Adenylyl cyclase alpha and cAMP signaling mediate Plasmodium sporozoite apical regulated exocytosis and hepatocyte infection

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.

Figure 1. 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.

Figure 2. 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.

Figure 3. Treatment with an inhibitor of PKA reduces sporozoite exocytosis and infection.

P. yoelii sporozoites were pre-incubated with H89 followed by addition of uracil derivatives to induce exocytosis (A) or followed by incubation with monolayers of Hepa1-6 cells to quantify infection (B) and migration though cells (C). (D) Sporozoites were pre-incubated with H89 before addition of 8Br-cAMP to induce exocytosis. (E) Sporozoites were pre-incubated with genistein (Gen) before addition of uracil derivatives. (F) P. yoelii sporozoites were pre-incubated with 2′, 5′-Dideoxyadenosine (DDA) or SQ22536 (SQ) to inhibit adenylyl cyclase activity or with cAMP Rp-isomer to inhibit PKA, before addition of uracil derivatives to induce exocytosis. Results are expressed as mean of triplicates±SD. * p<0.05; ** p<0.01 when compared to control by ANOVA.

Figure 4. Extracellular K⁺ is required for sporozoite apical regulated exocytosis.

(A) P. yoelii sporozoites were pre-incubated for 15 min in regular medium or K⁺-free medium before addition or not of uracil derivatives (UD) for 45 min. (B) Sporozoites were incubated with regular medium or K⁺-free medium for 45 min, followed by incubation in regular medium in the presence or absence of UD for another 45 min. (C,D) Sporozoites were pre-incubated with the K⁺-channel inhibitors charybdotoxin (C) or margatoxin (D) for 15 min before addition of UD for 45 min. (E,F) sporozoites were pre-incubated for 15 min in regular medium or K⁺-free medium before addition or not of forskolin (E) or 8Br-cAMP (F). (G) Sporozoites were incubated with UD, ionomycin or 8Br-cAMP for 45 min. (H) Sporozoites were pre-incubated for 15 min in regular medium or Ca⁺⁺-free medium before addition or not of UD for 45 min. (I) Sporozoites were pre-incubated with the membrane permeant calcium chelator BAPTA-AM for 15 min before addition of UD for 45 min. Results are expressed as mean of triplicates±SD. ** p<0.01 when compared to control by ANOVA. (J) Possible model consistent with the results. UD activate directly or indirectly the K+ channel domain of ACα (1) and trigger the activation of AC activity (2). The increase in cAMP activates PKA (3), which leads to the activation of exocytosis.

Figure 5. 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.

Figure 6. PbACα- has normal blood-stage growth rates and sporozoite motility.

(A) Growth curves of P. berghei WT (black squares), PbACα- C1 (black circles) and C2 (white circles) in mice. (B) Gliding motility of sporozoites from WT, PbACα- C1 and C2 in the presence (right panel) or absence (left panel) of mouse albumin. Percentage of sporozoites that do not glide or do less than a complete circle (black bars), gliding sporozoites exhibiting 1 (dark gray bars), 2 to 10 (light gray bars), or >10 (white bars) circles per trail. (C) Migration through Hepa1-6 cells was measured as the number of dextran positive cells per coverslip. The difference between C1 or C2 and WT is not significantly different (p>0.05).

Figure 7. 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.

Figure 8. 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.