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Cited by 2Malaria Research and ControlBird parasitology and diseasesToxoplasma gondii Research Studies

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A divergent cyclic nucleotide binding protein promotes Plasmodium ookinete infection of the mosquito

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Kwecka, Dominika, Wang, Zhishuo, Eigminas, Edvardas, Liu, Lijia, Regan, Jennifer C., Kim, Choel, Philip, Nisha·2025·PLOS Pathogens
DOI 10.1371/journal.ppat.1013467PMC PMC12422582

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Abstract

Colonisation of mosquitos by the malarial parasite is critically reliant on the invasive ookinete stage. Ookinete invasion of mosquito is coordinated by the apical complex, a specialised parasite structure containing components for secretion, attachment and penetration. While studies have investigated cytoskeletal and secretory elements, it is currently unknown if signalling modules are present or functional at the apical complex. Here we elucidate the role of a cryptic cyclic nucleotide-binding protein which we name CBP-O. PbCBP-O showed a marked localisation to the ookinete apex and disruption of the protein severely compromised ookinete invasion of mosquitos. Domain dissection analysis revealed that the N- and C-termini have distinct functions. Intriguingly, PbCBP-O exhibits dual binding specificity to both cGMP and cAMP. Our findings suggest the apical tip of the ookinete is a platform to transduce cyclic nucleotide signals essential for malaria parasite transmission.

