete recovery of exocytosis and infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Parasitology, New York University School of Medicine, New York, New York, United States of America.

ABSTRACT
Malaria starts with the infection of the liver of the host by Plasmodium sporozoites, the parasite form transmitted by infected mosquitoes. Sporozoites migrate through several hepatocytes by breaching their plasma membranes before finally infecting one with the formation of an internalization vacuole. Migration through host cells induces apical regulated exocytosis in sporozoites. Here we show that apical regulated exocytosis is induced by increases in cAMP in sporozoites of rodent (P. yoelii and P. berghei) and human (P. falciparum) Plasmodium species. We have generated P. berghei parasites deficient in adenylyl cyclase alpha (ACalpha), a gene containing regions with high homology to adenylyl cyclases. PbACalpha-deficient sporozoites do not exocytose in response to migration through host cells and present more than 50% impaired hepatocyte infectivity in vivo. These effects are specific to ACalpha, as re-introduction of ACalpha in deficient parasites resulted in complete recovery of exocytosis and infection. Our findings indicate that ACalpha and increases in cAMP levels are required for sporozoite apical regulated exocytosis, which is involved in sporozoite infection of hepatocytes.

Show MeSH
Related in: MedlinePlus