Apicomplexan Phosphodiesterases (PDEs)

Apicomplexan Phosphodiesterases (PDEs)

• Apicomplexa are intracellular parasites infecting animals, where cyclic nucleotide signaling is vital for life stages and cellular processes.
• Cyclases and kinases, which produce and respond to cyclic nucleotides (cGMP and cAMP), exhibit high conservation and are essential in the parasite phylum.
• PDEs regulate cyclic nucleotide signaling by hydrolyzing cyclic nucleotides.

Apicomplexan PDE Biology and Therapeutic Interventions

• Recent research focuses on apicomplexan PDE biology and the potential for therapeutic interventions.
• Focus is on Plasmodium, Toxoplasma, Cryptosporidium, and Babesia which are major human apicomplexan parasite genera.
• Apicomplexan PDEs display a flexible range and are linked to various cellular needs throughout parasite and lifecycle phases.
• Conserved motility, host cell egress, and invasion of apicomplexan PDEs despite phylogenetic differences.
• PDE diversity and redundancy are key for optimizing cyclic nucleotide turnover to adapt to different environments in each parasite and life stage.
• A deeper comprehension of apicomplexan PDE regulation and integration of multiple signaling systems provides opportunities for future research.

Apicomplexa

• Encompasses single-celled intracellular parasites posing worldwide threats to human and animal health.
• Plasmodium is the greatest risk to human health, causing 247 million malaria cases and 619,000 deaths in 2021.
• Toxoplasma gondii, which is more prevalent but less deadly, infects approximately one-third of humans and causes toxoplasmosis.
• Toxoplasmosis can be life-threatening in immunocompromised patients or during fetal development.
• Cryptosporidium is a waterborne parasite linked to outbreaks and a primary factor in diarrhea-related deaths among infants and young children.
• Theileria, Babesia, Eimeria, Neospora, and Besnoitia cause significant economic losses due to livestock or poultry infections.
• Babesia causes zoonotic ailments in humans, resembling a moderate form of malaria.
• Apicomplexans demonstrate lifecycles and undergo multiple differentiation stages into morphologically unique forms across various hosts and environments.
• Multiple signaling pathways, including cyclic nucleotides(cAMP) and (cGMP), facilitate apicomplexan parasitism.

Cyclic Nucleotide PDEs

• Found in organisms across the eukaryotic tree and exhibit diverse functions.
• Cyclic nucleotide signaling relies on fine-tuning local intracellular levels to activate effectors like cAMP-dependent protein kinase (PKA) or cGMP-dependent protein kinase (PKG).
• Adenylyl- and guanylyl cyclases synthesize Cyclic AMP and GMP from purines ATP and GTP, respectively.
• Apicomplexan parasites are purine auxotrophs, the availability of purines could impact cyclic nucleotide signaling.
• Stimulation of cellular effectors occurs after cellular cyclic nucleotide concentrations reach threshold levels.
• Cellular effectors in apicomplexans include PKA, PKG, and a cAMP-binding protein called "exchange protein directly activated by cAMP".
• Cyclic nucleotides are degraded by 3',5'-cyclic nucleotide PDEs to fine-tune cyclic nucleotide levels.
• Apicomplexan PDEs regulate critical cellular processes, development, and dissemination within various lifecycle stages.