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Figures

Fig 1 Domain architecture and cellular localization of PbCBP-O. (A) Schematic representation of the domains of mammalian EPAC isoforms composed of a regulatory and a catalytic portion. PbCBP-O shows an uncharacterised N-terminal domain, followed by five putative cyclic nucleotide binding domains (1-4 and X). DEP = Dishevelled, Egl-10/pleckstrin, cyclic nucleotide binding domain = CNB, Ras exchange motif = REM, RAS association = RA and CDC25-homology domain = CDC25HD. (B) Live imaging of GFP-tagged PbCBP-O shows expression at the ookinete’s apical tip. Scale bar = 5μm.
Fig 1 Domain architecture and cellular localization of PbCBP-O. (A) Schematic representation of the domains of mammalian EPAC isoforms composed of a regulatory and a catalytic portion. PbCBP-O shows an uncharacterised N-terminal domain, followed by five putative cyclic nucleotide binding domains (1-4 and X). DEP = Dishevelled, Egl-10/pleckstrin, cyclic nucleotide binding domain = CNB, Ras exchange motif = REM, RAS association = RA and CDC25-homology domain = CDC25HD. (B) Live imaging of GFP-tagged PbCBP-O shows expression at the ookinete’s apical tip. Scale bar = 5μm.CC BY · PubMed Central
Fig 2 PbCBP-O regulates parasite transmission into the mosquito. (A) Schematic of strategy used for deletion of pbcbp-o. (B) In asexual blood stages, both wild-type and PbΔCBP-O parasites grow at a similar rate. Parasitaemia over three replication cycles. Error bars, ± SD and n = 3 independent experiments. (C) Exflagellation is not affected in PbΔCBP-O parasites (n = 2 independent experiments, paired t-test). (D) Ookinetes are formed in the absence of PbΔCBP-O parasites (n = 1). (E) PbΔCBP-O parasites fail to infect the mosquito midgut. Images show a mosquito midgut and infected green oocysts (dissections performed at day 7 post infection, wild-type n = 40, PbΔCBP-O n = 43, unpaired t-test). In all relevant panels, statistical significance is indicated as: ns = not significant, and **** p < 0.0001. For panels B, C, D and E, medians are indicated.
Fig 2 PbCBP-O regulates parasite transmission into the mosquito. (A) Schematic of strategy used for deletion of pbcbp-o. (B) In asexual blood stages, both wild-type and PbΔCBP-O parasites grow at a similar rate. Parasitaemia over three replication cycles. Error bars, ± SD and n = 3 independent experiments. (C) Exflagellation is not affected in PbΔCBP-O parasites (n = 2 independent experiments, paired t-test). (D) Ookinetes are formed in the absence of PbΔCBP-O parasites (n = 1). (E) PbΔCBP-O parasites fail to infect the mosquito midgut. Images show a mosquito midgut and infected green oocysts (dissections performed at day 7 post infection, wild-type n = 40, PbΔCBP-O n = 43, unpaired t-test). In all relevant panels, statistical significance is indicated as: ns = not significant, and **** p < 0.0001. For panels B, C, D and E, medians are indicated.CC BY · PubMed Central
Fig 3 PbCBP-O is required for efficient midgut traversal. (A) PbΔCBP-O ookinetes show regular motility. Average speed is 3.07 and 3.2 μm/min for wild-type and ΔCBP-O, respectively (wild-type n = 38; PbΔCBP-O n = 48, representative data from 3 independent experiments, unpaired t-test). (B) Comparable ookinete maturation in the mosquito midgut is observed in PbΔCBP-O and wildtype parasites. Nineteen or twenty-one hours post-infection, six midguts were scored per replicate (n = 2). Significance was analysed using an unpaired t-test. (C) Expression of micronemal proteins including CTRP, WARP and chitinase are unaffected in the absence of CBP-O. Actin is used as loading control (left panel). Chitinase secretion is maintained in PbΔCBP-O ookinetes. Western blot of culture supernatants from wild type and PbΔCBP-O ookinetes show similar levels of chitinase secretion (right panel), representative data from 2 independent experiments, (D) Immunofluorescence showing decreased ookinete presence in the basal side of mosquito midguts. Midguts were fixed and stained with P25 (plasma membrane marker) and DAPI 24-25 hours post-infection. P25+ parasites were quantified, and single dots show number of ookinetes counted per midgut (n = 20 midguts per genotype, unpaired t-test.). Scale bar = 200μm. (E) Quantification of salivary gland sporozoites. Each data point is an average of five infected mosquitoes (representative data from two independent experiments). (F) Expression of An. stephensi SRPN6 mRNA relative to An. stephensi rps7 in mosquitoes that were fed uninfected blood (control), fed with wt or PbΔCBP-O infected blood (Data are mean ± SD, n = 25 midguts per condition, pooled from 4 independent experiments, unpaired t-test with Welch’s correction). (G) Quantification of sporozoites isolated from salivary glands nineteen days post ookinete injections. Each data point is an average of five infected mosquitoes (representative data from n = 2 independent experiments, unpaired t-test). In all relevant panels, statistical significance is indicated as: ns = not significant, ** = p < 0.01, *** = p < 0.001 and **** = p < 0.0001.
Fig 3 PbCBP-O is required for efficient midgut traversal. (A) PbΔCBP-O ookinetes show regular motility. Average speed is 3.07 and 3.2 μm/min for wild-type and ΔCBP-O, respectively (wild-type n = 38; PbΔCBP-O n = 48, representative data from 3 independent experiments, unpaired t-test). (B) Comparable ookinete maturation in the mosquito midgut is observed in PbΔCBP-O and wildtype parasites. Nineteen or twenty-one hours post-infection, six midguts were scored per replicate (n = 2). Significance was analysed using an unpaired t-test. (C) Expression of micronemal proteins including CTRP, WARP and chitinase are unaffected in the absence of CBP-O. Actin is used as loading control (left panel). Chitinase secretion is maintained in PbΔCBP-O ookinetes. Western blot of culture supernatants from wild type and PbΔCBP-O ookinetes show similar levels of chitinase secretion (right panel), representative data from 2 independent experiments, (D) Immunofluorescence showing decreased ookinete presence in the basal side of mosquito midguts. Midguts were fixed and stained with P25 (plasma membrane marker) and DAPI 24-25 hours post-infection. P25+ parasites were quantified, and single dots show number of ookinetes counted per midgut (n = 20 midguts per genotype, unpaired t-test.). Scale bar = 200μm. (E) Quantification of salivary gland sporozoites. Each data point is an average of five infected mosquitoes (representative data from two independent experiments). (F) Expression of An. stephensi SRPN6 mRNA relative to An. stephensi rps7 in mosquitoes that were fed uninfected blood (control), fed with wt or PbΔCBP-O infected blood (Data are mean ± SD, n = 25 midguts per condition, pooled from 4 independent experiments, unpaired t-test with Welch’s correction). (G) Quantification of sporozoites isolated from salivary glands nineteen days post ookinete injections. Each data point is an average of five infected mosquitoes (representative data from n = 2 independent experiments, unpaired t-test). In all relevant panels, statistical significance is indicated as: ns = not significant, ** = p < 0.01, *** = p < 0.001 and **** = p < 0.0001.CC BY · PubMed Central
