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

Generation of PbACα- parasite lines.(A) RNA from WT P. berghei sporozoites was reverse transcribed into cDNA and used as template to amplify ACα. Water was used as negative control (Neg) and wild type P. berghei genomic DNA (gDNA) as positive control. (B) Schematic representation of the ACα locus and the replacement vector. Correct integration of the construct results in the disrupted ACα gene as shown. Arrows indicate the position of the primers used for PCR in C. (C) Disruption of ACα was shown by PCR (left) and by Southern analysis (right). PCR on DNA of WT transfected population (before cloning) and PbACα- clones (C1 and C2) results in the amplification of two 0.7-kb WT fragments and a 0.8 and a 0.9-kb disrupted fragments when using the primers indicated in (B). Genomic Southern blot hybridization of WT and the PbACα- C1. The probe used for hybridization is represented in B. Integration of the targeting plasmid causes reduction in size of a 1.6-kb fragment in WT parasites to a 1.0-kb fragment in the PbACα- parasites. Similar results were found for PbACα- C2.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2279260&req=5

ppat-1000008-g005: Generation of PbACα- parasite lines.(A) RNA from WT P. berghei sporozoites was reverse transcribed into cDNA and used as template to amplify ACα. Water was used as negative control (Neg) and wild type P. berghei genomic DNA (gDNA) as positive control. (B) Schematic representation of the ACα locus and the replacement vector. Correct integration of the construct results in the disrupted ACα gene as shown. Arrows indicate the position of the primers used for PCR in C. (C) Disruption of ACα was shown by PCR (left) and by Southern analysis (right). PCR on DNA of WT transfected population (before cloning) and PbACα- clones (C1 and C2) results in the amplification of two 0.7-kb WT fragments and a 0.8 and a 0.9-kb disrupted fragments when using the primers indicated in (B). Genomic Southern blot hybridization of WT and the PbACα- C1. The probe used for hybridization is represented in B. Integration of the targeting plasmid causes reduction in size of a 1.6-kb fragment in WT parasites to a 1.0-kb fragment in the PbACα- parasites. Similar results were found for PbACα- C2.

Mentions: Microarray analysis had detected expression of PfACα in sporozoites [28]. To analyze the expression of PbACα, we isolated mRNA from P. berghei sporozoites and performed reverse transcription followed by PCR. We also found expression of this gene in sporozoites (Fig. 5A). Thus, we decided to pursue a targeted gene disruption at the blood stages to study the importance of ACα for the Plasmodium pre-erythrocytic life cycle stages. We created two independent cloned lines of P. berghei parasites that are deficient in ACα (PbACα-) by using targeted disruption of the ACα gene through double crossover homologous recombination (Fig. 5B). PbACα-deficiency of the mutant parasites was confirmed by RT-PCR and Southern Blotting (Fig. 5C).


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)

Generation of PbACα- parasite lines.(A) RNA from WT P. berghei sporozoites was reverse transcribed into cDNA and used as template to amplify ACα. Water was used as negative control (Neg) and wild type P. berghei genomic DNA (gDNA) as positive control. (B) Schematic representation of the ACα locus and the replacement vector. Correct integration of the construct results in the disrupted ACα gene as shown. Arrows indicate the position of the primers used for PCR in C. (C) Disruption of ACα was shown by PCR (left) and by Southern analysis (right). PCR on DNA of WT transfected population (before cloning) and PbACα- clones (C1 and C2) results in the amplification of two 0.7-kb WT fragments and a 0.8 and a 0.9-kb disrupted fragments when using the primers indicated in (B). Genomic Southern blot hybridization of WT and the PbACα- C1. The probe used for hybridization is represented in B. Integration of the targeting plasmid causes reduction in size of a 1.6-kb fragment in WT parasites to a 1.0-kb fragment in the PbACα- parasites. Similar results were found for PbACα- C2.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000008-g005: Generation of PbACα- parasite lines.(A) RNA from WT P. berghei sporozoites was reverse transcribed into cDNA and used as template to amplify ACα. Water was used as negative control (Neg) and wild type P. berghei genomic DNA (gDNA) as positive control. (B) Schematic representation of the ACα locus and the replacement vector. Correct integration of the construct results in the disrupted ACα gene as shown. Arrows indicate the position of the primers used for PCR in C. (C) Disruption of ACα was shown by PCR (left) and by Southern analysis (right). PCR on DNA of WT transfected population (before cloning) and PbACα- clones (C1 and C2) results in the amplification of two 0.7-kb WT fragments and a 0.8 and a 0.9-kb disrupted fragments when using the primers indicated in (B). Genomic Southern blot hybridization of WT and the PbACα- C1. The probe used for hybridization is represented in B. Integration of the targeting plasmid causes reduction in size of a 1.6-kb fragment in WT parasites to a 1.0-kb fragment in the PbACα- parasites. Similar results were found for PbACα- C2.
Mentions: Microarray analysis had detected expression of PfACα in sporozoites [28]. To analyze the expression of PbACα, we isolated mRNA from P. berghei sporozoites and performed reverse transcription followed by PCR. We also found expression of this gene in sporozoites (Fig. 5A). Thus, we decided to pursue a targeted gene disruption at the blood stages to study the importance of ACα for the Plasmodium pre-erythrocytic life cycle stages. We created two independent cloned lines of P. berghei parasites that are deficient in ACα (PbACα-) by using targeted disruption of the ACα gene through double crossover homologous recombination (Fig. 5B). PbACα-deficiency of the mutant parasites was confirmed by RT-PCR and Southern Blotting (Fig. 5C).

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