Limits...
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.

Show MeSH

Related in: MedlinePlus

Treatment with an inhibitor of PKA reduces sporozoite exocytosis and infection.P. yoelii sporozoites were pre-incubated with H89 followed by addition of uracil derivatives to induce exocytosis (A) or followed by incubation with monolayers of Hepa1-6 cells to quantify infection (B) and migration though cells (C). (D) Sporozoites were pre-incubated with H89 before addition of 8Br-cAMP to induce exocytosis. (E) Sporozoites were pre-incubated with genistein (Gen) before addition of uracil derivatives. (F) P. yoelii sporozoites were pre-incubated with 2′, 5′-Dideoxyadenosine (DDA) or SQ22536 (SQ) to inhibit adenylyl cyclase activity or with cAMP Rp-isomer to inhibit PKA, before addition of uracil derivatives to induce exocytosis. Results are expressed as mean of triplicates±SD. * p<0.05; ** p<0.01 when compared to control by ANOVA.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2279260&req=5

ppat-1000008-g003: Treatment with an inhibitor of PKA reduces sporozoite exocytosis and infection.P. yoelii sporozoites were pre-incubated with H89 followed by addition of uracil derivatives to induce exocytosis (A) or followed by incubation with monolayers of Hepa1-6 cells to quantify infection (B) and migration though cells (C). (D) Sporozoites were pre-incubated with H89 before addition of 8Br-cAMP to induce exocytosis. (E) Sporozoites were pre-incubated with genistein (Gen) before addition of uracil derivatives. (F) P. yoelii sporozoites were pre-incubated with 2′, 5′-Dideoxyadenosine (DDA) or SQ22536 (SQ) to inhibit adenylyl cyclase activity or with cAMP Rp-isomer to inhibit PKA, before addition of uracil derivatives to induce exocytosis. Results are expressed as mean of triplicates±SD. * p<0.05; ** p<0.01 when compared to control by ANOVA.

Mentions: The major downstream effector of cAMP is protein kinase A (PKA), a serine/threonine kinase that activates other kinases and transcription factors in the cell. This protein is likely to be present in Plasmodium because PKA activity has been detected in P. falciparum during the blood stage of the parasite [18],[19] and there is a gene sequence with high homology to PKA expressed in P. falciparum and conserved in all species of Plasmodium analyzed [20],[21], however no functional assays have yet determined the PKA activity of this putative protein. To investigate whether sporozoite exocytosis is mediated by PKA activity, we treated sporozoites with H89, a PKA inhibitor already shown to inhibit this kinase in a different stage of the parasite [18],[19]. We found that H89 inhibits sporozoite exocytosis induced by uracil derivatives (Fig. 3A), suggesting that this process is mediated by the activation of PKA. The infectivity of sporozoites pretreated with H89 is reduced, probably as a consequence of the inhibition of exocytosis (Fig. 3B), while parasite migration through host cells is not affected, confirming that H89 treatment is not toxic for sporozoites (Fig. 3C).


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)

Treatment with an inhibitor of PKA reduces sporozoite exocytosis and infection.P. yoelii sporozoites were pre-incubated with H89 followed by addition of uracil derivatives to induce exocytosis (A) or followed by incubation with monolayers of Hepa1-6 cells to quantify infection (B) and migration though cells (C). (D) Sporozoites were pre-incubated with H89 before addition of 8Br-cAMP to induce exocytosis. (E) Sporozoites were pre-incubated with genistein (Gen) before addition of uracil derivatives. (F) P. yoelii sporozoites were pre-incubated with 2′, 5′-Dideoxyadenosine (DDA) or SQ22536 (SQ) to inhibit adenylyl cyclase activity or with cAMP Rp-isomer to inhibit PKA, before addition of uracil derivatives to induce exocytosis. Results are expressed as mean of triplicates±SD. * p<0.05; ** p<0.01 when compared to control by ANOVA.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2279260&req=5

ppat-1000008-g003: Treatment with an inhibitor of PKA reduces sporozoite exocytosis and infection.P. yoelii sporozoites were pre-incubated with H89 followed by addition of uracil derivatives to induce exocytosis (A) or followed by incubation with monolayers of Hepa1-6 cells to quantify infection (B) and migration though cells (C). (D) Sporozoites were pre-incubated with H89 before addition of 8Br-cAMP to induce exocytosis. (E) Sporozoites were pre-incubated with genistein (Gen) before addition of uracil derivatives. (F) P. yoelii sporozoites were pre-incubated with 2′, 5′-Dideoxyadenosine (DDA) or SQ22536 (SQ) to inhibit adenylyl cyclase activity or with cAMP Rp-isomer to inhibit PKA, before addition of uracil derivatives to induce exocytosis. Results are expressed as mean of triplicates±SD. * p<0.05; ** p<0.01 when compared to control by ANOVA.
Mentions: The major downstream effector of cAMP is protein kinase A (PKA), a serine/threonine kinase that activates other kinases and transcription factors in the cell. This protein is likely to be present in Plasmodium because PKA activity has been detected in P. falciparum during the blood stage of the parasite [18],[19] and there is a gene sequence with high homology to PKA expressed in P. falciparum and conserved in all species of Plasmodium analyzed [20],[21], however no functional assays have yet determined the PKA activity of this putative protein. To investigate whether sporozoite exocytosis is mediated by PKA activity, we treated sporozoites with H89, a PKA inhibitor already shown to inhibit this kinase in a different stage of the parasite [18],[19]. We found that H89 inhibits sporozoite exocytosis induced by uracil derivatives (Fig. 3A), suggesting that this process is mediated by the activation of PKA. The infectivity of sporozoites pretreated with H89 is reduced, probably as a consequence of the inhibition of exocytosis (Fig. 3B), while parasite migration through host cells is not affected, confirming that H89 treatment is not toxic for sporozoites (Fig. 3C).

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