Fig 4 N- and C- terminal domains of PbCBP-O are important for ookinete function. (A) Schematic representation of full length and GFP tagged mutant PbCBP-O lacking N-terminus (ΔN-term) or cyclic nucleotide binding domains (ΔCNB). All mutants were generated in a background line constitutively expressing RFP. (B) Ookinete maturation of full length ΔCBP-O and the individual domain mutants (ΔN-term, ΔCNB1-4/X and ΔCNB4) are comparable to wildtype. n = 3 independent experiments. (C) Oocyst numbers in midguts infected with ΔCBP-O (null), ΔN-term, ΔCNB1-4/X, ΔCNB4 and wild-type 12 days post-infection. A dot represents the number of oocysts in a single midgut (n = number of midguts dissected, unpaired t-test). Pie chart shows infection prevalence (Fischer’s exact test). (D) Immunofluorescence imaging of ookinetes and early oocysts in mosquito midguts infected with ΔCBP-O (null), ΔN-term, ΔCNB1-4/X, ΔCNB4 and wild-type 24 hours post-infection. Scale bar = 200μM. (E) Quantification of ookinetes/early oocysts in the midgut. n = 10 midguts per genotype. (F) Quantification of salivary gland sporozoites. Each data point is an average of five infected mosquitoes. In all relevant panels, statistical significance is indicated as: ns = not significant, * p = 0.05, ** = p < 0.01, *** = p < 0.001 and **** = p < 0.0001. Experiments in C, D, E and F were independently repeated twice, and data is shown from one representative experiment.
Fig 4 N- and C- terminal domains of PbCBP-O are important for ookinete function. (A) Schematic representation of full length and GFP tagged mutant PbCBP-O lacking N-terminus (ΔN-term) or cyclic nucleotide binding domains (ΔCNB). All mutants were generated in a background line constitutively expressing RFP. (B) Ookinete maturation of full length ΔCBP-O and the individual domain mutants (ΔN-term, ΔCNB1-4/X and ΔCNB4) are comparable to wildtype. n = 3 independent experiments. (C) Oocyst numbers in midguts infected with ΔCBP-O (null), ΔN-term, ΔCNB1-4/X, ΔCNB4 and wild-type 12 days post-infection. A dot represents the number of oocysts in a single midgut (n = number of midguts dissected, unpaired t-test). Pie chart shows infection prevalence (Fischer’s exact test). (D) Immunofluorescence imaging of ookinetes and early oocysts in mosquito midguts infected with ΔCBP-O (null), ΔN-term, ΔCNB1-4/X, ΔCNB4 and wild-type 24 hours post-infection. Scale bar = 200μM. (E) Quantification of ookinetes/early oocysts in the midgut. n = 10 midguts per genotype. (F) Quantification of salivary gland sporozoites. Each data point is an average of five infected mosquitoes. In all relevant panels, statistical significance is indicated as: ns = not significant, * p = 0.05, ** = p < 0.01, *** = p < 0.001 and **** = p < 0.0001. Experiments in C, D, E and F were independently repeated twice, and data is shown from one representative experiment.CC BY · PubMed Central
Fig 5 The N-terminal domain guarantees accurate protein localisation. Live fluorescence microscopy with GFP-tagged CBP-O reveals requirement of the N-terminus for targeting CBP-O to the ookinete apex. The full-length and CNB domain mutant protein (ΔCNB1-4/X) maintain apical localisation. Scale bar = 5μM.
Fig 5 The N-terminal domain guarantees accurate protein localisation. Live fluorescence microscopy with GFP-tagged CBP-O reveals requirement of the N-terminus for targeting CBP-O to the ookinete apex. The full-length and CNB domain mutant protein (ΔCNB1-4/X) maintain apical localisation. Scale bar = 5μM.CC BY · PubMed Central
Fig 6 Structural conservation and binding properties of putative CNB domains. (A) Sequence alignment of the five CNBs of PbCBP-O with CNB-D of PfPKG and CNB2 of PfPKA-R illustrates that the domain organization is conserved. α-helices are shown in peach, β-barrels in green and the phosphate binding cleft (PBC) in magenta. Black arrows refer to the conserved glutamate (E), arginine (R) and threonine (T) in the PBC of PfCNB-D (E484, R493 and T494) and the PbCBP-O CNB1 (E1803, R1812, T1813), CNB3 (E2227, R2235, T2237) and CNB4 (E3014, R3023, T3024). The orange arrow marks the Tyrosine (Y) capping residue for PbCBP-O CNB1 (Y1853), CNB3 (Y2275) and CNB4 (Y3062) which is equivalent to the Tyrosine capping residue for PfPKA- R (Y424). Underlined residues highlight the conserved glycine triad. (B) Comparison of Alphafold model predictions of PbCBP-O-CNB1–4 and X to the crystal structure of PfCNB-D bound with cGMP (PDB ID: 4OFG) and CNB2 of PfPKA-R bound with cAMP (PDB ID: 5KBF). The PBC and αC helix of PfCNB-D/PfPKA-R -CNB2 are coloured in yellow and red respectively. The bound cGMP and cAMP are shown in sticks. A disordered insertion is predicted between β5 and β6 barrels of CNB3. Images were created in PyMOL. (C) cAMP and cGMP pull down assay with recombinantly expressed 6XHis tagged CNB-D from PbPKG and CNB1, 3 and 4 of PbCBP-O revealing that CNB3 binds both cAMP and cGMP. (D) Lysate from mature schizonts expressing GFP tagged ΔCNB1–4/X and ΔN-term isoforms of CBP-O were incubated with cAMP or cGMP agarose. Western blot analysis (α-GFP) revealed that upon removal of the N-terminal, the protein retains the capacity to bind cyclic nucleotides, while in the absence of CNB1–4/X, the mutant protein is unable to associate with cAMP or cGMP. n = 4 and 3 independent replicates for (C) and (D) respectively, and data is shown from a single representative experiment.
Fig 6 Structural conservation and binding properties of putative CNB domains. (A) Sequence alignment of the five CNBs of PbCBP-O with CNB-D of PfPKG and CNB2 of PfPKA-R illustrates that the domain organization is conserved. α-helices are shown in peach, β-barrels in green and the phosphate binding cleft (PBC) in magenta. Black arrows refer to the conserved glutamate (E), arginine (R) and threonine (T) in the PBC of PfCNB-D (E484, R493 and T494) and the PbCBP-O CNB1 (E1803, R1812, T1813), CNB3 (E2227, R2235, T2237) and CNB4 (E3014, R3023, T3024). The orange arrow marks the Tyrosine (Y) capping residue for PbCBP-O CNB1 (Y1853), CNB3 (Y2275) and CNB4 (Y3062) which is equivalent to the Tyrosine capping residue for PfPKA- R (Y424). Underlined residues highlight the conserved glycine triad. (B) Comparison of Alphafold model predictions of PbCBP-O-CNB1–4 and X to the crystal structure of PfCNB-D bound with cGMP (PDB ID: 4OFG) and CNB2 of PfPKA-R bound with cAMP (PDB ID: 5KBF). The PBC and αC helix of PfCNB-D/PfPKA-R -CNB2 are coloured in yellow and red respectively. The bound cGMP and cAMP are shown in sticks. A disordered insertion is predicted between β5 and β6 barrels of CNB3. Images were created in PyMOL. (C) cAMP and cGMP pull down assay with recombinantly expressed 6XHis tagged CNB-D from PbPKG and CNB1, 3 and 4 of PbCBP-O revealing that CNB3 binds both cAMP and cGMP. (D) Lysate from mature schizonts expressing GFP tagged ΔCNB1–4/X and ΔN-term isoforms of CBP-O were incubated with cAMP or cGMP agarose. Western blot analysis (α-GFP) revealed that upon removal of the N-terminal, the protein retains the capacity to bind cyclic nucleotides, while in the absence of CNB1–4/X, the mutant protein is unable to associate with cAMP or cGMP. n = 4 and 3 independent replicates for (C) and (D) respectively, and data is shown from a single representative experiment.CC BY · PubMed Central

