Peptidylarginine Deiminase (PAD) and Post-Translational Protein Deimination—Novel Insights into Alveolata Metabolism, Epigenetic Regulation and Host–Pathogen Interactions

SIMPLE SUMMARY: Alveolates are a major group of free living and parasitic organisms; some of which are serious pathogens of animals and humans. Apicomplexans and chromerids are two phyla belonging to the alveolates. Apicomplexans are obligate intracellular parasites; that cannot complete their life cycle without exploiting a suitable host. Chromerids are mostly photoautotrophs as they can obtain energy from sunlight; and are considered ancestors of the apicomplexans. The pathogenicity and life cycle strategies differ significantly between parasitic alveolates; with some causing major losses in host populations while others seem harmless to the host. As the life cycles of some are still poorly understood, a better understanding of the factors which can affect the parasitic alveolates’ life cycles and survival is of great importance and may aid in new biomarker discovery. This study assessed new mechanisms relating to changes in protein structure and function (so-called “deimination” or “citrullination”) in two key parasites—an apicomplexan and a chromerid—to assess the pathways affected by this protein modification. Our findings point to novel regulatory mechanisms in these parasites’ lifecycles via protein deimination and may provide novel insights into their adaptability to different environments and hosts as well as host–pathogen coevolution. ABSTRACT: The alveolates (Superphylum Alveolata) comprise a group of primarily single-celled eukaryotes that have adopted extremely diverse modes of nutrition, such as predation, photoautotrophy and parasitism. The alveolates consists of several major phyla including the apicomplexans, a large group of unicellular, spore forming obligate intracellular parasites, and chromerids, which are believed to be the phototrophic ancestors of the parasitic apicomplexans. Molecular pathways involved in Alveolata host–pathogen interactions, epigenetic regulation and metabolism in parasite development remain to be fully understood. Peptidylarginine deiminases (PADs) are a phylogenetically conserved enzyme family which causes post-translational protein deimination, affecting protein function through the conversion of arginine to citrulline in a wide range of target proteins, contributing to protein moonlighting in physiological and pathological processes. The identification of deiminated protein targets in alveolate parasites may therefore provide novel insight into pathogen survival and host-pathogen interactions. The current study assessed PAD homologues and deiminated protein profiles of two alveolate parasites, Piridium sociabile (Chromerida) and Merocystis kathae (Apicomplexa). Histological analysis verified strong cytoplasmic PAD expression in both Alveolates, detected deiminated proteins in nuclear and cytoplasmic compartments of the alveolate parasites and verified the presence of citrullinated histone H3 in Alveolata nucleus, indicating roles in epigenetic regulation. Histone H3 citrullination was also found significantly elevated in the host tissue, indicative of neutrophil extracellular trap formation, a host-defence mechanism against a range of pathogens, particularly those that are too large for phagocytosis. Proteomic analysis of deiminated proteins from both Alveolata identified GO and KEGG pathways strongly relating to metabolic and genetic regulation, with some species-specific differences between the apicomplexan and the chromerid. Our findings provide novel insights into roles for the conserved PAD/ADI enzyme family in the regulation of metabolic and epigenetic pathways in alveolate parasites, possibly also relating to their life cycle and host–pathogen interactions.