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

Stimulation of exocytosis increases sporozoite infection and decreases migration through host cells.P. yoelii sporozoites were pretreated with forskolin or 8Br-cAMP (A) or MDL-12.330A (B) before addition to monolayers of Hepa1-6 cells. Percentage of dextran-positive cells (white bars) and number of infected cells/coverslip (black bars) are shown 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-g002: Stimulation of exocytosis increases sporozoite infection and decreases migration through host cells.P. yoelii sporozoites were pretreated with forskolin or 8Br-cAMP (A) or MDL-12.330A (B) before addition to monolayers of Hepa1-6 cells. Percentage of dextran-positive cells (white bars) and number of infected cells/coverslip (black bars) are shown as mean of triplicates±SD. *, p<0.05; ** p<0.01 when compared to control by ANOVA.

Mentions: Migration through host cells induces sporozoite apical regulated exocytosis, which activates sporozoites for infection. Stimulation of exocytosis by other means, such as host cells lysate [9] or uracil derivatives [13], overcomes the need for extensive migration through cells and increases infection. To test whether stimulation of exocytosis by increases in intracellular cAMP in the sporozoite would also overcome the need for migration through host cells before infection, we incubated P. yoelii sporozoites with forskolin or 8Br-cAMP to induce regulated exocytosis before addition of sporozoites to intact Hepa1-6 cells. Migration through host cells is determined as the percentage of cells that are wounded by sporozoite migration and as a result become positive for a soluble impermeant tracer (dextran) [17]. We found an increase in the number of infected cells, indicating that stimulation of regulated exocytosis by cAMP in sporozoites increases their infectivity (Fig. 2A, black bars). In addition, activation of sporozoite exocytosis with increased cAMP levels reduces sporozoite migration through host cells, confirming that such extensive migration is no longer necessary when exocytosis is induced by elevations in the level of cAMP (Fig. 2A, white bars). These results indicate that cAMP-induced exocytosis contributes to the activation of sporozoites for infection.


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)

Stimulation of exocytosis increases sporozoite infection and decreases migration through host cells.P. yoelii sporozoites were pretreated with forskolin or 8Br-cAMP (A) or MDL-12.330A (B) before addition to monolayers of Hepa1-6 cells. Percentage of dextran-positive cells (white bars) and number of infected cells/coverslip (black bars) are shown 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-g002: Stimulation of exocytosis increases sporozoite infection and decreases migration through host cells.P. yoelii sporozoites were pretreated with forskolin or 8Br-cAMP (A) or MDL-12.330A (B) before addition to monolayers of Hepa1-6 cells. Percentage of dextran-positive cells (white bars) and number of infected cells/coverslip (black bars) are shown as mean of triplicates±SD. *, p<0.05; ** p<0.01 when compared to control by ANOVA.
Mentions: Migration through host cells induces sporozoite apical regulated exocytosis, which activates sporozoites for infection. Stimulation of exocytosis by other means, such as host cells lysate [9] or uracil derivatives [13], overcomes the need for extensive migration through cells and increases infection. To test whether stimulation of exocytosis by increases in intracellular cAMP in the sporozoite would also overcome the need for migration through host cells before infection, we incubated P. yoelii sporozoites with forskolin or 8Br-cAMP to induce regulated exocytosis before addition of sporozoites to intact Hepa1-6 cells. Migration through host cells is determined as the percentage of cells that are wounded by sporozoite migration and as a result become positive for a soluble impermeant tracer (dextran) [17]. We found an increase in the number of infected cells, indicating that stimulation of regulated exocytosis by cAMP in sporozoites increases their infectivity (Fig. 2A, black bars). In addition, activation of sporozoite exocytosis with increased cAMP levels reduces sporozoite migration through host cells, confirming that such extensive migration is no longer necessary when exocytosis is induced by elevations in the level of cAMP (Fig. 2A, white bars). These results indicate that cAMP-induced exocytosis contributes to the activation of sporozoites for infection.

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