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Metadata

Authors
Kwecka, D., Wang, Z., Eigminas, E., Liu, L., Regan, J. C., Kim, C., & Philip, N.
Affiliations
  • University of Edinburgh (GB)
  • Baylor College of Medicine (US)
Corresponding author
Philip, Nisha
ORCID
Venue
PLOS Pathogens
Publisher
Public Library of Science (PLoS)
Published
2025-09-02
Volume
21
Issue
9
Pages
e1013467
ISSN
  • 1553-7366 (ISSN-L)
  • 1553-7374
DOI
10.1371/journal.ppat.1013467
PMC
PMC12422582
OpenAlex
W4413924603
Cited by
2
References
53
Citations by year
2026: 2
FWCI
1.82
Type
journal article
Language
en
Update notices
new_version (2025-09-10)
Open access
Licence
Locations
  • PLoS Pathogens (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • Edinburgh Research Explorer (University of Edinburgh) (submittedVersion, cc-by)
  • Digital Commons-TMC (Texas Medical Center) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Wellcome Trust (212398/Z/18/Z)
  • Biotechnology and Biological Sciences Research Council (BB/M010996/1)
Topics
  • Health Sciences › Medicine › Public Health, Environmental and Occupational Health › Malaria Research and Control
  • Life Sciences › Immunology and Microbiology › Parasitology › Bird parasitology and diseases
  • Life Sciences › Immunology and Microbiology › Parasitology › Toxoplasma gondii Research Studies
Keywords
Plasmodium, mosquitoes, apical complex, cyclic GMP, cyclic AMP, malaria transmission, protein domain function
MeSH terms
Mosquito Vectors, Cyclic AMP, Animals, Cyclic GMP, Malaria, Culicidae, Plasmodium, Plasmodium berghei, Protozoan Proteins

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Kwecka, D., Wang, Z., Eigminas, E., Liu, L., Regan, J. C., Kim, C., & Philip, N. (2025). A divergent cyclic nucleotide binding protein promotes Plasmodium ookinete infection of the mosquito. PLOS Pathogens. https://doi.org/10.1371/journal.ppat.1013467

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