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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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PbACα- complemented sporozoites recover the WT phenotype.(A) Schematic representation of the complement replacement vector, the ACα- disrupted locus and the complemented ACα locus. Correct integration of the construct results in the reconstitution of the disrupted ACα gene as shown. Arrows indicate the position of the primers used for PCR in B. (B) Complementation of ACα was shown by PCR (left) and by Southern analysis (right). PCR on DNA of WT, PbACα- C1 and complemented ACα (Cmp) results in the amplification of a fragment of 1 kb when using the primers indicated in (A). Genomic Southern blot hybridization of WT, PbACα- C1 and complemented ACα. The probe used for hybridization is represented in A. Integration of the complementation plasmid causes reduction in size of a 4.3-kb fragment in PbACα- C1 parasites to a 2.0-kb fragment in the ACα− complemented parasites. (C) Exocytosis of WT (white bars), PbACα- C1 (black bars) and complemented ACα (stripped bars) sporozoites in response to uracil derivatives (UD). (D) Infection of Hepa1-6 cells in vitro by WT, ACα- C1 (black bars) and complemented ACα (stripped bars) sporozoites was determined by counting infected cells 24 h after addition of sporozoites. * significant difference (p<0.01, ANOVA) compared to WT and complemented ACα.
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ppat-1000008-g008: PbACα- complemented sporozoites recover the WT phenotype.(A) Schematic representation of the complement replacement vector, the ACα- disrupted locus and the complemented ACα locus. Correct integration of the construct results in the reconstitution of the disrupted ACα gene as shown. Arrows indicate the position of the primers used for PCR in B. (B) Complementation of ACα was shown by PCR (left) and by Southern analysis (right). PCR on DNA of WT, PbACα- C1 and complemented ACα (Cmp) results in the amplification of a fragment of 1 kb when using the primers indicated in (A). Genomic Southern blot hybridization of WT, PbACα- C1 and complemented ACα. The probe used for hybridization is represented in A. Integration of the complementation plasmid causes reduction in size of a 4.3-kb fragment in PbACα- C1 parasites to a 2.0-kb fragment in the ACα− complemented parasites. (C) Exocytosis of WT (white bars), PbACα- C1 (black bars) and complemented ACα (stripped bars) sporozoites in response to uracil derivatives (UD). (D) Infection of Hepa1-6 cells in vitro by WT, ACα- C1 (black bars) and complemented ACα (stripped bars) sporozoites was determined by counting infected cells 24 h after addition of sporozoites. * significant difference (p<0.01, ANOVA) compared to WT and complemented ACα.

Mentions: To confirm that the phenotype observed in the PbACα- sporozoites is caused specifically by depletion of the PbACα gene, we complemented one of the PbACα- parasite lines with ACα. The correct replacement event was confirmed by PCR and Southern blot hybridization (Fig. 8A). No differences were found between the complemented parasite line and WT or PbACα- parasites during blood stage infection in mice or in mosquito oocyst development and salivary gland sporozoite numbers (not shown). We found that apical regulated exocytosis response to uracil derivatives was recovered in the complemented sporozoites (Fig. 8B). The infectivity of sporozoites was restored by complementation of the PbACα gene (Fig. 8C), confirming the role of PbACα in sporozoite exocytosis and 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)

PbACα- complemented sporozoites recover the WT phenotype.(A) Schematic representation of the complement replacement vector, the ACα- disrupted locus and the complemented ACα locus. Correct integration of the construct results in the reconstitution of the disrupted ACα gene as shown. Arrows indicate the position of the primers used for PCR in B. (B) Complementation of ACα was shown by PCR (left) and by Southern analysis (right). PCR on DNA of WT, PbACα- C1 and complemented ACα (Cmp) results in the amplification of a fragment of 1 kb when using the primers indicated in (A). Genomic Southern blot hybridization of WT, PbACα- C1 and complemented ACα. The probe used for hybridization is represented in A. Integration of the complementation plasmid causes reduction in size of a 4.3-kb fragment in PbACα- C1 parasites to a 2.0-kb fragment in the ACα− complemented parasites. (C) Exocytosis of WT (white bars), PbACα- C1 (black bars) and complemented ACα (stripped bars) sporozoites in response to uracil derivatives (UD). (D) Infection of Hepa1-6 cells in vitro by WT, ACα- C1 (black bars) and complemented ACα (stripped bars) sporozoites was determined by counting infected cells 24 h after addition of sporozoites. * significant difference (p<0.01, ANOVA) compared to WT and complemented ACα.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2279260&req=5

ppat-1000008-g008: PbACα- complemented sporozoites recover the WT phenotype.(A) Schematic representation of the complement replacement vector, the ACα- disrupted locus and the complemented ACα locus. Correct integration of the construct results in the reconstitution of the disrupted ACα gene as shown. Arrows indicate the position of the primers used for PCR in B. (B) Complementation of ACα was shown by PCR (left) and by Southern analysis (right). PCR on DNA of WT, PbACα- C1 and complemented ACα (Cmp) results in the amplification of a fragment of 1 kb when using the primers indicated in (A). Genomic Southern blot hybridization of WT, PbACα- C1 and complemented ACα. The probe used for hybridization is represented in A. Integration of the complementation plasmid causes reduction in size of a 4.3-kb fragment in PbACα- C1 parasites to a 2.0-kb fragment in the ACα− complemented parasites. (C) Exocytosis of WT (white bars), PbACα- C1 (black bars) and complemented ACα (stripped bars) sporozoites in response to uracil derivatives (UD). (D) Infection of Hepa1-6 cells in vitro by WT, ACα- C1 (black bars) and complemented ACα (stripped bars) sporozoites was determined by counting infected cells 24 h after addition of sporozoites. * significant difference (p<0.01, ANOVA) compared to WT and complemented ACα.
Mentions: To confirm that the phenotype observed in the PbACα- sporozoites is caused specifically by depletion of the PbACα gene, we complemented one of the PbACα- parasite lines with ACα. The correct replacement event was confirmed by PCR and Southern blot hybridization (Fig. 8A). No differences were found between the complemented parasite line and WT or PbACα- parasites during blood stage infection in mice or in mosquito oocyst development and salivary gland sporozoite numbers (not shown). We found that apical regulated exocytosis response to uracil derivatives was recovered in the complemented sporozoites (Fig. 8B). The infectivity of sporozoites was restored by complementation of the PbACα gene (Fig. 8C), confirming the role of PbACα in sporozoite exocytosis and 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