Classes Of PDEs

• The 3',5'-cyclic nucleotide PDE superfamily is divided into two major classes.
• Class I PDEs contains the H-D-[LIVMFY]-x-H-x-[AG]-x(2)-[NQ]-x-[LIVMFY] signature, well conserved in eukaryotes, with examples in bacteria and archaea.
• Class II PDEs contains the H-x-H-L-D-H-[LIVM]- x-[GS]-[LIVMA]-LIVM-x-S-[AP] signature, mostly found in bacteria and fungi, with rare examples in amoeba and slime molds.
• All apicomplexan PDEs are Class I, but the number of PDEs varies widely.
• Piroplasms like Babesia and Theileria encode two PDEs.
• Cryptosporidium possesses three PDEs.
• Plasmodium contains four PDEs.
• Toxoplasma and Neospora have 18 PDEs.
• The disparity in PDE prompts the question of whether the coccidia expansion is due to recent expansion or the retention of redundant genes.
• Apicomplexan PDEs belong to at least three major groups or clades and are widely represented by eukaryotes, suggesting no PDE family is truly "apicomplexan-specific".
• Clade A is represented most widely by Apicomplexa and includes human dual-specific (hPDE3) or cAMP-specific (hPDE4,7,8) PDEs, with apicomplexan PDEs resembling hPDE3.
• Clade B includes human PDE1, representatives from free-living alveolates, and coccidians among the apicomplexans.
• Clade B partial conservation suggests that these PDEs were dispensable or detrimental to most apicomplexans.
• Clade B PDEs in Toxoplasma (TgPDE4 and TgPDE16) are only expressed in cat stages.
• Clade C is represented by sequences from coccidia and cryptosporidia and includes human hPDE2, 5, 6, 9, 10, and 11.
• Coccidian PDEs can either be dual-specific (hPDE2,10,11) or cGMP-specific (hPDE5,6,9).
• Inference of substrate preference is cautioned, as specificities may diverge over time, and PDE repertoires satisfy distinct requirements of cyclic nucleotide turnover.
• The common ancestor of apicomplexans had a large PDE repertoire, similar to free-living alveolates.

PDE Regulatory Domains and Structure

• Structurally, Class I PDEs possess an N-terminal regulatory domain and a C-terminal catalytic domain.
• Apicomplexan PDEs have retained this architecture, but their regulatory domains have not been defined or characterized.
• Trans-membrane domains precede the catalytic domain in apicomplexan PDEs, similar to mammalian PDE3.
• The regulatory domains found in Class I PDEs are absent/diverged beyond recognition in apicomplexan PDEs.
• Regulatory domains absent in apicomplexan PDEs include upstream conserved region, Per-ARNT-Sim, receiver, and calmodulin-binding domain.
• Class I PDE families contain dual GAF domains (cGMP-binding PDEs, Anabaena adenylyl cyclase, and Escherichia coli FhlA), mediating dimerization or allosteric cGMP /cAMP binding.
• A single GAF or GAF-like domain is present in cAMP-specific PDE2 in Toxoplasma, conserved in other coccidians.
• Its function has not been assessed in apicomplexans.
• Regulation of apicomplexan PDEs likely occurs via expression and kinase phosphorylation due to lack of regulatory domains.
• Phosphoproteomics studies have revealed phosphorylation sites on representative apicomplexan PDEs, yet not investigated.

Cyclic Nucleotide PDEs in Plasmodium

• Functional analyses have relied on reverse genetics and/or pharmacological approaches.
• Multiple PDE inhibitors have proved valuable in assessing hydrolysis roles across developmental stages.
• Asexual blood stages
• Malaria pathology is tied to the proliferation of asexual blood stages.
• Precise timing of egress is crucial for parasite survival.
• Premature or late egress leads to non-invasive merozoites.
• PDEs centrally coordinate egress and invasion through cAMP/cGMP levels.
• Studies on Plasmodium PDEs focused on Plasmodium falciparum (Pf) or on the parasites infecting rodents, Plasmodium berghei (Pb) and Plasmodium yoelii (Py).

Asexual Blood Stages

• P. berghei PlasmoGEM and P. falciparum transposon mutagenesis project identified PbPDEẞ and PfPDEẞ as PDE genes essential for asexual blood stages.
• The expression of PfPDEẞ peaks at late trophozoite and schizont stages.
• This enzyme is transported via the endoplasmic reticulum to an apical location (secretory apical organelle), then discharged to plasma membrane of merozoites within mature schizonts.
• Conditional deletion of PfPDEẞ reduces invasion and causes post-invasion death, does not affect egress from the host erythrocyte.
• Treatment of early ring stages with the PDE inhibitor BIPPO phenocopied the PfPDEẞ-null post-invasion phenotype, suggesting that BIPPO targets PfPDEẞ.
• PfPDEẞ deletion reduced schizont cAMP and cGMP hydrolytic activity.
• Immunoprecipitated PDEẞ from schizonts is dual-specific, hydrolyzing both cAMP and cGMP.
• BIPPO abolished cAMP and cGMP hydrolytic activity confirmed the targeting of PDEẞ.
• Zaprinast, a precursor to human PDE5 inhibitors triggers cGMP-dependent merozoite egress, as a highly effective inhibitor of cAMP activity.
• PfPDEB deletion led to elevated cAMP levels and PKA activation but not PKG(Egress depends PKG activation)
• Suggests another PDE degrading cGMP is active in schizonts
• Plasmodium PDEẞ shows dual cAMP and cGMP activity and essential for asexual blood stages for controlling cAMP levels.
• Other active cGMP-specific PDEs likely complement the loss of cGMP hydrolytic activity that does not hydrolyze cAMP in schizonts.

