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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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Related in: MedlinePlus

PbACα- sporozoites have defective exocytosis and infection.Exocytosis and infectivity of P. berghei WT (white bars), PbACα- C1 (black bars) and C2 (gray bars) sporozoites was analyzed. (A, B) Sporozoites were incubated or not with uracil derivatives (UD) or forskolin (FSK) (A) or 8Br-cAMP (B) for 1 h before fixation and quantification of exocytosis. (C) Sporozoites were added to filter insets containing confluent Hepa1-6 cells and collected on empty coverslips placed underneath the filters in the lower chamber. Percentage of sporozoites in coverslips showing apical-regulated exocytosis is shown. (D) Infection of Hepa1-6 cell by sporozoites in vitro was determined by counting the number of infected cells after 24 h incubation. (E) Infection of mice was determined by real-time PCR amplification of 18S rRNA in the liver 40 h after inoculation of sporozoites.
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ppat-1000008-g007: PbACα- sporozoites have defective exocytosis and infection.Exocytosis and infectivity of P. berghei WT (white bars), PbACα- C1 (black bars) and C2 (gray bars) sporozoites was analyzed. (A, B) Sporozoites were incubated or not with uracil derivatives (UD) or forskolin (FSK) (A) or 8Br-cAMP (B) for 1 h before fixation and quantification of exocytosis. (C) Sporozoites were added to filter insets containing confluent Hepa1-6 cells and collected on empty coverslips placed underneath the filters in the lower chamber. Percentage of sporozoites in coverslips showing apical-regulated exocytosis is shown. (D) Infection of Hepa1-6 cell by sporozoites in vitro was determined by counting the number of infected cells after 24 h incubation. (E) Infection of mice was determined by real-time PCR amplification of 18S rRNA in the liver 40 h after inoculation of sporozoites.

Mentions: We then tested whether apical regulated exocytosis was affected in PbACα-sporozoites. Activation of exocytosis by the mix of uracil derivatives or by forskolin, was greatly reduced in the two different clones of PbACα- sporozoites analyzed (Fig. 7A). Addition of a membrane permeant analogue of cAMP (8-Br-cAMP), which induces exocytosis in WT parasites, also stimulated exocytosis in PbACα- sporozoites (Fig. 7B). This result indicates that all sporozoite components required for exocytosis downstream of cAMP are functional in PbACα- sporozoites; however, the lack of ACα inhibits proper response upon activation with uracil derivatives or activators of AC activity. Migration through host cells induces apical regulated exocytosis in Plasmodium sporozoites [9]. To confirm that ACα is also required for exocytosis stimulated by migration through hepatocytes, we measured the response of WT and PbACα- sporozoites after migration through Hepa1-6 cells. We found that regulated exocytosis was not activated in sporozoites deficient in ACα (Fig. 7C).


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)

PbACα- sporozoites have defective exocytosis and infection.Exocytosis and infectivity of P. berghei WT (white bars), PbACα- C1 (black bars) and C2 (gray bars) sporozoites was analyzed. (A, B) Sporozoites were incubated or not with uracil derivatives (UD) or forskolin (FSK) (A) or 8Br-cAMP (B) for 1 h before fixation and quantification of exocytosis. (C) Sporozoites were added to filter insets containing confluent Hepa1-6 cells and collected on empty coverslips placed underneath the filters in the lower chamber. Percentage of sporozoites in coverslips showing apical-regulated exocytosis is shown. (D) Infection of Hepa1-6 cell by sporozoites in vitro was determined by counting the number of infected cells after 24 h incubation. (E) Infection of mice was determined by real-time PCR amplification of 18S rRNA in the liver 40 h after inoculation of sporozoites.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000008-g007: PbACα- sporozoites have defective exocytosis and infection.Exocytosis and infectivity of P. berghei WT (white bars), PbACα- C1 (black bars) and C2 (gray bars) sporozoites was analyzed. (A, B) Sporozoites were incubated or not with uracil derivatives (UD) or forskolin (FSK) (A) or 8Br-cAMP (B) for 1 h before fixation and quantification of exocytosis. (C) Sporozoites were added to filter insets containing confluent Hepa1-6 cells and collected on empty coverslips placed underneath the filters in the lower chamber. Percentage of sporozoites in coverslips showing apical-regulated exocytosis is shown. (D) Infection of Hepa1-6 cell by sporozoites in vitro was determined by counting the number of infected cells after 24 h incubation. (E) Infection of mice was determined by real-time PCR amplification of 18S rRNA in the liver 40 h after inoculation of sporozoites.
Mentions: We then tested whether apical regulated exocytosis was affected in PbACα-sporozoites. Activation of exocytosis by the mix of uracil derivatives or by forskolin, was greatly reduced in the two different clones of PbACα- sporozoites analyzed (Fig. 7A). Addition of a membrane permeant analogue of cAMP (8-Br-cAMP), which induces exocytosis in WT parasites, also stimulated exocytosis in PbACα- sporozoites (Fig. 7B). This result indicates that all sporozoite components required for exocytosis downstream of cAMP are functional in PbACα- sporozoites; however, the lack of ACα inhibits proper response upon activation with uracil derivatives or activators of AC activity. Migration through host cells induces apical regulated exocytosis in Plasmodium sporozoites [9]. To confirm that ACα is also required for exocytosis stimulated by migration through hepatocytes, we measured the response of WT and PbACα- sporozoites after migration through Hepa1-6 cells. We found that regulated exocytosis was not activated in sporozoites deficient in ACα (Fig. 7C).

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