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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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Increases in cytosolic cAMP induce Plasmodium sporozoite exocytosis.(A–B) P. yoelii sporozoites were pre-incubated for 15 min with 8Br-cAMP, forskolin (FSK) or MDL-12.330A to activate or inhibit adenylate cyclase respectively, followed by addition or not of uracil derivatives (UD). Sporozoites were incubated for 1 h before fixation and quantification of exocytosis. (C) P. berghei wt (white bars) or spect 1-deficient (black bars) sporozoites were pre-incubated with the different activators and inhibitors as in (A,B). (D) P. falciparum sporozoites were pre-incubated with the different activators and inhibitors as in (A,B). (E) Intracellular levels of cAMP in P. yoelii sporozoites incubated or not with uracil derivatives for 45 min. Same number of uninfected salivary glands were processed in a similar way and used as a control (uninfected). Results are expressed as mean of triplicates±SD. *, p<0.05; ** p<0.01 when compared to control by ANOVA.
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ppat-1000008-g001: Increases in cytosolic cAMP induce Plasmodium sporozoite exocytosis.(A–B) P. yoelii sporozoites were pre-incubated for 15 min with 8Br-cAMP, forskolin (FSK) or MDL-12.330A to activate or inhibit adenylate cyclase respectively, followed by addition or not of uracil derivatives (UD). Sporozoites were incubated for 1 h before fixation and quantification of exocytosis. (C) P. berghei wt (white bars) or spect 1-deficient (black bars) sporozoites were pre-incubated with the different activators and inhibitors as in (A,B). (D) P. falciparum sporozoites were pre-incubated with the different activators and inhibitors as in (A,B). (E) Intracellular levels of cAMP in P. yoelii sporozoites incubated or not with uracil derivatives for 45 min. Same number of uninfected salivary glands were processed in a similar way and used as a control (uninfected). Results are expressed as mean of triplicates±SD. *, p<0.05; ** p<0.01 when compared to control by ANOVA.

Mentions: We first investigated whether cAMP induces or modulates sporozoite regulated exocytosis by preincubating P. yoelii sporozoites with a membrane permeant analogue of cAMP. Exocytosis is quantified as the percentage of sporozoites that present a defined accumulation of extracellular TRAP/SSP2 in their apical end [9]. We found that 8Br-cAMP induces sporozoite exocytosis to a similar level than uracil derivatives. Addition of both stimuli to sporozoites did not increase the level of exocytosis (Fig. 1A), suggesting that both stimuli may be using the same pathway to induce exocytosis. As an alternative way to increase cytosolic cAMP in sporozoites, we used forskolin, an activator of the enzyme that synthesizes cAMP, adenylyl cyclase (AC). This treatment also induced apical regulated exocytosis in sporozoites (Fig. 1B). Incubation of sporozoites with MDL-12,330A, an inhibitor of AC [16] prevented activation of exocytosis by uracil derivatives (Fig. 1B). We confirmed that these treatments did not increased sporozoite lysis compared to control (Table S1 and Fig. S2).


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)

Increases in cytosolic cAMP induce Plasmodium sporozoite exocytosis.(A–B) P. yoelii sporozoites were pre-incubated for 15 min with 8Br-cAMP, forskolin (FSK) or MDL-12.330A to activate or inhibit adenylate cyclase respectively, followed by addition or not of uracil derivatives (UD). Sporozoites were incubated for 1 h before fixation and quantification of exocytosis. (C) P. berghei wt (white bars) or spect 1-deficient (black bars) sporozoites were pre-incubated with the different activators and inhibitors as in (A,B). (D) P. falciparum sporozoites were pre-incubated with the different activators and inhibitors as in (A,B). (E) Intracellular levels of cAMP in P. yoelii sporozoites incubated or not with uracil derivatives for 45 min. Same number of uninfected salivary glands were processed in a similar way and used as a control (uninfected). 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-g001: Increases in cytosolic cAMP induce Plasmodium sporozoite exocytosis.(A–B) P. yoelii sporozoites were pre-incubated for 15 min with 8Br-cAMP, forskolin (FSK) or MDL-12.330A to activate or inhibit adenylate cyclase respectively, followed by addition or not of uracil derivatives (UD). Sporozoites were incubated for 1 h before fixation and quantification of exocytosis. (C) P. berghei wt (white bars) or spect 1-deficient (black bars) sporozoites were pre-incubated with the different activators and inhibitors as in (A,B). (D) P. falciparum sporozoites were pre-incubated with the different activators and inhibitors as in (A,B). (E) Intracellular levels of cAMP in P. yoelii sporozoites incubated or not with uracil derivatives for 45 min. Same number of uninfected salivary glands were processed in a similar way and used as a control (uninfected). Results are expressed as mean of triplicates±SD. *, p<0.05; ** p<0.01 when compared to control by ANOVA.
Mentions: We first investigated whether cAMP induces or modulates sporozoite regulated exocytosis by preincubating P. yoelii sporozoites with a membrane permeant analogue of cAMP. Exocytosis is quantified as the percentage of sporozoites that present a defined accumulation of extracellular TRAP/SSP2 in their apical end [9]. We found that 8Br-cAMP induces sporozoite exocytosis to a similar level than uracil derivatives. Addition of both stimuli to sporozoites did not increase the level of exocytosis (Fig. 1A), suggesting that both stimuli may be using the same pathway to induce exocytosis. As an alternative way to increase cytosolic cAMP in sporozoites, we used forskolin, an activator of the enzyme that synthesizes cAMP, adenylyl cyclase (AC). This treatment also induced apical regulated exocytosis in sporozoites (Fig. 1B). Incubation of sporozoites with MDL-12,330A, an inhibitor of AC [16] prevented activation of exocytosis by uracil derivatives (Fig. 1B). We confirmed that these treatments did not increased sporozoite lysis compared to control (Table S1 and Fig. S2).

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