Homeostasis

• Treatment with zaprinast rises schizont cGMP levels by inducing merozoite egress.
• As conditional deletion of PDEẞ did not impact schizont egress or global cGMP levels, this indicates that at least another cGMP-hydrolyzing PDE is active in schizont. -Conversely, BIPPO triggers egress in a cGMP-dependent manner, it targets a second cGMP PDE.
• Transcriptions of PDEa, PDEY, and PDES peak a late trophozoite/schizont stages, but reverse genetics reveals their non-essential roles for blood replication (Plasmodium species).
• Dual KO of PbPDEa and PbPDES showed and defect occurred in asexual blood stages.
• Expressed PyPDEY showed low expression levels in blood stages .

Sexual Blood Stages

• Transmission from humans to mosquito is mediated by an obligatory sexual lifecycle.
  • Differentiation from asexually replicating stages into non-dividing male and female gametocytes takes place inside red blood cells.
• P. falciparum gametocytes remodel the membrane of their erythrocyte host for maturation.
• Environmental signals trigger ready sexual precursors following maturation of sexual precursors (ingested by a mosquito).
• There are roles for cAMP-dependent signaling during gametocytogenesis.
  • For modification of the erythrocyte host
  • The prevention of premature activation of gametocytes in the mammalian host.
• High relative PDES suggests regulation of gametocytes by epistatic interactions, transcriptional studies show stage-specific expression patterns

Regulation in Gametocytes

• Activity is important for gametocyte maturation
• Gametocytes modify mechanical properties of their erythrocyte host to persist for circulation in the blood.
• PKA leads to GIE stiffness, rising PfPDES correlated to deformability.
• Characterization indicates PfPDEs is correlated with a cAMP increase (hydrolytic)
• Sildenafil impaired both retention in in-vitro circulation -Treatment increased gametocyte

Mosquito Stages

• Gametocytes need gliding motility for host infection
• Ookinete is banana-shaped, formed when fertilizing the female gamete
• Sporozoites are formed and move faster than ookinetes
• PbPDES led to cGMP-dependent motility, rounding, and gut invasion
• Deletion of PyPDEy increase non-infective Sporozoites levels
• CAMP leads to elevation

Cyclic Nucleotide Regulation in Cryptosporidium

• Cyclic nucleotide signaling insights are gained from other Apicomplexa, including Plasmodium and Toxoplasma.
• Cryptosporidium cyclic nucleotide signaling.
  • Cryptosporidium is intestinal parasite and cause diarrhea
  • Undergoes asexual and sexual lifecycles in a single host
  • Infections occur via ingestion of thick-walled oocysts, causing infection
• After 3 cycles, becomes undifferentiated Gametes
• CPKac is expressed through sporozoites

Babesia microti

• Sequence analysis of B. microti genome revealed conserved cyclic nucleotide signaling genes. -encodes one GC (BMR1_03g00165) with P4-ATPase-GCa architecture
  • contains N-terminal cyclase homology domains, resembling ACẞs.
  • one PKG (BMR1_04g05785).
  • two catalytic subunits
• 4. for cAMP
• BmPDEa is one gene

Apicomplexan PDE's As Drug Targets

• Potential drug are valid that are being pursued.
• Therapeutic target for human diseases
• PDE targeted therapeutics
• These molecules will provide simultaneous interactions
• Cyclic Camp is a promising PDE target
• There has been exploration in new anti-complexan drugs

Specificity Challenge

• Molecules that are used are applied to humans, which can lead to issues
• Human scaffolds will be required for apicomplexian use
• Four plasmodium PDE's will need to be targetted